ORCID Profile
0000-0001-9098-3626
Current Organisation
University of South Australia
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Publisher: Wiley
Date: 25-01-2022
Abstract: An optical redox ratio can potentially be used to report on the dynamics of cell and tissue metabolism and define altered metabolic conditions for different pathologies. While there are methods to measure the optical redox ratio, they are not particularly suited to real‐time in situ or in vivo analysis. Here, we have developed a fiber‐optic system to measure redox ratios in cells and tissues and two mathematical models to enable real‐time, in vivo redox measurements. The optical redox ratios in tissue explants are correlated directly with endogenous NADH/FAD fluorescence emissions. We apply the mathematical models to the two‐photon microscopy data and show consistent results. We also used our fiber‐optic system to measure redox in different tissues and show consistent results between the two models, hence demonstrating proof‐of‐principle. This innovative redox monitoring system will have practical applications for defining different metabolic disease states.
Publisher: Springer Science and Business Media LLC
Date: 20-03-2019
DOI: 10.1038/S41598-019-41375-Y
Abstract: Dipeptidyl peptidase-4 inhibitors (DPP4i) are a class of orally available, small molecule inhibitors for the management of Type-II diabetes. A rapid, real-time, functional breath test for DPP4 enzyme activity could help to define DPP4i efficacy in patients that are refractory to treatment. We aimed to develop a selective, non-invasive, stable-isotope 13 C-breath test for DPP4. In vitro experiments were performed using high (Caco-2) and low (HeLa) DPP4 expressing cells. DPP gene expression was determined in cell lines by qRT-PCR. A DPP4 selective 13 C-tripeptide was added to cells in the presence and absence of the DPP4 inhibitor Sitagliptin. Gas s les were collected from the cell headspace and 13 CO 2 content quantified by isotope ratio mass spectrometry (IRMS). DPP4 was highly expressed in Caco-2 cells compared to HeLa cells and using the 13 C-tripeptide, we detected a high 13 CO 2 signal from Caco2 cells. Addition of Sitaglitpin to Caco2 cells significantly inhibited this 13 CO 2 signal. 13 C-assay DPP4 activity correlated positively with the enzyme activity detected using a colorimetric substrate. We have developed a selective, non-invasive, 13 C-assay for DPP4 that could have broad translational applications in diabetes and gastrointestinal disease.
Publisher: Springer Science and Business Media LLC
Date: 18-10-2019
DOI: 10.1038/S41598-019-51549-3
Abstract: Patient-derived explant (PDE) culture of solid tumors is increasingly being applied to preclinical evaluation of novel therapeutics and for biomarker discovery. In this technique, treatments are added to culture medium and penetrate the tissue via a gelatin sponge scaffold. However, the penetration profile and final concentrations of small molecule drugs achieved have not been determined to date. Here, we determined the extent of absorption of the clinical androgen receptor antagonist, enzalutamide, into prostate PDEs, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser/desorption ionisation (MALDI) mass spectrometry imaging (MSI). In a cohort of 11 PDE tissues from eight in idual patients, LC-MS/MS quantification of PDE homogenates confirmed enzalutamide (10 µM) uptake by all PDEs, which reached maximal average tissue concentration of 0.24–0.50 ng/µg protein after 48 h culture. Time dependent uptake of enzalutamide (50 µM) in PDEs was visualized using MALDI MSI over 24–48 h, with complete penetration throughout tissues evident by 6 h of culture. Drug signal intensity was not homogeneous throughout the tissues but had areas of markedly high signal that corresponded to drug target (androgen receptor)-rich epithelial regions of tissue. In conclusion, application of MS-based drug quantification and visualization in PDEs, and potentially other 3-dimensional model systems, can provide a more robust basis for experimental study design and interpretation of pharmacodynamic data.
Publisher: Elsevier BV
Date: 04-2004
Publisher: Portland Press Ltd.
Date: 04-1989
DOI: 10.1042/BJ2590199
Abstract: alpha-L-Iduronidase from human liver was purified by a three-step five-column procedure and by immunoaffinity chromatography with a monoclonal antibody raised against purified enzyme. Seven bands identified by staining with Coomassie Blue had molecular masses of 74, 65, 60, 49, 44, 18 and 13 kDa and were present in both preparations of the liver enzyme. However, relative to the immunopurification procedure, alpha-L-iduronidase purified by the five-column procedure was considerably enriched in the 65 kDa polypeptide band. The seven bands were identified by Western-blot analysis with two different monoclonal antibodies raised against alpha-L-iduronidase. The chromatographic behaviour of alpha-L-iduronidase on the antibody column was dependent upon the quantity of enzyme loaded. Above a particular load concentration a single peak of enzyme activity was eluted, whereas at load concentrations below the critical value alpha-L-iduronidase was eluted in two peaks of activity, designated form I (eluted first) and form II (eluted second). The following properties of the two forms of alpha-L-iduronidase were determined. (1) The two forms from liver were composed of different proportions of the same seven polypeptides. (2) When in idually rechromatographed on the antibody column, each form from liver shifted to a more retarded elution position but essentially retained its chromatographic behaviour relative to the other form. (3) Forms I and II of liver alpha-L-iduronidase showed no difference in their activities towards disaccharide substrates derived from two glycosaminoglycan sources, heparan sulphate and dermatan sulphate. (4) The native molecular size of forms I and II of liver alpha-L-iduronidase was 65 kDa as determined by gel-permeation chromatography. (5) Immunoaffinity chromatography of extracts of human lung and kidney resulted in the separation of alpha-L-iduronidase into two forms, each with different proportions of the seven common polypeptide species. (6) Lung forms I and II were taken up readily into cultured skin fibroblasts taken from a patient with alpha-L-iduronidase deficiency. Liver forms I and II were not taken up to any significant extent. Lung form II gave intracellular contents of alpha-L-iduronidase that were more than double those of normal control fibroblasts, whereas lung form I gave contents approximately equal to normal control values. We propose that all seven polypeptides are derived from a single alpha-L-iduronidase gene product, and that different proportions of these polypeptides can function as a single alpha-L-iduronidase entity.(ABSTRACT TRUNCATED AT 400 WORDS)
Publisher: Wiley
Date: 09-2016
Abstract: Live cell imaging can provide important information on cellular dynamics however, the full utilisation of this technology has been h ered by the limitations of imaging reagents. Metal-based complexes have the potential to overcome many of the issues common to many current imaging agents. The rhenium (I)-based complex fac-[Re(CO)3 (1,10-phenanthroline)(4-pyridyltetrazolate)], herein referred to as ReZolve-ER(™) , shows promise as a live cell imaging agent with rapid cell uptake, low cytotoxicity, resistance to photobleaching and compatibility with multicolour imaging. ReZolve-ER(™) localised to the nuclear membrane/endoplasmic reticulum (ER) and allowed the detection of exocytotic events at the plasma membrane. Thus, we present a new imaging agent for monitoring live cell events in real time, which is ideal for imaging either short- or long-time courses.
Publisher: Springer Science and Business Media LLC
Date: 20-02-2019
DOI: 10.1038/S41598-019-38864-5
Abstract: Toll-like receptor 7 (TLR7) is a pattern recognition receptor that recognizes viral RNA following endocytosis of the virus and initiates a powerful immune response characterized by Type I IFN production and pro-inflammatory cytokine production. Despite this immune response, the virus causes very significant pathology, which may be inflammation-dependent. In the present study, we examined the effect of intranasal delivery of the TLR7 agonist, imiquimod or its topical formulation Aldara, on the inflammation and pathogenesis caused by IAV infection. In mice, daily intranasal delivery of imiquimod prevented peak viral replication, bodyweight loss, airway and pulmonary inflammation, and lung neutrophils. Imiquimod treatment also resulted in a significant reduction in pro-inflammatory neutrophil chemotactic cytokines and prevented the increase in viral-induced lung dysfunction. Various antibody isotypes (IgG1, IgG2a, total IgG, IgE and IgM), which were increased in the BALF following influenza A virus infection, were further increased with imiquimod. While epicutaneous application of Aldara had a significant effect on body weight, it did not reduce neutrophil and eosinophil airway infiltration indicating less effective drug delivery for this formulation. We concluded that intranasal imiquimod facilitates a more effective immune response, which can limit the pathology associated with influenza A virus infection.
Publisher: Wiley
Date: 16-01-2014
DOI: 10.1002/PROS.22777
Publisher: CRC Press
Date: 08-05-1981
Publisher: Elsevier BV
Date: 2005
DOI: 10.1016/J.YMGME.2004.09.004
Abstract: Mucopolysaccharidosis I is a lysosomal storage disorder caused by a deficiency of the lysosomal hydrolase alpha-l-iduronidase, which is required for the degradation of heparan sulphate and dermatan sulphate. Given the wide spectrum of disease severity in mucopolysaccharidosis I patients, one of the challenges for managing the disorder is to accurately predict clinical phenotype. Enzyme replacement therapy by intravenous infusion is unlikely to make a significant impact on central nervous system pathology and patients displaying this clinical manifestation may respond better to bone marrow transplantation. In order to predict whether mucopolysaccharidosis I patients are going to develop central nervous system pathology, we investigated a number of biochemical parameters in cultured skin fibroblasts from patients of different genotype henotype. Residual levels of alpha-l-iduronidase activity and protein were determined using sensitive immune-quantification assays and fibroblast cell extracts from patients with central nervous system pathology generally had lower levels of alpha-l-iduronidase than patients with no evidence of central nervous system disease. A total of 15 oligosaccharides, derived from heparan sulphate and dermatan sulphate, was measured in fibroblast extracts using electrospray-ionisation tandem mass spectrometry and all were shown to discriminate mucopolysaccharidosis I from controls. Of these, two trisaccharides were able to group patients based on the presence/absence of central nervous system disease. Moreover, a ratio of alpha-l-iduronidase activity to these trisaccharides provided clear discrimination between mucopolysaccharidosis I patients with and without central nervous system pathology. We suggest that this type of analysis may be very useful for predicting disease severity in mucopolysaccharidosis I patients.
Publisher: Wiley
Date: 10-05-1993
DOI: 10.1007/BF00711520
Publisher: Elsevier BV
Date: 10-1992
DOI: 10.1016/0022-1759(92)90279-3
Abstract: A method for the purification of mouse monoclonal antibodies from hybridoma culture supernatants is described. The protocol involves the use of a combination of three previously described methods for the concentration and purification of monoclonal antibodies. Firstly, hybridomas were grown in a Diacult dialysis system (Inter Med Laboratory, Denmark) to yield milligram quantities of monoclonal antibody in a culture supernatant. Monoclonal antibodies were then purified from the culture supernatant by precipitation with polyethylene glycol 6000 (PEG 6000) and finally reprecipitated using an ammonium sulphate procedure. The PEG 6000 treatment caused a density change in the ammonium sulphate immunoglobulin precipitate, and resulted in the formation of a pellicle which contained pure mouse monoclonal antibody. The protocol removed contaminating bovine serum immunoglobulin as well as other serum and cellular protein from the monoclonal antibody preparations.
Publisher: Elsevier BV
Date: 02-2023
DOI: 10.1016/J.PATHOL.2022.08.001
Abstract: Diagnosis and assessment of patients with prostate cancer is dependent on accurate interpretation and grading of histopathology. However, morphology does not necessarily reflect the complex biological changes occurring in prostate cancer disease progression, and current biomarkers have demonstrated limited clinical utility in patient assessment. This study aimed to develop biomarkers that accurately define prostate cancer biology by distinguishing specific pathological features that enable reliable interpretation of pathology for accurate Gleason grading of patients. Online gene expression databases were interrogated and a pathogenic pathway for prostate cancer was identified. The protein expression of key genes in the pathway, including adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (Appl1), Sortilin and Syndecan-1, was examined by immunohistochemistry (IHC) in a pilot study of 29 patients with prostate cancer, using monoclonal antibodies designed against unique epitopes. Appl1, Sortilin, and Syndecan-1 expression was first assessed in a tissue microarray cohort of 112 patient s les, demonstrating that the monoclonal antibodies clearly illustrate gland morphologies. To determine the impact of a novel IHC-assisted interpretation (the utility of Appl1, Sortilin, and Syndecan-1 labelling as a panel) of Gleason grading, versus standard haematoxylin and eosin (H&E) Gleason grade assignment, a radical prostatectomy s le cohort comprising 114 patients was assessed. In comparison to H&E, the utility of the biomarker panel reduced subjectivity in interpretation of prostate cancer tissue morphology and improved the reliability of pathology assessment, resulting in Gleason grade redistribution for 41% of patient s les. Importantly, for equivocal IHC-assisted labelling and H&E staining results, the cancer morphology interpretation could be more accurately applied upon re-review of the H&E tissue sections. This study addresses a key issue in the field of prostate cancer pathology by presenting a novel combination of three biomarkers and has the potential to transform clinical pathology practice by standardising the interpretation of the tissue morphology.
Publisher: American Society of Hematology
Date: 08-1981
DOI: 10.1182/BLOOD.V58.2.406.406
Abstract: A new monoclonal antibody, FMC7, was studied in 68 patients with chronic B-cell leukemia. All 17 cases of prolymphocytic leukemia (B- PLL) and 8 of 9 of hairy-cell leukemia were positive. In contrast, FMC7 was negative in 32 of 38 chronic lymphocytic leukemias (B-CLL p less than 0.001) and 4 cases of B-cell lymphoma. Four of the 6 positive B- CLL cases were in “prolymphocytoid” transformation two of them had bright membrane Ig (SmIg) staining and may represent an intermediate form between B-CLL and B-PLL. Although there was a tendency for the intensity of the immunofluorescence reaction with FMC7 and SmIg to change in parallel, FMC7 did not correlate with any Ig class. In addition, almost all FMC7-negative B-CLL had weak expression of SmIg. FMC7 is different from other monoclonal antibodies raised against B- lineage cells in that it recognizes only some subsets, presumably those at a late stage of maturation. This property confers diagnostic potential to this reagent and may contribute to the better characterization of the B-cell neoplasias.
Publisher: Portland Press Ltd.
Date: 15-02-1991
DOI: 10.1042/BJ2740263
Abstract: alpha-L-Iduronidase synthesis and maturation were analysed in fibroblasts from normal controls and from alpha-L-iduronidase-deficient mucopolysaccharidosis-type-I (MPS-I) patients. Fibroblasts were radiolabelled with [3H]leucine and alpha-L-iduronidase was isolated from cell lysates or culture medium by monoclonal-antibody affinity chromatography. Pulse-chase labelling of normal control fibroblasts showed that alpha-L-iduronidase was synthesized as an 81 kDa precursor and processed within 24 h via intermediates of 76 kDa and 70 kDa to a 69 kDa species. The incorporation of radiolabel into alpha-L-iduronidase in fibroblasts from three of four MPS-I patients was at levels that were either very low or undetectable. Fibroblasts from one MPS-I patient, however, exhibited levels of incorporation of radiolabelled amino acid into alpha-L-iduronidase similar to those shown by normal control fibroblasts, despite having undetectable alpha-L-iduronidase enzyme activity. The maturation of alpha-L-iduronidase in fibroblasts from this patient was delayed compared with normal controls and showed accumulation of the 76 kDa intermediate, as well as the major 69 kDa, form of the enzyme.
Publisher: Springer Science and Business Media LLC
Date: 31-03-2021
DOI: 10.1186/S12943-021-01347-1
Abstract: Cancer cells that transit from primary tumours into the circulatory system are known as circulating tumour cells (CTCs). These cancer cells have unique phenotypic and genotypic characteristics which allow them to survive within the circulation, subsequently extravasate and metastasise. CTCs have emerged as a useful diagnostic tool using “liquid biopsies” to report on the metastatic potential of cancers. However, CTCs by their nature interact with components of the blood circulatory system on a constant basis, influencing both their physical and morphological characteristics as well as metastatic capabilities. These properties and the associated molecular profile may provide critical diagnostic and prognostic capabilities in the clinic. Platelets interact with CTCs within minutes of their dissemination and are crucial in the formation of the initial metastatic niche. Platelets and coagulation proteins also alter the fate of a CTC by influencing EMT, promoting pro-survival signalling and aiding in evading immune cell destruction. CTCs have the capacity to directly hijack immune cells and utilise them to aid in CTC metastatic seeding processes. The disruption of CTC clusters may also offer a strategy for the treatment of advance staged cancers. Therapeutic disruption of these heterotypical interactions as well as direct CTC targeting hold great promise, especially with the advent of new immunotherapies and personalised medicines. Understanding the molecular role that platelets, immune cells and the coagulation cascade play in CTC biology will allow us to identify and characterise the most clinically relevant CTCs from patients. This will subsequently advance the clinical utility of CTCs in cancer diagnosis rognosis.
Publisher: Elsevier BV
Date: 09-2002
DOI: 10.1016/S1096-7192(02)00148-8
Abstract: Immune response to replacement therapy has been reported for a range of therapeutic strategies being developed for the treatment of patients with genetic disease. The potential problem of immune response to enzyme replacement therapy has been investigated in alpha-L-iduronidase immunized rats, representing a model of the lysosomal storage disorder Hurler syndrome (alpha-L-iduronidase deficiency). The antibody response to alpha-L-iduronidase showed that the positional location of antibody reactivity was similar for different immunized rats, but the precise linear sequence epitopes identified, varied between rats. A monoclonal antibody reacting to an epitope in close proximity to one high antigenicity site on alpha-L-iduronidase was used to reproduce the in vivo effect of altered enzyme tissue distribution, previously observed in immunized rats infused with alpha-L-iduronidase. The study demonstrated that during an immune response, antibody reacting to a single epitope could partially control the tissue distribution of antigen from circulation.
Publisher: Wiley
Date: 07-02-2019
Publisher: Elsevier BV
Date: 07-2005
DOI: 10.1016/J.CELLIMM.2005.08.024
Abstract: CD107a, also known as the lysosome associated membrane protein-1 (LAMP-1), is expressed largely in the endosome-lysosome membranes of cells, but is also found on the plasma membrane (1-2% of total LAMP-1). LAMP-1 has been implicated in a variety of cellular functions, including cancer metastasis. It has been proposed as a therapeutic agent for some cancers, and is a marker for lysosomal storage disorders and different cell types such as cytotoxic T cells. In light of this ersity of applications, it is important to have well characterized immune-reagents for the detection and quantification of LAMP-1. We have compared a new monoclonal antibody 80280 against LAMP-1 to an existing monoclonal antibody BB6 and a rabbit polyclonal antibody. While all antibodies gave similar results by immunofluorescence, the monoclonal antibody 80280 showed no epitope reactivity to LAMP-1 peptides, suggesting the possibility of a carbohydrate epitope. Western blotting revealed a weaker activity of the monoclonal antibody 80280 relative to either the BB6 monoclonal or the polyclonal antibodies. The monoclonal antibody 80280 is distinct from BB6, providing an additional reagent for CD107a analysis.
Publisher: Mary Ann Liebert Inc
Date: 05-2020
Publisher: American Physiological Society
Date: 03-2020
DOI: 10.1152/PHYSIOLGENOMICS.00092.2019
Abstract: There are critical molecular mechanisms that can be activated to induce myocardial repair, and in humans this is most efficient during fetal development. The timing of heart development in relation to birth and the size/electrophysiology of the heart are similar in humans and sheep, providing a model to investigate the repair capacity of the mammalian heart and how this can be applied to adult heart repair. Myocardial infarction was induced by ligation of the left anterior descending coronary artery in fetal (105 days gestation when cardiomyocytes are proliferative) and adolescent sheep (6 mo of age when all cardiomyocytes have switched to an adult phenotype). An ovine gene microarray was used to compare gene expression in sham and infarcted (remote, border and infarct areas) cardiac tissue from fetal and adolescent hearts. The gene response to myocardial infarction was less pronounced in fetal compared with adolescent sheep hearts and there were unique gene responses at each age. There were also region-specific changes in gene expression between each age, in the infarct tissue, tissue bordering the infarct, and tissue remote from the infarction. In total, there were 880 genes that responded to MI uniquely in the adolescent s les compared with 170 genes in the fetal response, as well as 742 overlap genes that showed concordant direction of change responses to infarction at both ages. In response to myocardial infarction, there were specific changes in genes within pathways of mitochondrial oxidation, muscle contraction, and hematopoietic cell lineages, suggesting that the control of energy utilization and immune function are critical for effective heart repair. The more restricted gene response in the fetus may be an important factor in its enhanced capacity for cardiac repair.
Publisher: MDPI AG
Date: 05-02-2020
DOI: 10.3390/CELLS9020372
Abstract: Innate immunity is critical for host defence against pathogen and environmental challenge and this involves the production and secretion of immune mediators, such as antimicrobial peptides and pro-inflammatory cytokines. However, when dysregulated, innate immunity can contribute to multifactorial diseases, including inflammatory rheumatic disorders, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and even septic shock. During an innate immune response, antimicrobial peptides and cytokines are trafficked via Rab11 multivesicular endosomes, and then sorted into Rab11 vesicles for traffic to the plasma membrane and secretion. In this study, a cyclin-dependent kinase inhibitor CDKI-73 was used to determine its effect on the innate immune response, based on previously identified targets for this compound. Our results showed that CDKI-73 inhibited the delivery of Rab11 vesicles to the plasma membrane, resulting in the accumulation of large multivesicular Rab11 endosomes near the cell periphery. In addition to the effect on endosome delivery, CDKI-73 down-regulated the amount of innate immune cargo, including the antimicrobial peptide Drosomycin and pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFα). We concluded that CDKI-73 has the potential to regulate the delivery and secretion of certain innate immune cargo, which could be used to control inflammation.
Publisher: Elsevier BV
Date: 2003
DOI: 10.1016/S1096-7192(02)00200-7
Abstract: In this study we have investigated a group of 29 Brazilian patients, who had been diagnosed with the lysosomal storage disorder, Mucopolysaccharidosis type I (MPS-I). MPS I is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase. Ninety percent of the MPS I patients in this study were genotyped and revealed 10 recurrent and thirteen novel IDUA gene mutations. Eight of these new mutations and three common mutations W402X, P533R, and R383H were in idually expressed in CHO-K1 cells and analyzed for alpha-L-iduronidase protein and enzyme activity. A correlation was observed between the MPS I patient clinical phenotype and the associated mutant alpha-L-iduronidase protein/enzyme activity expressed in CHO-K1 cells. This was the first time that Brazilian MPS I patients had been thoroughly analyzed and highlighted the difficulties of mutation screening and clinical phenotype assessment in populations with high numbers of unique mutations.
Publisher: Elsevier BV
Date: 08-2006
DOI: 10.1016/J.YMGME.2006.02.013
Abstract: Lysosomal storage disorders (LSD) are chronic progressive diseases that have a devastating impact on the patient and family. Most patients are clinically normal at birth but develop symptoms early in childhood. Despite no curative treatment, a number of therapeutic options are available to improve quality of life. To achieve this, there is a pressing need for newborn screening to identify affected in iduals early, before the onset of severe irreversible pathology. We have developed a multiplexed immune-quantification assay of 11 different lysosomal proteins for the identification of in iduals with an LSD and evaluated this assay in a retrospective study using blood-spots from newborns subsequently diagnosed with an LSD (n=19, six different LSD), in iduals s led after diagnosis of an LSD (n=92, 11 different LSD), newborn controls (n=433), and adult controls (n=200). All patients with mucopolysaccharidosis type I (MPS I), MPS II, MPS IIIA, MPS VI, metachromatic leukodystrophy, Niemann-Pick disease type A/B, and multiple sulfatase deficiency could be identified by reduced enzyme levels compared to controls. All mucolipidosis type II/III patients were identified by the elevation of several lysosomal enzymes, above the control range. Most Fabry, Pompe, and Gaucher disease patients were identified from either single protein differences or profiles of multiple protein markers. Newborn screening for multiple LSD is achievable using multiplexed immune-quantification of a panel of lysosomal proteins. With further validation, this method could be readily incorporated into existing screening laboratories and will have a substantial impact on patient management and counseling of families.
Publisher: Mary Ann Liebert Inc
Date: 05-2020
Publisher: Impact Journals, LLC
Date: 14-10-2015
Publisher: Wiley
Date: 18-03-2019
DOI: 10.1111/RESP.13524
Publisher: Elsevier BV
Date: 02-2022
DOI: 10.1016/J.EXPHEM.2021.12.193
Abstract: Many cancers rely on glucose as an energy source, but it is becoming increasingly apparent that some cancers use alternate substrates to fuel their proliferation. Chronic lymphocytic leukaemia (CLL) is one such cancer. Through the use of flow cytometry and confocal microscopy, low levels of glucose uptake were observed in the OSU-CLL and HG3 CLL cell lines relative to highly glucose-avid Raji cells (Burkitt's lymphoma). Glucose uptake in CLL cells correlated with low expression of the GLUT1 and GLUT3 receptors. In contrast, both CLL cell lines and primary CLL cells, but not healthy B cells, were found to rapidly internalise medium- and long-chain, but not short-chain, fatty acids (FAs). Differential FA uptake was also observed in primary cells taken from patients with unmutated immunoglobulin heavy variable chain usage (IGHV) compared with patients with mutated IGHV. Delipidation of serum in the culture medium slowed the proliferation and significantly reduced the viability of OSU-CLL and HG3 cells, effects that were partially reversed by supplementation with a chemically defined lipid concentrate. These observations highlight the potential importance of FAs in the pathogenesis of CLL and raise the possibility that targeting FA utilisation may represent a novel therapeutic and prognostic approach in this disease.
Publisher: Elsevier BV
Date: 05-2003
Publisher: Frontiers Media SA
Date: 23-03-2022
DOI: 10.3389/FPHAR.2022.870156
Abstract: There is an urgent need to develop effective therapeutic strategies including immunomodulators to combat influenza A virus (IAV) infection. Influenza A viruses increase ROS production, which suppress anti-viral responses and contribute to pathological inflammation and morbidity. Two major cellular sites of ROS production are endosomes via the NOX2-oxidase enzyme and the electron transport chain in mitochondria. Here we examined the effect of administration of Cgp91ds-TAT, an endosome-targeted NOX2 oxidase inhibitor, in combination with mitoTEMPO, a mitochondrial ROS scavenger and compared it to monotherapy treatment during an established IAV infection. Mice were infected with IAV (Hkx31 strain 10 4 PFU/mouse) and 24 h post infection were treated with Cgp91ds-TAT (0.2 mg/kg), mitoTEMPO (100 μg) or with a combination of these inhibitors [Cgp91ds-TAT (0.2 mg/kg)/mitoTEMPO (100 μg)] intranasally every day for up to 2 days post infection (pi). Mice were euthanized on Days 3 or 6 post infection for analyses of disease severity. A combination of Cgp91ds-TAT and mitoTEMPO treatment was more effective than the ROS inhibitors alone at reducing airway and neutrophilic inflammation, bodyweight loss, lung oedema and improved the lung pathology with a reduction in alveolitis following IAV infection. Dual ROS inhibition also caused a significant elevation in Type I IFN expression at the early phase of infection (day 3 pi), however, this response was suppressed at the later phase of infection (day 6 pi). Furthermore, combined treatment with Cgp91ds-TAT and mitoTEMPO resulted in an increase in IAV-specific CD8 + T cells in the lungs. In conclusion, this study demonstrates that the reduction of ROS production in two major subcellular sites, i.e. endosomes and mitochondria, by intranasal delivery of a combination of Cgp91ds-TAT and mitoTEMPO, suppresses the severity of influenza infection and highlights a novel immunomodulatory approach for IAV disease management.
Publisher: MDPI AG
Date: 16-06-2023
Abstract: Gleason scoring is used within a five-tier risk stratification system to guide therapeutic decisions for patients with prostate cancer. This study aimed to compare the predictive performance of routine H& E or biomarker-assisted ISUP (International Society of Urological Pathology) grade grouping for assessing the risk of biochemical recurrence (BCR) and clinical recurrence (CR) in patients with prostate cancer. This retrospective study was an assessment of 114 men with prostate cancer who provided radical prostatectomy s les to the Australian Prostate Cancer Bioresource between 2006 and 2014. The prediction of CR was the primary outcome (median time to CR 79.8 months), and BCR was assessed as a secondary outcome (median time to BCR 41.7 months). The associations of (1) H& E ISUP grade groups and (2) modified ISUP grade groups informed by the Appl1, Sortilin and Syndecan-1 immunohistochemistry (IHC) labelling were modelled with BCR and CR using Cox proportional hazard approaches. IHC-assisted grading was more predictive than H& E for BCR (C-statistic 0.63 vs. 0.59) and CR (C-statistic 0.71 vs. 0.66). On adjusted analysis, IHC-assisted ISUP grading was independently associated with both outcome measures. IHC-assisted ISUP grading using the biomarker panel was an independent predictor of in idual BCR and CR. Prospective studies are needed to further validate this biomarker technology and to define BCR and CR associations in real-world cohorts.
Publisher: Elsevier BV
Date: 02-2015
DOI: 10.1016/J.YMGME.2014.11.005
Abstract: Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from deficiency of α-galactosidase A (GLA). Traditionally, heterozygotes were considered asymptomatic carriers of FD, but it is now apparent that the asymptomatic female carrier is the exception and most heterozygotes suffer significant multisystemic disease. To determine why the process of cross-correction does not occur effectively in FD heterozygotes, we investigated GLA production and secretion in cultured skin fibroblasts as well as GLA levels in plasma. The maturation of GLA was similar in FD heterozygotes and control fibroblasts, confirming that both produce the 46kDa mature form the same as that present in control plasma. However, the proportion of GLA secreted into the culture media was substantially less than eight other lysosomal proteins. Artificial generation of FD heterozygotes in cellulo, along with another lysosomal storage disorder, mucopolysaccharidosis type II, revealed no cross-correction in the FD system, whereas MPS II fibroblasts were able to cross-correct. In plasma, GLA was present as the 46kDa mature form, which lacks the mannose 6-phosphorylated moiety and is not able to be efficiently endocytosed by affected cells. Our evidence shows that fibroblasts secrete minimal amounts of GLA and consequently normal fibroblasts are unable to cross-correct FD fibroblasts. We suggest that symptomatic FD heterozygotes arise due to the secretion of primarily the mature form, with only small amounts of the mannose 6-phosphorylated form of GLA from unaffected cells. This limits capacity for enzyme cross correction of affected cells, despite uptake of exogenous recombinant GLA.
Publisher: Elsevier BV
Date: 08-2006
DOI: 10.1016/J.MOLMED.2006.06.001
Abstract: Lysosomal storage disorders are a group of inherited diseases that can result in severe and progressive pathology due to a specific lysosomal dysfunction. Current treatment strategies include bone-marrow transplantation, substrate reduction, chemical-chaperone and enzyme-replacement therapy. However, each of these treatments has its limitations. Enhanced stop-codon read-through is a potential alternative or adjunct therapeutic strategy for treating lysosomal-storage-disorder patients. Premature stop-codon mutations have been identified in a large cohort of patients with a lysosomal storage disorder, making stop-codon read-through a possible treatment for this disease. In lysosomal-storage-disorder cells (mucopolysaccharidosis type I, alpha-L-iduronidase deficient), preclinical studies have shown that gentamicin induced the read-through of premature stop codons, resulting in enzyme activity that reduced substrate storage.
Publisher: Oxford University Press (OUP)
Date: 11-2004
DOI: 10.1373/CLINCHEM.2004.037937
Abstract: Background: Fabry disease is an X-linked inborn error of glycosphingolipid catabolism resulting from a deficiency of the lysosomal exoglycohydrolase, α-galactosidase. Enzyme replacement therapy is currently available for Fabry disease, but early diagnosis before the onset of irreversible pathology will be mandatory for successful treatment. Presymptomatic detection would be possible through the use of a newborn-screening program. We report on the use of sensitive assays for the measurement of α-galactosidase protein and activity and for the protein saposin C, which are diagnostic markers for Fabry disease. Methods: Two sensitive immunoassays for the measurement of α-galactosidase activity and protein were used to determine the concentrations of α-galactosidase in dried filter-paper blood spots and plasma s les from control patients and patients with a lysosomal storage disorder (LSD). Results: Fabry hemizygous in iduals were clearly identified from control populations by decreases in both α-galactosidase activity and protein. Fabry heterozygotes generally fell between the hemizygotes and controls. Including the measurement of saposin C enabled differentiation between Fabry heterozygotes and controls. In blood spots, all Fabry in iduals could be distinguished from control blood spots as well as from 16 other LSD patients. Conclusions: The determination of α-galactosidase activity or protein in dried filter-paper blood spots could be used for the diagnosis of Fabry patients. With further validation, these assays could be used for the identification of Fabry patients in newborn-screening programs and may also be suitable for screening high-risk populations.
Publisher: Wiley
Date: 08-1983
Publisher: Wiley
Date: 27-02-1991
DOI: 10.1007/BF01804381
Abstract: Using cDNA microarrays we determined the gene expression patterns in the human acute promyelocytic leukemia (APL) cell line NB4 during all-trans retinoic acid (ATRA)-induced differentiation. We analyzed the expression of 12,288 genes in the NB4 cells after 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours of ATRA exposure. During this time course, we found 168 up-regulated and more than 179 down-regulated genes, most of which have not been reported before. Many of the altered genes encode products that participate in signaling pathways, cell differentiation, programmed cell death, transcription regulation, and production of cytokines and chemokines. Of interest, the CD52 and protein kinase A regulatory subunit alpha (PKA-Rlalpha) genes, whose products are being used as therapeutic targets for certain human neoplasias in currently ongoing clinical trials, were among the genes observed to be markedly up-regulated after ATRA treatment. The present study provides valuable data to further understand the mechanism of ATRA-induced APL cell differentiation and suggests potential therapeutic alternatives for this leukemia.
Publisher: MDPI AG
Date: 26-03-2013
DOI: 10.3390/CELLS2020188
Publisher: Elsevier BV
Date: 04-2000
Abstract: Enzyme replacement therapy (ERT) has been developed and trialed for the treatment of human lysosomal storage disorder patients. The viability of ERT for the treatment of these severe multiple pathology disorders has subsequently been established. However, in both animal model studies and human clinical trials, some in iduals have been shown to develop an immune response to the replacement protein. This potential complication for treatment has been investigated by the infusion of recombinant human alpha-L-iduronidase (rh-alpha-L-iduronidase) into nonimmune and immunized rats to simulate mucopolysaccharidosis type I ERT in the presence of different levels of antibody. In rats with high antibody titers to rh-alpha-L-iduronidase (titer 1,024,000) there was evidence of altered organ distribution and subcellular targeting when compared to either lower titer immunized rats (titers less than 64,000) or nonimmune rats (titers 512-1024). In addition, hypersensitivity reactions were observed for high titer rats (titer 1,024,000) during rh-alpha-L-iduronidase infusion, but not for the other two treatment groups. A rat with an antibody titer of 64,000 had only minor changes in subcellular targeting and organ distribution when infused with rh-alpha-L-iduronidase. This implied that a high level of antibody was required to effect changes in alpha-L-iduronidase enzyme targeting and distribution. Notably, in the high titer rats, the antibody produced appeared to increase the tissue and subcellular level of rh-alpha-L-iduronidase specific activity. This suggested that antibody production may not always result in an adverse effect on ERT.
Publisher: Elsevier BV
Date: 08-1998
DOI: 10.1016/S0925-4439(98)00034-9
Abstract: Immune responses to enzyme replacement therapy (ERT) have been reported and can result in a hypersensitivity/anaphylactic reaction during or immediately after enzyme infusion. We have investigated the infusion of the lysosomal enzyme N-acetylgalactosamine 4-sulphatase (4-sulphatase) into immunized, high titre rats as a model of immune response to ERT. To simulate ERT, high and low titre rats were infused with different doses of radiolabelled recombinant human 4-sulphatase (3H-rh4S). There was evidence of altered targeting, inactivation and degradation of 4-sulphatase in high titre (titre 1024000) compared to low titre (titre 64) rats. There was more 4-sulphatase enzyme activity detected in 5 mg/kg high titre rats when compared to 1 mg/kg high titre rats, suggesting that the antibodies could be saturable in vivo. However, the rats treated with 5 mg/kg 3H-rh4S all had clinical signs of hypersensitivity reactions to 4-sulphatase infusion. There were no apparent signs of adverse reactions in either the high titre 1 mg/kg rats or the low titre rats (1, 5 mg/kg). The high titre 5 mg/kg rats also had changes in 3H-rh4S distribution, with lower levels delivered to the liver and a marked increase in the level remaining in plasma, when compared to either 1 mg/kg high titre rats or low titre rats (1, 5 mg/kg).
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1038/MT.2012.9
Publisher: The Company of Biologists
Date: 07-2011
DOI: 10.1242/JCS.080598
Abstract: The secretion of anti-microbial peptides is recognised as an essential step in innate immunity, but there is limited knowledge of the molecular mechanism controlling the release of these effectors from immune response cells. Here, we report that Drosophila 14-3-3ε mutants exhibit reduced survival when infected with either Gram-positive or Gram-negative bacteria, indicating a functional role for 14-3-3ε in innate immunity. In 14-3-3ε mutants, there was a reduced release of the anti-microbial peptide Drosomycin into the haemolymph, which correlated with an accumulation of Drosomycin-containing vesicles near the plasma membrane of cells isolated from immune response tissues. Drosomycin appeared to be delivered towards the plasma membrane in Rab4- and Rab11-positive vesicles and smaller Rab11-positive vesicles. RNAi silencing of Rab11 and Rab4 significantly blocked the anterograde delivery of Drosomycin from the perinuclear region to the plasma membrane. However, in 14-3-3ε mutants there was an accumulation of small Rab11-positive vesicles near the plasma membrane. This vesicular phenotype was similar to that observed in response to the depletion of the vesicular Syntaxin protein Syx1a. In wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, and partially overlapping with Syx1a, on vesicles near the plasma membrane. We conclude that 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response.
Publisher: American Physiological Society
Date: 02-2011
Abstract: Fructose is a hexose sugar that is being increasingly consumed in its monosaccharide form. Patients who exhibit fructose malabsorption can present with gastrointestinal symptoms that include chronic diarrhea and abdominal pain. However, with no clearly established gastrointestinal mechanism for fructose malabsorption, patient analysis by the proxy of a breath hydrogen test (BHT) is controversial. The major transporter for fructose in intestinal epithelial cells is thought to be the facilitative transporter GLUT5. Consistent with a facilitative transport system, we show here by analysis of past studies on healthy adults that there is a significant relationship between fructose malabsorption and fructose dose ( r = 0.86, P 0.001). Thus there is a dose-dependent and limited absorption capacity even in healthy in iduals. Changes in fructose malabsorption with age have been observed in human infants, and this may parallel the developmental regulation of GLUT5 expression. Moreover, a GLUT5 knockout mouse has displayed the hallmarks associated with profound fructose malabsorption. Fructose malabsorption appears to be partially modulated by the amount of glucose ingested. Although solvent drag and passive diffusion have been proposed to explain the effect of glucose on fructose malabsorption, this could possibly be a result of the facilitative transporter GLUT2. GLUT5 and GLUT2 mRNA have been shown to be rapidly upregulated by the presence of fructose and GLUT2 mRNA is also upregulated by glucose, but in humans the distribution and role of GLUT2 in the brush border membrane are yet to be definitively decided. Understanding the relative roles of these transporters in humans will be crucial for establishing a mechanistic basis for fructose malabsorption in gastrointestinal patients.
Publisher: Oxford University Press (OUP)
Date: 09-2001
Abstract: Mucopolysaccharidosis type I (MPS I McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were in idually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.
Publisher: Oxford University Press (OUP)
Date: 09-2006
DOI: 10.1373/CLINCHEM.2005.064915
Abstract: Background: Lysosomal storage disorders are a group of genetic diseases, each with a broad spectrum of clinical presentation that ranges from attenuated to severe. The immunochemical analysis of patient s les is aimed at several key aspects of patient management, including early detection of the disorder, prediction of clinical severity, determining the most appropriate therapeutic regimen, and monitoring of patients on therapy. Methods: In this study, we review the current and emerging technology available to achieve these assessments. Results: Immune assays have direct practical application for the early detection, diagnosis and prognosis of lysosomal storage disorder patients. Multiplexing of these assays may provide a platform to allow newborn screening for multiple lysosomal storage disorders. Conclusions: We have reviewed the immunochemical techniques available for the analysis of lysosomal storage disorder patient s les and advise that these may be used in conjunction with other technologies for effective patient management.
Publisher: Elsevier BV
Date: 11-2013
DOI: 10.1016/J.METABOL.2013.06.013
Abstract: Intrauterine growth restriction that results in low birth weight (LBW) has been linked to the onset of pathological cardiac hypertrophy. An altered transition from a fetal to an adult energy metabolism phenotype, with increased reliance on glucose rather than fatty acids for energy production, could help explain this connection. We have therefore investigated cardiac metabolism in relation to left ventricular hypertrophy in LBW lambs, at 21days after birth. The expression of regulatory molecules involved in cardiac glucose and fatty acid metabolism was measured using real-time PCR and Western blotting. A section of the left ventricle was fixed for Periodic Acid Schiff staining to determine tissue glycogen content. There was increased abundance of insulin signalling pathway proteins (phospho-insulin receptor, insulin receptor and phospho-Akt) and the glucose transporter (GLUT)-1, but no change in GLUT-4 or glycogen content in the heart of LBW compared to ABW lambs. There was, however, increased abundance of cardiac pyruvate dehydrogenase kinase 4 (PDK-4) in LBW compared to ABW lambs. There were no significant changes in the mRNA expression of components of the peroxisome proliferator activated receptor regulatory complex or proteins involved in fatty acid metabolism. We concluded that LBW induced left ventricular hypertrophy was associated with increased GLUT-1 and PDK-4, suggesting increased glucose uptake, but decreased efficacy for the conversion of glucose to ATP. A reduced capacity for energy conversion could have significant implications for vulnerability to cardiovascular disease in adults who are born LBW.
Publisher: Elsevier BV
Date: 02-2000
Publisher: Springer Science and Business Media LLC
Date: 12-07-2017
DOI: 10.1038/S41467-017-00057-X
Abstract: The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease.
Publisher: Elsevier BV
Date: 07-1999
Publisher: Wiley
Date: 28-08-2012
DOI: 10.1111/J.1440-1681.2011.05649.X
Abstract: 1. World-wide epidemiological and experimental animal studies demonstrate that adversity in fetal life, resulting in intrauterine growth restriction, programmes the offspring for a greater susceptibility to ischaemic heart disease and heart failure in adult life. 2. After cardiogenesis, cardiomyocyte endowment is determined by a range of hormones and signalling pathways that regulate cardiomyocyte proliferation, apoptosis and the timing of multinucleation/terminal differentiation. 3. The small fetus may have reduced cardiomyocyte endowment owing to the impact of a suboptimal intrauterine environment on the signalling pathways that regulate cardiomyocyte proliferation, apoptosis and the timing of terminal differentiation.
Publisher: Elsevier BV
Date: 08-2022
Publisher: Elsevier BV
Date: 12-2018
Publisher: S. Karger AG
Date: 1984
DOI: 10.1159/000212661
Abstract: Peripheral blood from aging and young humans and patients with Down's syndrome, and from age- and sex-matched controls, was studied for the proportions of surface immunoglobulin (SIg+) bearing and monoclonal antibodies FMC1, and FMC7 defined B lymphocytes and B lymphocyte subsets using fluorescent-activated cell sorter. In aging humans, the proportion of SIg+ and FMC1+ (that detect all B lymphocytes) were comparable to simultaneously studied healthy young controls. However, FMC7+ (that detects a subset of B cells) B cells were significantly (p less than 0.05) increased when compared to young subjects. In aging subjects, the proportions of FMC7+ B cells were comparable to their FMC1+ B cells, whereas in young subjects FMC7+ B cells were a subset of FMC1+ B cells. In Down's syndrome, a phenomenon similar to aging humans was observed, that is the proportions of FMC7+ were increased when compared to age- and sex-matched controls and were comparable to their own FMC1+ B cells. This study demonstrates the abnormality of B lymphocytes in human aging and Down's syndrome. The significance of these findings is discussed.
Publisher: Elsevier BV
Date: 02-2012
Abstract: Glycogen storage in the α-glucosidase knockout((6neo/6neo)) mouse recapitulates the biochemical defect that occurs in the human condition as such, this mouse serves as a model for the inherited metabolic deficiency of lysosomal acid α-glucosidase known as Pompe disease. Although this model has been widely used for the assessment of therapies, the time course of glycogen accumulation that occurs as untreated Pompe mice age has not been reported. To address this, we developed a quantitative method involving amyloglucosidase digestion of glycogen and quantification of the resulting free glucose by liquid chromatography/electrospray ionization-tandem mass spectrometry. The method was sensitive enough to measure as little as 0.1 μg of glycogen in tissue extracts with intra- and interassay coefficients of variation of less than 12%. Quantification of glycogen in tissues from Pompe mice from birth to 26 weeks of age showed that, in addition to the accumulation of glycogen in the heart and skeletal muscle, glycogen also progressively accumulated in the brain, diaphragm, and skin. Glycogen storage was also evident at birth in these tissues. This method may be particularly useful for longitudinal assessment of glycogen reduction in response to experimental therapies being trialed in this model.
Publisher: Oxford University Press (OUP)
Date: 16-04-2013
DOI: 10.1111/NURE.12020
Abstract: Fructose malabsorption came to prominence in the pediatric arena as so-called "apple juice diarrhea," with excess consumption of fructose being linked to gastrointestinal symptoms such as diarrhea and abdominal pain. Over the past two decades the amount of fructose in children's diets has been increasing in the United States. A test for fructose malabsorption has yet to be fully validated, due mainly to the lack of an established etiology. In animal models, however, the fructose transporter GLUT5 is developmentally regulated, and this could be consistent with the greater susceptibility of children, especially toddlers, to fructose malabsorption. Additionally, the available evidence indicates the fructose breath hydrogen test has no apparent diagnostic utility in infants younger than 1 year it may, therefore, be advisable to test for malabsorption by dietary exclusion in these patients. The present review aims to expound on the biological basis for fructose malabsorption in children and evaluate the current evidence for diagnostic procedures in order to identify clinical testing strategies that can be recommended and areas where further investigation is required.
Publisher: American Physiological Society
Date: 04-2015
DOI: 10.1152/AJPREGU.00346.2014
Abstract: The cardiac insulin-like growth factor 2 receptor (IGF-2R) can induce cardiomyocyte hypertrophy in a heterotrimeric G protein receptor-coupled manner involving αq (G αq ) or αs (G αs ). We have previously shown increased left ventricular weight and cardiac IGF-2 and IGF-2R gene expression in low-birth-weight (LBW) compared with average-birth-weight (ABW) lambs. Here, we have investigated the cardiac expression of IGF-2 gene variants, the degree of histone acetylation, and the abundance of proteins in the IGF-2R downstream signaling pathway in ABW and LBW lambs. S les from the left ventricle of ABW and LBW lambs were collected at 21 days of age. There was increased phospho-CaMKII protein with decreased HDAC 4 abundance in the LBW compared with ABW lambs. There was increased GATA 4 and decreased phospho-troponin I abundance in LBW compared with ABW lambs, which are markers of pathological cardiac hypertrophy and impaired or reduced contractility, respectively. There was increased histone acetylation of H3K9 at IGF-2R promoter and IGF-2R intron 2 differentially methylated region in the LBW lamb. In conclusion, histone acetylation of IGF-2R may lead to increased IGF-2R mRNA expression and subsequently mediate G αq signaling early in life via CaMKII, resulting in an increased risk of left ventricular hypertrophy and cardiovascular disease in adult life.
Publisher: Wiley
Date: 28-06-2018
DOI: 10.1113/JP276072
Publisher: American Association for Cancer Research (AACR)
Date: 12-2014
DOI: 10.1158/1541-7786.MCR-14-0074
Abstract: Prostate cancer is the second most common form of cancer in males, affecting one in eight men by the time they reach the age of 70 years. Current diagnostic tests for prostate cancer have significant problems with both false negatives and false positives, necessitating the search for new molecular markers. A recent investigation of endosomal and lysosomal proteins revealed that the critical process of endosomal biogenesis might be altered in prostate cancer. Here, a panel of endosomal markers was evaluated in prostate cancer and nonmalignant cells and a significant increase in gene and protein expression was found for early, but not late endosomal proteins. There was also a differential distribution of early endosomes, and altered endosomal traffic and signaling of the transferrin receptors (TFRC and TFR2) in prostate cancer cells. These findings support the concept that endosome biogenesis and function are altered in prostate cancer. Microarray analysis of a clinical cohort confirmed the altered endosomal gene expression observed in cultured prostate cancer cells. Furthermore, in prostate cancer patient tissue specimens, the early endosomal marker and adaptor protein APPL1 showed consistently altered basement membrane histology in the vicinity of tumors and concentrated staining within tumor masses. These novel observations on altered early endosome biogenesis provide a new avenue for prostate cancer biomarker investigation and suggest new methods for the early diagnosis and accurate prognosis of prostate cancer. Implications: This discovery of altered endosome biogenesis in prostate cancer may lead to novel biomarkers for more precise cancer detection and patient prognosis. Mol Cancer Res 12(12) 1851–62. ©2014 AACR.
Publisher: Elsevier BV
Date: 09-1998
DOI: 10.1016/S0925-4439(98)00046-5
Abstract: Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype henotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.
Publisher: Wiley
Date: 09-06-1997
DOI: 10.1016/S0014-5793(97)00423-7
Abstract: Genetic diseases associated with an enzyme deficiency frequently have reduced intracellular levels of the mutant protein, despite apparently normal levels of message and protein synthesis. It has been suggested that the endoplasmic reticulum (ER) can recognise mutant protein as incorrectly folded and invoke 'quality control' processes which cause the retention and degradation of this protein. This process may occur, even for mutations which do not abrogate protein activity, contributing directly to pathophysiology. Genetic diseases associated with defects in ER and Golgi processing proteins have also been reported and generally result in impaired processing of multiple protein products. In this review the role of the ER and Golgi in the pathogenesis of genetic diseases relating to the vacuolar network are discussed.
Publisher: Elsevier BV
Date: 03-1999
Abstract: The lysosome-associated membrane protein (LAMP-1) is elevated in the cells and plasma from lysosomal storage disorder-affected in iduals however, the mechanism of this elevation is not well defined. In this study we have investigated the synthesis, glycoprocessing, trafficking, and turnover of LAMP-1 in human skin fibroblasts from Pompe disease patients and control in iduals. There were similar levels of LAMP-1 synthesis in both cell types, but glycoprocessing was retarded in Pompe (T1/2 = 25 min) compared to control (T1/2 = 17 min) fibroblasts. There was also a marked delay in trafficking of LAMP-1 to lysosomes of Pompe (T1/2 = 200 min) compared to control (T1/2 = 100 min) cells. A proportion of newly synthesized LAMP-1 (5.4% in Pompe and 8.5% in controls) was trafficked out of the cell (T1/2 = 3.5 h in controls) and, although significantly smaller than the lysosomal form, still had a transmembrane domain and cytoplasmic tail. In contrast, a soluble lysosomal pool of LAMP-1 had no tail sequence, suggesting that it had been clipped from the membrane. In turnover studies, LAMP-1 was more stable in Pompe (T1/2 = 4.9 days) compared to control (T1/2 = 1. 6 days) cells, implying either reduced proteolysis or lysosomal function, in Pompe cells. These results indicate altered traffic and turnover of LAMP-1 in storage disorders and identify different intracellular and extracellular pools of soluble LAMP-1, suggesting alternative trafficking pathways.
Publisher: Elsevier BV
Date: 02-2007
DOI: 10.1016/J.CCA.2006.08.030
Abstract: Mucopolysaccharidosis type IVA (MPS IVA Morquio syndrome) is a lysosomal storage disorder caused by a deficiency in the activity of the lysosomal hydrolase N-acetylgalactosamine-6-sulfatase (GALNS). MPS IVA patients can present with severe myelopathy, hearing loss, heart valve involvement, short trunk/dwarfism and corneal clouding. Early diagnosis of MPS IVA will allow potential treatments to be implemented before the onset of irreversible pathology. We have developed a sensitive immune-quantification assay for the accurate detection of GALNS protein in skin fibroblasts, blood and plasma from unaffected control and MPS IVA patients. MPS IVA patient fibroblast extracts (n=11) had non-detectable (ND)-10 ng/mg of 6-sulfatase protein compared to 3-82 ng/mg for normal controls (n=19). Dried blood-spots from MPS IVA patients (n=4) contained ND-1.3 ng/L of 6-sulfatase protein compared to 18-145 ng/L for normal controls (n=49). Plasma from MPS IVA patients (n=7) contained ND 6-sulfatase protein compared to 1-9 ng/L for normal controls (n=49). The immune assay described here had the capacity to accurately measure the amount of GALNS protein in various biological s les, providing the basis of an assay that could be further developed to enable newborn and high-risk population screening for MPS IVA patients.
Publisher: Elsevier BV
Date: 10-1994
Abstract: Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI MPS VI) is a disorder which results from a deficiency in the lysosomal associated enzyme N-acetylgalactosamine 4-sulfatase (4-sulfatase). A feline model of human MPS VI has previously been described and provides a system for the evaluation of enzyme replacement therapy protocols. As a preliminary study to human 4-sulfatase enzyme replacement therapy in feline we have compared the immunochemical properties of human and feline 4-sulfatase. By SDS-PAGE the molecular mass of purified feline and human 4-sulfatase were similar under both reducing and nonreducing conditions. There was, however, a detectable conformation difference between human and feline 4-sulfatase indicating some structural variation. Feline 4-sulfatase reacted weakly with a panel of monoclonal antibodies in an immunobinding assay (interacting with 4-sulfatase in free solution), but the same monoclonal antibodies reacted strongly with feline 4-sulfatase in an immunoquantification assay where the feline 4-sulfatase was bound to a polyclonal antibody (which presumably induces a conformation change in the feline 4-sulfatase to closer approximate the structure of human 4-sulfatase). A monoclonal antibody which selectively reacts with human 4-sulfatase has been used to develop an assay suitable for evaluating human 4-sulfatase enzyme replacement in cat tissues.
Publisher: Elsevier BV
Date: 09-2023
Publisher: Oxford University Press (OUP)
Date: 04-2006
DOI: 10.1373/CLINCHEM.2005.061838
Abstract: Background: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder related to a deficiency in the enzyme iduronate-2-sulfatase (IDS). Clinical trials of enzyme replacement therapy are in progress, but effective treatment will require screening assays to enable early detection and diagnosis of MPS II. Our study evaluated the diagnostic accuracy of IDS protein and enzyme activity measurements in dried blood spots and plasma. Methods: We collected dried-blood-spot and plasma s les from unaffected control in iduals and from MPS II patients. We measured IDS protein concentration with a 2-step time-delayed dissociation-enhanced lanthanide fluorescence immunoassay. To measure enzyme activity, we immobilized anti-IDS antibody on microtiter plates to capture the enzyme and measured its activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. Results: Dried-blood-spot s les from MPS II patients showed an almost total absence of IDS activity (0–0.075 μmol · h−1 · L−1) compared with control blood spots (0.5–4.7 μmol · h−1 · L−1) and control plasma (0.17–8.1 μmol · h−1 · L−1). A dried-blood-spot s le from only 1 of 12 MPS II patients had detectable concentrations of IDS protein (24.8 μg/L), but no IDS protein was detected in plasma from MPS II patients. Ranges for IDS protein in control s les were 25.8–153 μg/L for blood spots and 22.8–349.4 μg/L for plasma. Conclusion: Measurement of the IDS protein concentration and enzyme activity (as measured by a simple fluorogenic assay with an immune capture technique) enables identification of the majority of MPS II patient s les from both dried blood spots and plasma s les.
Publisher: Hindawi Limited
Date: 2004
DOI: 10.1002/HUMU.10313
Abstract: Mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each in idual exon of the ARSB gene was lified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356_358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient.
Publisher: Scitechnol Biosoft Pvt. Ltd.
Date: 2015
Publisher: MDPI AG
Date: 06-12-2017
DOI: 10.3390/IJMS18122628
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4RA00050A
Abstract: Neutral Re( i ) tetrazolato complexes exhibit labeling of lipid droplets with high specificity.
Publisher: Wiley
Date: 31-07-2013
DOI: 10.1111/PBI.12096
Publisher: MDPI AG
Date: 29-07-2022
Abstract: Macrophages undergo a metabolic switch from oxidative phosphorylation to glycolysis when exposed to gram-negative bacterial lipopolysaccharide (LPS), which modulates antibacterial host defence mechanisms. Here, we show that LPS treatment of macrophages increased the classical oxidative burst response via the NADPH oxidase (NOX) 2 enzyme, which was blocked by 2-deoxyglucose (2-DG) inhibition of glycolysis. The inhibition of the pentose phosphate pathway with 6-aminonicotinamide (6-AN) also suppressed the LPS-induced increase in NOX2 activity and was associated with a significant reduction in the mRNA expression of NOX2 and its organizer protein p47phox. Notably, the LPS-dependent enhancement in NOX2 oxidase activity was independent of both succinate and mitochondrial reactive oxygen species (ROS) production. LPS also increased type I IFN-β expression, which was suppressed by 2-DG and 6-AN and, therefore, is dependent on glycolysis and the pentose phosphate pathway. The type I IFN-β response to LPS was also inhibited by apocynin pre-treatment, suggesting that NOX2-derived ROS promotes the TLR4-induced response to LPS. Moreover, recombinant IFN-β increased NOX2 oxidase-dependent ROS production, as well as NOX2 and p47phox expression. Our findings identify a previously undescribed molecular mechanism where both glycolysis and the pentose phosphate pathway are required to promote LPS-induced inflammation in macrophages.
Publisher: American Society for Clinical Investigation
Date: 15-04-1996
DOI: 10.1172/JCI118617
Publisher: Elsevier BV
Date: 07-1997
Abstract: Lysosomal biogenesis is an orchestration of the structural and functional elements of the lysosome to form an integrated organelle and involves the synthesis, targeting, functional residence, and turnover of the proteins that comprise the lysosome. We have investigated lysosomal biogenesis during the formation and dissipation of storage vacuoles in two model systems. One involves the formation of sucrosomes in normal skin fibroblasts and the other utilizes storage disorder-affected skin fibroblasts both of these systems result in an increase in the size and the number of lysosomal vacuoles. Lysosomal proteins, beta-hexosaminidase, alpha-mannosidase, N-acetylgalactosamine-4-sulfatase, acid phosphatase, and the lysosome-associated membrane protein, LAMP-1, were shown to be elevated between 2- and 28-fold above normal during lysosomal storage. Levels of mRNA for the lysosome-associated membrane proteins LAMP-1 and LAMP-2, N-acetylgalactosamine-4-sulfatase, and the 46- and 300-kDa mannose-6-phosphate receptors were also elevated 2- to 8-fold. The up-regulation of protein and mRNA lagged 2-4 days behind the formation of lysosomal storage vacuoles. Correction of storage, in both systems, resulted in the rapid decline of the mRNA to basal levels, with a slower decrease in the levels of lysosomal proteins. Lysosomal biogenesis in storage disorders is shown to be a regulated process which is partially controlled at, or prior to, the level of mRNA. Although lysosomal proteins were differentially regulated, the coordination of these events in lysosomal biogenesis would suggest that a common mechanism(s) may be in operation.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6MB00242K
Abstract: Localisation of a neutral rhenium( i ) tricarbonyl phenanthroline species to regions of high polar lipid concentrations is demonstrated by Fourier transform infrared (FTIR) microspectroscopy.
Publisher: Elsevier BV
Date: 06-2017
Publisher: Oxford University Press (OUP)
Date: 06-2012
DOI: 10.1095/BIOLREPROD.112.100388
Abstract: Activation of the insulin-like growth factor-1 receptor (IGF-1R) is known to play a role in cardiomyocyte hypertrophy. While IGF-2R is understood to be a clearance receptor for IGF-2, there is also evidence that it may play a role in the induction of pathological cardiomyocyte hypertrophy. It is not known whether IGF-2R activates cardiomyocyte hypertrophy during growth of the fetal heart. Fetal sheep hearts (125 ± 0.4 days gestation) were dissected, and the cardiomyocytes isolated from the left and right ventricles for culturing. Cultured cardiomyocytes were treated with either LONG R(3)IGF-1, an IGF-1R agonist picropodophyllin, an IGF-1R autophosphorylation inhibitor U0126, an inhibitor of extracellular signal-regulated protein kinase (ERK) Leu(27)IGF-2, an IGF-2R agonist Gö6976, a protein kinase C inhibitor KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) or KN-92, an L-type calcium channel inhibitor and negative control for KN-93. The cross-sectional area of cultured cardiomyocytes was determined relative to control cardiomyocytes treated with serum-free culture medium. IGF-1R and IGF-2R activation each resulted in ERK signaling, but IGF-2R activation alone induced CaMKII signaling, resulting in hypertrophy of cardiomyocytes in the late gestation sheep fetus. These data suggest that changes in the intrauterine environment that result in increased cardiac IGF-2R may also lead to cardiomyocyte hypertrophy in the fetus and potentially an increased risk of cardiovascular disease in adult life.
Publisher: The Company of Biologists
Date: 09-09-2015
DOI: 10.1242/BIO.013979
Abstract: Autophagy is an intracellular recycling and degradation process, which is important for energy metabolism, lipid metabolism, physiological stress response and organism development. During Drosophila development, autophagy is up-regulated in fat body and midgut cells, to control metabolic function and to enable tissue remodelling. Atg9 is the only transmembrane protein involved in the core autophagy machinery and is thought to have a role in autophagosome formation. During Drosophila development, Atg9 co-located with Atg8 autophagosomes, Rab11 endosomes and L 1 endosomes-lysosomes. RNAi silencing of Atg9 reduced both the number and the size of autophagosomes during development and caused morphological changes to hisomes/autolysosomes. In control cells there was compartmentalised acidification corresponding to intraluminal Rab11/L -1 vesicles, but in Atg9 depleted cells there were no intraluminal vesicles and the acidification was not compartmentalised. We concluded that Atg9 is required to form intraluminal vesicles and for localised acidification within hisomes/autolysosomes, and consequently when depleted, reduced the capacity to degrade and remodel gut tissue during development.
Publisher: Wiley
Date: 16-09-2021
Abstract: Intrauterine growth restriction (IUGR) is a result of limited substrate supply to the developing fetus in utero, and can be caused by either placental, genetic or environmental factors. Babies born IUGR can have poor long‐term health outcomes, including being at higher risk of developing cardiovascular disease. Limited substrate supply in the IUGR fetus not only changes the structure of the heart but may also affect metabolism and function of the developing heart. We have utilised two imaging modalities, two‐photon microscopy and phase‐contrast MRI (PC‐MRI), to assess alterations in cardiac metabolism and function using a sheep model of IUGR. Two‐photon imaging revealed that the left ventricle of IUGR fetuses (at 140–141 d GA) had a reduced optical redox ratio, suggesting a reliance on glycolysis for ATP production. Concurrently, the use of PC‐MRI to measure foetal left ventricular cardiac output (LVCO) revealed a positive correlation between LVCO and redox ratio in IUGR, but not control fetuses. These data suggest that altered heart metabolism in IUGR fetuses is indicative of reduced cardiac output, which may contribute to poor cardiac outcomes in adulthood.
Publisher: Oxford University Press (OUP)
Date: 2017
DOI: 10.1039/C6MT00243A
Abstract: Optical epifluorescence microscopy was used in conjunction with X-ray fluorescence imaging to monitor the stability and intracellular distribution of the luminescent rhenium(i) complex fac-[Re(CO)
Publisher: American Chemical Society (ACS)
Date: 22-03-2002
DOI: 10.1021/BI0121149
Abstract: The lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (4-sulfatase) is required for the degradation of the glycosaminoglycan substrates dermatan and chondroitin sulfate. A 4-sulfatase deficiency results in the accumulation of undegraded substrate and causes the severe lysosomal storage disorder mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. A wide variation in clinical severity is observed between MPS VI patients and reflects the number of different 4-sulfatase mutations that can cause the disorder. The most common 4-sulfatase mutation, Y210C, was detected in approximately 10% of MPS VI patients and has been associated with an attenuated clinical phenotype when compared to the archetypical form of MPS VI. To define the molecular defect caused by this mutation, Y210C 4-sulfatase was expressed in Chinese hamster ovary (CHO-K1) cells for protein and cell biological analysis. Biosynthetic studies revealed that Y210C 4-sulfatase was synthesized at a comparable molecular size and amount to wild-type 4-sulfatase, but there was evidence of delayed processing, traffic, and stability of the mutant protein. Thirty-three percent of the intracellular Y210C 4-sulfatase remained as a precursor form, for at least 8 h post labeling and was not processed to the mature lysosomal form. However, unlike other 4-sulfatase mutations causing MPS VI, a significant amount of Y210C 4-sulfatase escaped the endoplasmic reticulum and was either secreted from the expression cells or underwent delayed intracellular traffic. Sixty-seven percent of the intracellular Y210C 4-sulfatase was processed to the mature form (43, 8, and 7 kDa molecular mass forms) by a proteolytic processing step known to occur in endosomes-lysosomes. Treatment of Y210C CHO-K1 cells with the protein stabilizer glycerol resulted in increased amounts of Y210C 4-sulfatase in endosomes, which was eventually trafficked to the lysosome after a long, 24 h chase time. This demonstrated delayed traffic of Y210C 4-sulfatase to the lysosomal compartment. The endosomal Y210C 4-sulfatase had a low specific activity, suggesting that the mutant protein also had problems with stability. Treatment of Y210C CHO-K1 cells with the protease inhibitor ALLM resulted in an increased amount of mature Y210C 4-sulfatase localized in lysosomes, but this protein had a very low level of activity. This indicated that the mutant protein was being inactivated and degraded at an enhanced rate in the lysosomal compartment. Biochemical analysis of Y210C 4-sulfatase revealed a normal pH optimum for the mutant protein but demonstrated a reduced enzyme activity with time, also consistent with a protein stability problem. This study indicated that multiple subcellular and biochemical processes can contribute to the biogenesis of mutant protein and may in turn influence the clinical phenotype of a patient. In MPS VI patients with a Y210C allele, the composite effect of different stages of intracellular processing/handling and environment has been shown to cause a reduced level of Y210C 4-sulfatase protein and activity, resulting in an attenuated clinical phenotype.
Publisher: Wiley
Date: 10-07-2006
DOI: 10.1016/J.FEBSLET.2006.06.096
Abstract: alpha-Glucosidase (EC 3.2.1.3) is a lysosomal enzyme that hydrolyses alpha-1,4- and alpha-1,6-linkages of glycogen to produce free glucose. A deficiency in alpha-glucosidase activity results in glycogen storage disorder type II (GSD II), also called Pompe disease. Here, d-glucose was shown to be a competitive inhibitor of alpha-glucosidase and when added to culture medium at 6.0 g/L increased the production of this protein by CHO-K1 expression cells and stabilised the enzyme activity. D-Glucose also prevented alpha-glucosidase aggregation recipitation and increased protein yield in a modified purification scheme. In fibroblast cells, from adult-onset GSD II patients, D-glucose increased the residual level of alpha-glucosidase activity, suggesting that a structural analogue of d-glucose may be used for enzyme enhancement therapy.
Publisher: Springer Berlin Heidelberg
Date: 2014
Publisher: Hindawi Limited
Date: 1992
Abstract: Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the glycosidase alpha-L-iduronidase which is required for the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. Patients with MPS-I store forms of these partially degraded glycosaminoglycans in their lysosomes. MPS-I patients present with a wide range of clinical phenotypes, which makes prognostic predictions and genetic counselling difficult, therefore impeding the selection and evaluation of patients undergoing experimental therapy, such as bone marrow transplantation. We report the presence of two mutations, one that introduces a stop codon at position 70 (Q70X), and the other that alters the proline at position 533 to an arginine (P533R) in the 653 amino acid alpha-L-iduronidase protein. These mutations were originally detected by chemical cleavage and then by direct PCR sequencing. Allele specific oligonucleotides were used to detect the mutations in a group of 73 MPS-I patients and Q70X was found to account for 15% of all MPS-I alleles and P533R for 3% of MPS-I alleles. Both mutations are associated with an extremely severe clinical phenotype in homozygotes. MPS-I patients heterozygous for either mutation may have a wide range of clinical phenotypes. We have now described three mutations, W402X (Scott et al., 1992c), Q70X, and P533R totalling 53% of MPS-I alleles which together define 28% of MPS-I genotypes.
Publisher: Informa Healthcare
Date: 12-2002
Abstract: Mucopolysaccharidosis I (McKusick 25280, Hurler syndrome, Scheie syndrome) is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase (EC 3.2.1.76) and results in a failure to degrade the glycosaminoglycans, dermatan sulfate and heparan sulfate. Mucopolysaccharidosis I patients present within a spectrum of clinical phenotypes, where Hurler and Scheie syndromes represent the two extremes. In the 80 or more years since the discovery of mucopolysaccharidosis I, the molecular defect has been defined, the alpha-L-iduronidase protein purified and characterised, the alpha-L-iduronidase (IDUA) gene cloned, molecular genetic studies performed and expression systems developed. These advances have allowed the development of alpha-L-iduronidase enzyme replacement therapy as a treatment strategy for mucopolysaccharidosis I patients. Using animal models of mucopolysaccharidosis I, the efficacy of alpha-L-iduronidase replacement therapy has been evaluated and justified the initiation of human clinical trials in mucopolysaccharidosis I patients. Phase I/II and Phase III clinical trials have recently been conducted and demonstrated that this therapy is effective in treating patients with the attenuated forms of mucopolysaccharidosis I (that is, little or no neuronal involvement). Further development of this technology is required to effectively treat the problem sites of neuronal and skeletal pathology, present in severe Hurler syndrome patients.
Publisher: Elsevier BV
Date: 02-2007
DOI: 10.1016/J.YMGME.2006.10.008
Abstract: Mucopolysaccharidosis type VI (MPS VI Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-4-sulfatase (ARSB) gene. These mutations result in a deficiency of ARSB activity. Ten MPS VI patients were involved in a phase II clinical study of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each in idual exon of the ARSB gene was lified by PCR and subsequently sequenced. Thirteen substitutions (c.215T>G [p.L72R] c.284G>A [p.R95Q], c.305G>A [p.R102H], c.323G>T [p.G108V], c.389C>T [p.P130L], c.511G>A [p.G171S], c.904G>A [p.G302R], c.944G>A [p.R315Q], c.1057T>C [p.W353R], c.1151G>A [p.S384N], c.1178A>C [p.H393P], c.1289A>G [p.H430R] and c.1336G>C [p.G446R]), one deletion (c.238delG), and two intronic mutations (c.1213+5G>A and c.1214-2A>G) were identified. Nine of the 16 mutations identified were novel (R102H, G108V, P130L, G171S, W353R, H430R, G446R, c.1213+5G>A and c.1214-2A>G). The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified in some of the patients, along with the silent mutations c.972A>G and c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient and, together with genotype information, used to predict the expected clinical severity of each patient.
Publisher: Frontiers Media SA
Date: 05-03-2019
Publisher: Wiley
Date: 13-08-2008
DOI: 10.1111/J.1523-5378.2008.00625.X
Abstract: Helicobacter pylori infection is an important health problem, as it involves approximately 50% of the world's population, causes chronic inflammatory disease and increases the risk of gastric cancer development. H. pylori infection elicits a vigorous immune response, but this does not usually result in bacterial clearance. We have investigated whether the persistence of H. pylori in the host could be partly due to an inability of macrophages to kill this bacterium. Monocytes and macrophages isolated from the peripheral blood of normal human controls were infected in vitro with five H. pylori isolates. The isolates were characterized for known H. pylori virulence factors vacuolating cytotoxin (VacA), the cag pathogenicity island (cagPAI), urease, and catalase by Western blot and polymerase chain reaction analysis. The ability of primary human monocytes and macrophages to kill each of these H. pylori strains was then defined at various time points after cellular infection. The five H. pylori strains showed contrasting patterns of the virulence factors. There were different rates of killing for the bacterial strains. Macrophages had less capacity than monocytes to kill three H. pylori strains. There appeared to be no correlation between the virulence factors studied and differential killing in monocytes. Primary human monocytes had a higher capacity to kill certain strains of H. pylori when compared to macrophages. The VacA, cagPAI, urease, and catalase virulence factors were not predictive of the capacity to avoid monocyte and macrophage killing, suggesting that other factors may be important in H. pylori intracellular pathogenicity.
Publisher: Elsevier BV
Date: 07-2005
DOI: 10.1016/J.YMGME.2005.02.008
Abstract: Mucopolysaccharidosis type VI (MPS VI, Maroteaux-Lamy syndrome, McKusick #253200) is a lysosomal storage disorder that is caused by a deficiency in the lysosomal exohydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase, EC 3.1.6.1). We report a patient with no obvious clinical signs of MPS VI that has 5% of normal 4-sulphatase catalytic capacity. This patient represents an index case for the attenuated end of the MPS VI clinical spectrum.
Publisher: Elsevier BV
Date: 12-2012
DOI: 10.1016/J.NEUROSCIENCE.2012.09.034
Abstract: Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in the activity of the lysosomal hydrolase, sulphamidase, an enzyme involved in the degradation of heparan sulphate. MPS IIIA patients exhibit progressive mental retardation and behavioural disturbance. While neuropathology is the major clinical problem in MPS IIIA patients, there is little understanding of how lysosomal storage generates this phenotype. As reduced neuronal communication can underlie cognitive deficiencies, we investigated whether the secretion of neurotransmitters is altered in MPS IIIA mice utilising adrenal chromaffin cells, a classical model for studying secretion via exocytosis. MPS IIIA chromaffin cells displayed heparan sulphate storage and electron microscopy revealed large electron-lucent storage compartments. There were also increased numbers of large/elongated chromaffin granules, with a morphology that was similar to immature secretory granules. Carbon fibre erometry illustrated a significant decrease in the number of exocytotic events for MPS IIIA, when compared to control chromaffin cells. However, there were no changes in the kinetics of release, the amount of catecholamine released per exocytotic event, or the amount of Ca(2+) entry upon stimulation. The increased number of large/elongated granules and reduced number of exocytotic events suggests that either the biogenesis and/or the cell surface docking and fusion potential of these vesicles is impaired in MPS IIIA. If this also occurs in central nervous system neurons, the reduction in neurotransmitter release could help to explain the development of neuropathology in MPS IIIA.
Publisher: American Physiological Society
Date: 15-07-2012
Abstract: The vigorous host immune response that is mounted against Helicobacter pylori is unable to eliminate this pathogenic bacterium from its niche in the human gastric mucosa. This results in chronic inflammation, which can develop into gastric or duodenal ulcers in 10% of infected in iduals and gastric cancer in 1% of infections. The determinants for these more severe pathologies include host (e.g., high IL-1β expression polymorphisms), bacterial [e.g., cytotoxicity-associated gene ( cag) pathogenicity island], and environmental (e.g., dietary nitrites) factors. However, it is the failure of host immune effector cells to eliminate H. pylori that underlies its persistence and the subsequent H. pylori-associated disease. Here we discuss the mechanisms used by H. pylori to survive the host immune response and, in particular, the role played by altered phagosome maturation.
Publisher: Wiley
Date: 10-1985
DOI: 10.1111/J.1432-1033.1985.TB09158.X
Abstract: Human alpha-L-iduronidase from liver was purified about 20 000-fold with a new rapid three-step, five-column procedure which consisted of a Concanavalin-A-Sepharose/Blue-A-Agarose coupled step, a CM-Sepharose/Bio-Gel HT coupled step followed by a cupric-ion-chelating Sepharose 6B step. The behaviour of alpha-L-iduronidase on gel permeation chromatography was dependent upon both pH and ionic strength of the eluting buffer. The formation of species with enzyme activity which behaved as large-molecular-mass aggregates was favoured under conditions of low ionic strength and neutral pH. The amount of high-Mr species diminished as the pH decreased or the ionic strength increased to favour a single active species of Mr 65 000. A specific monoclonal antibody was generated against liver alpha-L-iduronidase. The antibody specifically immunoprecipitated enzyme activity from both crude and purified sources. The subunit Mr of liver alpha-L-iduronidase was estimated to be 65 000 using SDS-PAGE. Monoclonal antibody immunoprecipitation of radiolabelled enzyme was used to provide definitive confirmation of this subunit size.
Publisher: Wiley
Date: 07-05-2010
DOI: 10.1111/J.1523-5378.2010.00750.X
Abstract: Helicobacter pylori infection can lead to the development of gastritis, peptic ulcers and gastric cancer, which makes this bacterium an important concern for human health. Despite evoking a strong immune response in the host, H. pylori persists, requiring complex antibiotic therapy for eradication. Here we have studied the impact of a patient's immune serum on H. pylori in relation to macrophage uptake, phagosome maturation, and bacterial killing. Primary human macrophages were infected in vitro with both immune serum-treated and control H. pylori. The ability of primary human macrophages to kill H. pylori was characterized at various time points after infection. H. pylori phagosome maturation was analyzed by confocal immune fluorescence microscopy using markers specific for H. pylori, early endosomes (EEA1), late endosomes (CD63) and lysosomes (LAMP-1). Immune serum enhanced H. pylori uptake into macrophages when compared to control bacteria. However, a sufficient inoculum remained for recovery of viable H. pylori from macrophages, at 8 hours after infection, for both the serum-treated and control groups. Both serum-treated and control H. pylori phagosomes acquired EEA1 (15 minutes), CD63 and LAMP-1 (30 minutes). These markers were then retained for the rest of an 8 hour time course. While immune sera appeared to have a slight positive effect on bacterial uptake, both serum-treated and control H. pylori were not eliminated by macrophages. Furthermore, the same disruptions to phagosome maturation were observed for both serum-treated and control H. pylori. We conclude that to eliminate H. pylori, a strategy is required to restore the normal process of phagosome maturation and enable effective macrophage killing of H. pylori, following a host immune response.
Publisher: American Medical Association (AMA)
Date: 2021
Publisher: Hindawi Limited
Date: 09-2007
DOI: 10.1002/HUMU.20534
Abstract: Mucopolysaccharidosis type VI (MPS VI Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB) gene. ARSB is a lysosomal enzyme involved in the degradation of the glycosaminoglycans (GAG) dermatan and chondroitin sulfate. ARSB mutations reduce enzyme function and GAG degradation, causing lysosomal storage and urinary excretion of these partially degraded substrates. Disease onset and rate of progression is variable, producing a spectrum of clinical presentation. In this study, 105 MPS VI patients-representing about 10% of the world MPS VI population-were studied for molecular genetic and biochemical parameters. Direct sequencing of patient genomic DNA was used to identify ARSB mutations. In total, 83 different disease-causing mutations were found, 62 of which were previously unknown. The novel sequence changes included: 38 missense mutations, five nonsense mutations, 11 deletions, one insertion, seven splice-site mutations, and four polymorphisms. ARSB mutant protein and residual activity were determined on fibroblast extracts for each patient. The identification of many novel mutations unique to in iduals/their families highlighted the genetic heterogeneity of the disorder and provided an appropriate cohort to study the MPS VI phenotypic spectrum. This mutation analysis has identified a clear correlation between genotype and urinary GAG that can be used to predict clinical outcome.
Publisher: Springer US
Date: 2007
Publisher: Cambridge University Press (CUP)
Date: 12-07-2017
DOI: 10.1017/S2040174417000496
Abstract: Cardiometabolic diseases exhibit changes in lipid biology, which is important as lipids have critical roles in membrane architecture, signalling, hormone synthesis, homoeostasis and metabolism. However, Developmental Origins of Health and Disease studies of cardiometabolic disease rarely include analysis of lipids. This short review highlights some ex les of lipid pathology and then explores the technology available for analysing lipids, focussing on the need to develop imaging modalities for intracellular lipids. Analytical methods for studying interactions between the complex endocrine and intracellular signalling pathways that regulate lipid metabolism have been critical in expanding our understanding of how cardiometabolic diseases develop in association with obesity and dietary factors. Biochemical methods can be used to generate detailed lipid profiles to establish links between lifestyle factors and metabolic signalling pathways and determine how changes in specific lipid subtypes in plasma and homogenized tissue are associated with disease progression. New imaging modalities enable the specific visualization of intracellular lipid traffic and distribution in situ . These techniques provide a dynamic picture of the interactions between lipid storage, mobilization and signalling, which operate during normal cell function and are altered in many important diseases. The development of methods for imaging intracellular lipids can provide a dynamic real-time picture of how lipids are involved in complex signalling and other cell biology pathways and how they ultimately regulate metabolic function/homoeostasis during early development. Some imaging modalities have the potential to be adapted for in vivo applications, and may enable the direct visualization of progression of pathogenesis of cardiometabolic disease after poor growth in early life.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-2011
Publisher: Mary Ann Liebert Inc
Date: 09-1995
DOI: 10.1089/HUM.1995.6.9-1153
Abstract: Mucopolysaccharidose type I is a lysosomal storage disease caused by a deficiency in the enzyme alpha-L-iduronidase (IDUA). The existence of a secretory pathway for lysosomal enzymes and the capture of secreted molecules by distant cells through binding to mannose-6-phosphate receptors have provided a rationale for enzyme replacement therapy in lysosomal storage diseases. We have used genetically modified fibroblasts implanted into neo-organs as an in vivo delivery system for IDUA. The human IDUA cDNA was isolated and inserted into a retroviral vector where it was expressed from the phosphoglycerate kinase 1 gene promoter. MPS I fibroblasts transduced with this vector showed high levels of IDUA activity and secreted phosphorylated molecules that could be internalized by naive deficient cells. Neo-organs containing 2 x 10(7) IDUA-secreting cells were implanted into nude mice. Human and murine IDUA activities were measured in the liver and spleen of animals sacrificed 35-77 days after implantation. Human IDUA activity corresponded to 0.6-2.3% of the murine enzyme activity in the liver and to 0.1-0.3% in the spleen. These data indicated that human IDUA was secreted from neo-organs and internalized in distant tissues.
Publisher: Wiley
Date: 02-05-2018
Abstract: Coronary heart disease is one of the largest causes of death worldwide, making this a significant health care issue. A critical problem for the adult human heart is that it does not undergo effective repair in response to damage, leaving patients with a poor prognosis. Unlike the adult, fetal hearts have the ability to repair after myocardial damage. Using two-photon microscopy, we have visualised the morphological and metabolic changes following myocardial infarction in sheep fetuses, to characterise response to cardiac injury in a mammalian model. Following myocardial infarction, fetal hearts showed no significant increase in collagen deposition in the region of the infarction, when compared to either the surrounding tissue or shams. In contrast, metabolic activity (i. e. NAD(P)H and FAD) was significantly reduced in the region of myocardial infarction, when compared to either the surrounding tissue or sham hearts. For comparison, we also imaged two hearts from preadolescent sheep (sham and myocardial infarction) and showed highly ordered collagen deposition with decreased metabolic activity within the infarcted area. Therefore, two-photon imaging had the capacity to image both morphological and metabolic changes in response to myocardial infarction and showed differences in the response with age. Picture: Two-photon imaging of myocardial infarction (b and d) enabled the visualisation of increased collagen (blue Em=431 nm) and changes in other tissue autofluorescence (green Em=489-606 nm) in fetal (a and b) and preadolescent (c and d) hearts, compared to shams (a and c). The excitation wavelength was 840 nm. Scale bars: 10 μm.
Publisher: Springer Science and Business Media LLC
Date: 2005
DOI: 10.2165/00063030-200519010-00001
Abstract: The lysosomal storage disorder (LSD) mucopolysaccharidosis type I (MPS I, McKusick 25280, Hurler syndrome, Hurler-Scheie syndrome, Scheie syndrome) is caused by a deficiency in the lysosomal enzyme, alpha-L-iduronidase (EC 3.2.1.76). MPS I patients can present within a erse clinical spectrum, ranging from classical Hurler syndrome to attenuated Scheie syndrome. Laronidase (Aldurazyme) enzyme replacement therapy has been developed as a treatment strategy for MPS I patients and has been approved for clinical practice. Here we review the pre-clinical studies and clinical trials that have been used to demonstrate that intravenous laronidase therapy is well tolerated and effective for treating MPS I patients who do not have neuronal pathology. Current challenges for a viable treatment strategy for all MPS I patients include development of an early screening protocol that identifies patients before the onset of irreversible pathology, methods to predict disease severity, appropriate treatment for neuropathology, and an effective patient monitoring regimen.
Publisher: American Chemical Society (ACS)
Date: 13-04-2017
DOI: 10.1021/ACS.JPROTEOME.6B01032
Abstract: The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking, and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle, and lipid metabolic pathways. The ecdysone receptor (EcR), which is responsible for initiating the larval-to-adult transition, colocalized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1, and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or direct interaction with metabolic proteins.
Publisher: American Chemical Society (ACS)
Date: 19-05-2020
Publisher: Informa UK Limited
Date: 05-2009
DOI: 10.1586/EOG.09.8
Publisher: Bioscientifica
Date: 05-2017
DOI: 10.1530/JOE-17-0039
Abstract: Intrauterine insults, such as poor nutrition and placental insufficiency, can alter cardiomyocyte development, and this can have significant long-term implications for heart health. Consequently, epidemiological studies have shown that low-birth-weight babies have an increased risk of death from cardiovascular disease in adult life. In addition, intrauterine growth restriction can result in increased left ventricular hypertrophy, which is the strongest predictor for poor health outcomes in cardiac patients. The mechanisms responsible for these associations are not clear, but a suboptimal intrauterine environment can program alternative expression of genes such as cardiac IGF-2/H19, IGF-2R and AT 1 R through either an increase or decrease in DNA methylation or histone acetylation at specific loci. Furthermore, hypoxia and other intrauterine insults can also activate the IGF-1 receptor via IGF-1 and IGF-2, and the AT 1 receptor via angiotensin signaling pathways both of which can result in the phosphorylation of Akt and the activation of a range of downstream pathways. In turn, Akt activation can increase cardiac angiogenesis and cardiomyocyte apoptosis and promote a reversion of metabolism in postnatal life to a fetal phenotype, which involves increased reliance on glucose. Cardiac Akt can also be indirectly regulated by microRNAs and conversely can target microRNAs that will eventually affect other specific cardiac genes and proteins. This review aims to discuss our understanding of this complex network of interactions, which may help explain the link between low birth weight and the increased risk of cardiovascular disease in adult life.
Publisher: Elsevier BV
Date: 10-2003
DOI: 10.1016/S0925-4439(03)00129-7
Abstract: Mucopolysaccharidosis type I (MPS I McKusick 25280 Hurler syndrome, Hurler-Scheie syndrome and Scheie syndrome) is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase (EC 3.2.1.76). MPS I patients present within a clinical spectrum bounded by the extremes of Hurler and Scheie syndromes. The alpha-L-iduronidase missense mutations R89Q and R89W were investigated and altered an important arginine residue proposed to be a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase. The R89Q alpha-L-iduronidase mutation was shown to result in a reduced level of alpha-L-iduronidase protein (< or =10% of normal control) compared to a normal control level of alpha-L-iduronidase protein that was detected for the R89W alpha-L-iduronidase mutation. When taking into account alpha-L-iduronidase specific activity, the R89W mutation had a greater effect on alpha-L-iduronidase activity than the R89Q mutation. However, overall the R89W mutation produced more residual alpha-L-iduronidase activity than the R89Q mutation. This was consistent with MPS I patients, with an R89W allele, having a less severe clinical presentation compared to MPS I patients with either a double or single allelic R89Q mutation. The effects of the R89Q and R89W mutations on enzyme activity supported the proposed role of R89 as a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase.
Publisher: Public Library of Science (PLoS)
Date: 23-08-2016
Publisher: Elsevier BV
Date: 1984
DOI: 10.3109/00313028409067914
Abstract: Three monoclonal antibodies, FMC24, 25 and 27, reactive with human platelets, are described. In normal blood the 3 antibodies are specific for platelets, but FMC27 reacts with leukemic blast cells in a proportion of acute leukemias. FMC25 precipitates the platelet membrane glycoprotein lb and a glycoprotein of molecular weight 22,000. The antibodies show different effects on platelet aggregation.
Publisher: Wiley
Date: 17-06-2021
Abstract: Re(I) complexes have potential in biomedical sciences as imaging agents, diagnostics and therapeutics. Thus, it is crucial to understand how Re(I) complexes interact with carrier proteins, like serum albumins. Here, two neutral Re(I) complexes were used ( fac ‐[Re(CO) 3 (1,10‐phenanthroline)L], in which L is either 4‐cyanophenyltetrazolate (1) or 4‐methoxycarbonylphenyltetrazole ester (2) , to study the interactions with bovine serum albumin (BSA). Spectroscopic measurements, calculations of thermodynamic and Förster resonance energy transfer parameters, as well as molecular modelling, were performed to study differential binding between BSA and complex 1 and 2 . Induced‐fit docking combined with quantum‐polarised ligand docking were employed in what is believed to be a first for a Re(I) complex as a ligand for BSA. Our findings provide a basis for other molecular interaction studies and suggest that subtle functional group alterations at the terminal region of the Re(I) complex have a significant impact on the ability of this class of compounds to interact with BSA.
Publisher: Cold Spring Harbor Laboratory
Date: 12-2017
Abstract: The oncogenic transcription factor TAL1/SCL induces an aberrant transcriptional program in T-cell acute lymphoblastic leukemia (T-ALL) cells. However, the critical factors that are directly activated by TAL1 and contribute to T-ALL pathogenesis are largely unknown. Here, we identified AT-rich interactive domain 5B ( ARID5B ) as a collaborating oncogenic factor involved in the transcriptional program in T-ALL. ARID5B expression is down-regulated at the double-negative 2–4 stages in normal thymocytes, while it is induced by the TAL1 complex in human T-ALL cells. The enhancer located 135 kb upstream of the ARID5B gene locus is activated under a superenhancer in T-ALL cells but not in normal T cells. Notably, ARID5B-bound regions are associated predominantly with active transcription. ARID5B and TAL1 frequently co-occupy target genes and coordinately control their expression. ARID5B positively regulates the expression of TAL1 and its regulatory partners. ARID5B also activates the expression of the oncogene MYC . Importantly, ARID5B is required for the survival and growth of T-ALL cells , and forced expression of ARID5B in immature thymocytes results in thymus retention, differentiation arrest, radioresistance, and tumor formation in zebrafish. Our results indicate that ARID5B reinforces the oncogenic transcriptional program by positively regulating the TAL1-induced regulatory circuit and MYC in T-ALL, thereby contributing to T-cell leukemogenesis.
Publisher: Wiley
Date: 23-12-2020
Abstract: The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability. We utilized molecular and histological techniques to define the metabolic status in foetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two-photon microscopy. Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with oxygen demanding oxidative phosphorylation activity being utilized for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterization with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism.
Publisher: Hindawi Limited
Date: 18-12-2013
DOI: 10.1155/2013/498598
Abstract: With the aim of directing lanthanide complex uptake via the mannose receptor, a first generation of luminescent lanthanide complexes has been developed with an α -D-mannose targeting motif. Four complexes were produced to investigate photophysical properties and determine the effect of the coordinated mannose residue on emission intensity. The free hydroxyls of the α -D-mannose residue quenched lanthanide phosphorescence due to their close proximity, though they did not bind the lanthanide centre as observed by q -values ≈ 1.0 for all complexes between pH 3 and 10. Fluorescent emission was found to vary significantly with pH, though phosphorescent emission was relatively insensitive to pH. This lack of pH sensitivity has the potential to provide stable emission for the visualisation of the endosome-lysosome system where acidic pH is often encountered.
Publisher: Hindawi Limited
Date: 09-2004
DOI: 10.1002/HUMU.20081
Abstract: Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase (IDUA). Mutations in the gene are responsible for the enzyme deficiency, which leads to the intralysosomal storage of the partially degraded glycosaminoglycans dermatan sulfate and heparan sulfate. Molecular characterization of MPS I patients has resulted in the identification of over 70 distinct mutations in the IDUA gene. The high degree of molecular heterogeneity reflects the wide clinical variability observed in MPS I patients. Six novel mutations, c.1087C>T (p.R363C), c.1804T>A (p.F602I), c.793G>C, c.712T>A (p.L238Q), c.1727+2T>A, and c.1269C>G (p.S423R), in a total of 14 different mutations, and 13 different polymorphic changes, including the novel c.246C>G (p.H82Q), were identified in a cohort of 10 MPS I patients enrolled in a clinical trial of enzyme-replacement therapy. Five novel amino acid substitutions and c.236C>T (p.A79V) were engineered into the wild-type IDUA cDNA and expressed. A p.G265R read-through mutation, arising from the c.793G>C splice mutation, was also expressed. Each mutation reduced IDUA protein and activity levels to varying degrees with the processing of many of the mutant forms also affected by IDUA. The varied properties of the expressed mutant forms of IDUA reflect the broad range of biochemical and clinical phenotypes of the 10 patients in this study. IDUA kinetic data derived from each patient's cultured fibroblasts, in combination with genotype data, was used to predict disease severity. Finally, residual IDUA protein concentration in cultured fibroblasts showed a weak correlation to the degree of immune response to enzyme-replacement therapy in each patient.
Publisher: Elsevier BV
Date: 07-2007
DOI: 10.1016/J.YGENO.2007.03.005
Abstract: The Aristaless-related homeobox gene (ARX) is one of the major genes causing X-linked mental retardation. We have been interested in the pathogenic mechanism of expanded polyalanine tract mutations in ARX. We showed that the c.304ins(GCG)7 mutation causing an increase from 16 to 23 alanines increased the propensity of ARX protein aggregation and a shift from nuclear to cytoplasmic localization. We proposed that mislocalization of ARX via cytoplasmic aggregation and subsequent degradation leads to a partial loss of function, contributing to the pathogenesis. We identified importin 13 (IPO13), a mediator of nuclear import for a variety of proteins, as a novel ARX interacting protein. We predicted that the transport of ARX by IPO13 from the cytoplasm to the nucleus might be disrupted by expanded polyalanine tract mutations, but our data showed that in both yeast and mammalian cells these mutant ARX proteins were still able to interact with IPO13. We established the nuclear localization regions of the ARX homeodomain that were required for the interaction with IPO13 and correct localization of the full-length ARX transcription factor to the nucleus.
Publisher: Public Library of Science (PLoS)
Date: 28-12-2020
DOI: 10.1371/JOURNAL.PONE.0243715
Abstract: Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alterative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcome and prevent the development of chemoresistance.
Publisher: Elsevier BV
Date: 03-2005
DOI: 10.1016/J.CCCN.2004.10.009
Abstract: Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disorder (LSD), which is caused by a deficiency in the enzyme N-acetylgalactosamine 4-sulfatase (4-sulfatase). MPS VI is characterized by severe skeletal abnormalities, somatic tissue pathology and early death. Treatment possibilities include bone marrow transplantation (BMT) and enzyme replacement therapy (ERT currently in phase III clinical trial). Early diagnosis of MPS VI will allow treatment before the onset of irreversible pathology. Sensitive immune assays have been developed to detect 4-sulfatase protein and activity in normal control and MPS VI blood-spots. Dried blood-spots from MPS VI patients contained no detectable 4-sulfatase protein and activity, compared to 3.5-21 microg/L of 4-sulfatase protein and 291-1298 nmol/min/L of activity for normal human controls. In this evaluation study, the assay was sensitive and 100% specific, allowing reliable detection of in iduals with MPS VI. The assays reported here have the potential to detect MPS VI patients using dried blood-spots.
Publisher: Springer Science and Business Media LLC
Date: 19-08-2007
DOI: 10.1038/NG2100
Publisher: Elsevier BV
Date: 2014
Publisher: Wiley
Date: 03-1983
DOI: 10.1111/J.1600-0609.1983.TB01479.X
Abstract: In all 11 patients with hairy-cell leukaemia (HCL) tested, a high percentage of peripheral blood and splenic (3 cases) hairy cells (HCs) reacted with 2 B-cell-specific monoclonal antibodies (FMC1 and FMC7), but failed to stain with 5 monocyte/macrophage-specific (FMC17, UCHM1, SM1, SMø and OKM1) and with 4 T-cell-specific antibodies (UCHT1, Leu1, 2a and 3a). The data provide further evidence in support of the B-cell nature of the HCs of HCL.
Publisher: MDPI AG
Date: 23-12-2021
Abstract: Fluorescence microscopy has become a critical tool for researchers to understand biological processes at the cellular level. Micrographs from fixed and live-cell imaging procedures feature in a plethora of scientific articles for the field of cell biology, but the complexities of fluorescence microscopy as an imaging tool can sometimes be overlooked or misunderstood. This review seeks to cover the three fundamental considerations when designing fluorescence microscopy experiments: (1) hardware availability (2) amenability of biological models to fluorescence microscopy and (3) suitability of imaging agents for intended applications. This review will help equip the reader to make judicious decisions when designing fluorescence microscopy experiments that deliver high-resolution and informative images for cell biology.
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.JINORGBIO.2017.10.003
Abstract: In this work we have developed a series of highly emissive europium(III) and terbium(III) complexes tethered to either folic acid (FA) or methotrexate (MTX), with the aim of developing visual probes that enable the imaging of folate receptors in cancer cells. The synthesis, photophysical properties and cellular behaviour are reported for four new lanthanide Ln(III) complexes, where either FA or MTX are tethered to 1,4,7-tris(carbonylmethyl)-10-(4'-quinolineacetic acid, (7'-acetamido)-1',2'-dihydro-2'-oxo)-1,4,7,10-tetraazacyclododecane Ln(III) complex, and Ln(III)=Eu(III) or Tb(III) herein referred to as Eu-FA, Eu-MTX, Tb-FA or Tb-MTX. All four complexes were found to be sensitive to the presence of the folate receptor in a range of cell lines. The MTX conjugates showed different cellular specificity in an oral adenosquamous carcinoma cell line (CAL-27) compared with the analogous FA conjugates. This suggests that it is viable to explore differences in folate receptors using folate vs. anti-folate probes, with labels that have different emissive properties (e.g. Eu-FA vs. Tb-MTX). The MTX complexes were found to be the most cytotoxic, with Eu-MTX showing greater cytotoxicity than free MTX or the isostructural Tb-MTX. This suggested that there could be a synergistic effect on toxicity for the Eu(III) chelate and the MTX components of the complex.
Publisher: Elsevier BV
Date: 05-2021
Publisher: American Society for Cell Biology (ASCB)
Date: 09-2000
Abstract: Protein traffic from the cell surface or thetrans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, α and γ SNAP, and a Rab GTPase based on inhibition by Rab GDI. InSaccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE V 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.
Publisher: Wiley
Date: 20-04-1990
DOI: 10.1007/BF01799338
Abstract: A biochemical and cytochemical study has been made of the distribution of ATPase in mature and differentiating phloem cells of Nicotiana tabacum and of the substrate specificity and effects of fixation on enzyme activity. Homogenates of unfixed leaf midveins and midveins fixed in formaldehyde-glutaraldehyde were assayed for enzyme activity by determining the amount of P(i), liberated per milligram of protein from various substrates in a 30 min period at pH 7.2. In fresh homogenates, hydrolysis of ATP was not significantly different from that of ITP, CTP, and UTP. Hydrolysis of GTP was slightly higher than that of ATP. ATP hydrolysis by fresh homogenates was 17% more extensive than that of ADP, 76% more extensive than that of 5'-AMP, and was inhibited by fluoride and p-chloromercuribenzoate (PCMB). There was little or no hydrolysis of the competitive inhibitors 2'- and 3'-AMP nor with the alternate substrates p-nitrophenylphosphate (PNP) or beta-glycerophosphate (beta-GP). In homogenates of material fixed in formaldehyde-glutaraldehyde for 1(1/4) h, ATPase activity was 13% preserved. Hydrolysis of ATP by fixed homogenates was not significantly different from that of ADP, 5'-AMP, ITP, CTP, and GTP. Hydrolysis of UTP was lower. Fluoride and PCMB inhibited fixed ATPase activity. The results of cytochemical localization experiments using a lead phosphate precipitation technique were in agreement with the biochemical results. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP. Activity was also localized with ADP and 5'-AMP but not with the competitive inhibitors 2'- and 3'-AMP, nor with PNP or beta-GP. Little or no reaction product was deposited in other controls incubated without substrate or with substrate plus fluoride, PCMB, or N-ethylmaleimide. ATPase activity was demonstrated chiefly at the plasma membrane of mature and differentiating phloem cells and was associated with the P-protein of mature sieve elements. It is suggested that the phloem transport system derives its energy from the demonstrated nucleoside triphosphatase activity.
Publisher: Elsevier BV
Date: 07-1997
DOI: 10.1016/S0005-2736(97)00053-9
Abstract: A monoclonal antibody designated MBR 39 has been generated against a membrane associated protein found selectively on lysosomes. MBR 39 reacts with the cytosolic face of the lysosome and was used to develop an organelle binding assay which reacted with high density organelles characteristic of lysosomes. These organelles contained lysosomal enzyme markers which included the integral membrane protein acetyl-CoA:alpha-glucosaminide N-acetyltransferase and the soluble lysosomal enzyme markers acid phosphatase (mature form), beta-hexosaminidase, arylsulfatase, and alpha-L-iduronidase. Under conditions which disrupt lysosomes the release of the latter soluble lysosomal enzymes was demonstrated from MBR 39 bound organelles. Immunoblots of MBR 39 with purified fibroblast lysosomal membrane, demonstrated reactivity with polypeptides of molecular mass 63 kDa (major species) and 73 kDa (minor species).
Publisher: Wiley
Date: 10-01-2021
Publisher: Wiley
Date: 29-10-2012
DOI: 10.1111/J.1440-1681.2012.05743.X
Abstract: Epidemiological studies indicate that poor growth before birth is associated with left ventricular hypertrophy and an increased risk of death from heart disease later in life. In fetal life, the insulin-like growth factor (IGF) system has been implicated in physiological growth of the heart, whereas in postnatal life IGFs can be involved in both physiological and pathological cardiac hypertrophy. A reduction in substrate supply in fetal life, resulting in chronic hypoxaemia and intrauterine growth restriction, results in increased cardiac IGF-1R, IGF-2 and IGF-2R gene expression and there is also evidence for a role of the IGF-2 receptor in the ensuing cardiac hypertrophy. The persistent high level of cardiac IGF-2R gene expression from fetal to postnatal life may be due to epigenetic changes in key cardiac hypertrophy regulatory pathways.
Publisher: Wiley
Date: 25-01-1993
DOI: 10.1007/BF00711309
Publisher: Proceedings of the National Academy of Sciences
Date: 21-09-2020
Abstract: Influenza infection during pregnancy is associated with increased maternal and perinatal complications. Here, we show that, during pregnancy, influenza infection leads to viral dissemination into the aorta, resulting in a peripheral “vascular storm” characterized by enhanced inflammatory mediators the influx of Ly6C monocytes, neutrophils, and T cells and impaired vascular function. The ensuing vascular storm induced hypoxia in the placenta and fetal brain and caused an increase in circulating cell free fetal DNA and soluble Flt1 release. We demonstrate that vascular dysfunction occurs in response to viral infection during pregnancy, which may explain the high rates of morbidity and mortality in pregnant dams, as well as the downstream perinatal complications associated with influenza infection.
Publisher: Elsevier BV
Date: 2004
DOI: 10.1016/J.YMGME.2003.11.002
Abstract: The lysosomal storage disorder mucopolysaccharidosis type II (MPS II) is caused by a deficiency in the activity of the lysosomal exohydrolase iduronate-2-sulphatase (IDS). MPS II patients present within a spectrum of clinical phenotypes, which reflects the dynamic balance between the level of mutant protein, its residual enzyme activity and the resultant level of storage product. In this study, we have developed an immunoquantification assay for the accurate detection of iduronate-2-sulphatase protein and applied this methodology to the analysis of mutant iduronate-2-sulphatase protein in plasma s les from MPS II patients. The detection limit for the assay was defined as 20 ng/ml for wild type iduronate-2-sulphatase, but could be extended to a detection limit of 0.3 ng/ml by heat denaturation of the protein lasma s le. The mutant protein detected in plasma from MPS II patients displayed similar properties to heat denatured wild type iduronate-2-sulphatase, suggesting an altered protein conformation. The ratio of heat denatured to native ELISA reactivity could be used to confirm the diagnosis of MPS II (i.e., a ratio of >1 for normal protein and <or=1 for mutant protein). Notably, four of the 20 patients tested had either normal or higher than normal levels of iduronate-2-sulphatase protein, but this protein also showed evidence of conformation change. The iduronate-2-sulphatase protein level detected in plasma from MPS II patients showed little or no direct correlation with the severity of the clinical phenotype observed in these patients.
Publisher: Springer Science and Business Media LLC
Date: 02-2018
DOI: 10.1038/S41598-018-19871-4
Abstract: The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for the folding, stabilization and maturation of multiple oncoproteins, which are implicated in PCa progression. Compared to first-in-class Hsp90 inhibitors such as 17-allylamino-demethoxygeldanamycin (17-AAG) that were clinically ineffective, second generation inhibitor AUY922 has greater solubility and efficacy. Here, transcriptomic and proteomic analyses of patient-derived PCa explants identified cytoskeletal organization as highly enriched with AUY922 treatment. Validation in PCa cell lines revealed that AUY922 caused marked alterations to cell morphology, and suppressed cell motility and invasion compared to vehicle or 17-AAG, concomitant with dysregulation of key extracellular matrix proteins such as fibronectin (FN1). Interestingly, while the expression of FN1 was increased by AUY922, FN1 secretion was significantly decreased. This resulted in cytosolic accumulation of FN1 protein within late endosomes, suggesting that AUY922 disrupts vesicular secretory trafficking pathways. Depletion of FN1 by siRNA knockdown markedly reduced the invasive capacity of PCa cells, phenocopying AUY922. These results highlight a novel mechanism of action for AUY922 beyond its established effects on cellular mitosis and survival and, furthermore, identifies extracellular matrix cargo delivery as a potential therapeutic target for the treatment of aggressive PCa.
Publisher: Wiley
Date: 07-08-2017
Abstract: A family of five neutral cyclometalated iridium(III) tetrazolato complexes and their methylated cationic analogues have been synthesised and characterised. The complexes are distinguished by variations of the substituents or degree of π conjugation on either the phenylpyridine or tetrazolato ligands. The photophysical properties of these species have been evaluated in organic and aqueous media, revealing predominantly a solvatochromic emission originating from mixed metal-to-ligand and ligand-to-ligand charge transfer excited states of triplet multiplicity. These emissions are characterised by typically long excited-state lifetimes (∼hundreds of ns), and quantum yields around 5-10 % in aqueous media. Methylation of the complexes caused a systematic red-shift of the emission profiles. The behaviour and the effects of the different complexes were then examined in cells. The neutral species localised mostly in the endoplasmic reticulum and lipid droplets, whereas the majority of the cationic complexes localised in the mitochondria. The amount of complexes found within cells does not depend on lipophilicity, which potentially suggests erse uptake mechanisms. Methylated analogues were found to be more cytotoxic compared to the neutral species, a behaviour that might to be linked to a combination of uptake and intracellular localisation.
Publisher: MDPI AG
Date: 19-09-2023
Publisher: SPIE
Date: 02-01-2018
DOI: 10.1117/12.2283352
Publisher: Portland Press Ltd.
Date: 06-1990
DOI: 10.1042/BJ2680379
Abstract: The biosynthesis and maturation of N-acetylgalactosamine-4-sulphatase (4-sulphatase) was studied in normal fibroblasts and in fibroblasts from patients with either mucopolysaccharidosis type VI (MPS VI Maroteaux-Lamy syndrome) or multiple sulphatase deficiency (MSD). Fibroblasts were incubated in culture medium containing [3H]leucine or [35S]methionine, and radiolabelled 4-sulphatase was isolated by immunoaffinity chromatography using 4-sulphatase-specific monoclonal antibodies. In normal fibroblasts a precursor of 66 kDa, detected intracellularly after 3 h and in NH4Cl-induced secretions, was processed intracellularly, within an additional 3 h, to a polypeptide of 57 kDa composed of disulphide-linked polypeptides of 43 kDa and 8 kDa. All fibroblast lines obtained from MPS VI patients, exhibiting clinical characteristics ranging from no clearly recognized symptoms to the severe classical phenotype, incorporated radioactivity into immune-purified 4-sulphatase at a rate less than 10% of that seen in normal fibroblasts. Maturation of the residual 4-sulphatase showed, variously, features which may be indicative of delayed intracellular transport, decreased intracellular stability, failure of lysosomal targetting or resistance to enzyme processing. Although some features of the residual enzyme synthesis and maturation were consistent with the patient's clinical phenotype, this was infrequent. The maturation of 4-sulphatase in fibroblasts from MSD patients was indistinguishable from that in normal fibroblasts, and the half-life of 4-sulphatase in these fibroblasts, determined after a 24 h pulse and prolonged chase, was only slightly less than that in normal fibroblasts.
Publisher: Wiley
Date: 06-12-2006
DOI: 10.1016/J.FEBSLET.2005.11.053
Abstract: Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites.
Publisher: Wiley
Date: 06-1982
DOI: 10.1111/J.1600-0765.1982.TB01151.X
Abstract: The history of cosmetics goes back to early Egyptian times for hygiene and health benefits while the history of topical applications that provide a medicinal treatment to combat dermal aging is relatively new. For ex le, the term cosmeceutical was first coined by Albert Kligman in 1984 to describe topical products that afford both cosmetic and therapeutic benefits. However, beauty comes from the inside. Therefore, for some time scientists have considered how nutrition reflects healthy skin and the aging process. The more recent link between nutrition and skin aging began in earnest around the year 2000 with the demonstrated increase in peer-reviewed scientific journal reports on this topic that included biochemical and molecular mechanisms of action. Thus, the application of: (a) topical administration from outside into the skin and (b) inside by oral consumption of nutritionals to the outer skin layers is now common place and many journal reports exhibit significant improvement for both on a variety of dermal parameters. Therefore, this review covers, where applicable, the history, chemical structure, and sources such as biological and biomedical properties in the skin along with animal and clinical data on the oral applications of: (a) collagen, (b) ceramide, (c) β-carotene, (d) astaxanthin, (e) coenzyme Q
Publisher: Springer Science and Business Media LLC
Date: 02-2007
Publisher: Elsevier BV
Date: 10-1999
Publisher: Elsevier BV
Date: 10-2003
DOI: 10.1016/J.MOLMED.2003.08.004
Abstract: Lysosomal storage disorders are collectively important because they cause significant morbidity and mortality. Patients can present with severe symptoms that include somatic tissue and bone pathology, developmental delay and neurological impairment. Enzyme-replacement therapy has been developed as a treatment strategy for patients with a lysosomal storage disorder, and for many of these disorders this treatment is either in clinical trial or clinical practice. One major complication arising from enzyme infusion into patients with a lysosomal storage disorder is an immune response to the replacement protein. From clinical trials, it is clear that there is considerable variability in the level of immune response to enzyme-replacement therapy, dependent upon the replacement protein being infused and the in idual patient. Hypersensitivity reactions, neutralizing antibodies to the replacement protein and altered enzyme targeting or turnover are potential concerns for patients exhibiting an immune response to enzyme-replacement therapy. The relative occurrence and significance of these issues have been appraised.
Publisher: Elsevier BV
Date: 1983
DOI: 10.3109/00313028309061401
Abstract: Three monoclonal antibodies, FMC17, FMC32, and FMC33 directed against human cells of the monocyte-macrophage lineage are described. The antibodies react strongly with blood monocytes and weakly, if at all, with granulocytes. Lymphoid cells are not stained. In tissue sections macrophages and interdigitating reticulum cells are stained. Lymphoid leukemia cells generally do not react with the antibodies, while myeloid leukemia cells give a variable pattern, with relatively differentiated cells more likely to react than undifferentiated cells. Differences between the 3 antibodies in their reactivity with leukemic cells and tissue macrophages indicate that they are directed against distinct antigens, which may serve as differentiation markers in the monocyte/macrophage lineage.
Publisher: MDPI AG
Date: 26-03-2020
DOI: 10.3390/INORGANICS8040023
Abstract: Five cyclometalated Ir(III) tetrazolato complexes functionalised with fatty acid chains (octanoic, palmitic, stearic, palmitoleic, and oleic) have been synthesised. The fatty acids were chosen to evaluate the potential effect of the length and degree of unsaturation on the biological properties of the complexes for use as cellular imaging agents. The complexes were analysed in both organic and aqueous media to determine if the presence and nature of the fatty acid chains had a significant effect on their photophysical properties. The complexes display green–yellow emission in dichloromethane solutions with relatively long excited state decays, within the range 360–393 ns, and quantum yields between 5.4% and 6.7% (from degassed solutions). Temperature-dependent photophysical studies suggest that the emitting excited states of the complexes might be quenched by the thermal population of dark states. In water, the quantum yields drop within the range of 0.5%–2.4%, and the photophysical measurements are influenced by the variable degrees of aggregation. In general, the entire series displayed low cytotoxicity and relatively high photostability, which are favourable attributes in the design of cellular imaging agents. Images of live HeLa cells were obtained for all the complexes, but those functionalised with palmitic and stearic acids had limitations due the lower solubility conferred by the saturated aliphatic chains. The complexes were mainly detected within the endoplasmic reticulum.
Publisher: Portland Press Ltd.
Date: 22-02-2005
DOI: 10.1042/BJ20040739
Abstract: Mammalian sulphatases (EC 3.1.6) are a family of enzymes that have a high degree of similarity in amino acid sequence, structure and catalytic mechanism. IDS (iduronate-2-sulphatase EC 3.1.6.13) is a lysosomal exo-sulphatase that belongs to this protein family and is involved in the degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. An IDS deficiency causes the lysosomal storage disorder MPS II (mucopolysaccharidosis type II). To examine the structural alterations in heat-denatured and mutant IDS, a panel of four monoclonal antibodies was raised to the denatured protein and used as probes of protein conformation. The linear sequence epitope reactivity of a polyclonal antibody raised against the native protein and the monoclonal antibodies were defined and mapped to distinct regions on the IDS protein. The antigenicity of native IDS was higher in regions without glycosylation, but reactivity was not restricted to protein surface epitopes. One monoclonal epitope was relatively surface accessible and in close proximity to an N-linked glycosylation site, while three others required additional thermal energy to expose the epitopes. The monoclonal antibodies demonstrated the capacity to differentiate progressive structural changes in IDS and could be used to characterize the severity of MPS type II in patients based on variable denatured microstates.
Publisher: Hindawi Limited
Date: 16-06-2021
DOI: 10.1155/2021/2414897
Abstract: Introduction. Afatinib is a first-line treatment option for patients with an advanced nonsmall cell lung cancer (NSCLC) expressing an epidermal growth factor receptor (EGFR) activating mutation. This study aimed to evaluate the association between early adverse events induced by afatinib and overall survival (OS) and progression free survival (PFS) in patients with advanced NSCLC. Methods. The study was a pooled post hoc analysis of the randomized trials LUX-Lung 3 and LUX-Lung 6 which evaluated afatinib versus pemetrexed-cisplatin or gemcitabine-cisplatin, respectively. Cox proportional hazard analysis was used to assess the impact of adverse events occurring within the first 28 days of afatinib therapy on the PFS and OS outcomes in treatment-naïve advanced NSCLC patients harbouring an EGFR activating mutation. Results. There were 468 patients who initiated first-line afatinib therapy within LUX-Lung 3 and LUX-Lung 6. A significant association between early rash and improved OS (hazard ratio (HR 95% CI) grade 1 = 0.74 [0.56–0.97] grade 2+ = 0.64 [0.46–0.89]) ( P = 0.018) was observed, although no significant association with PFS was present ( P = 0.732). A significant association was identified between early diarrhoea and improved PFS (grade 1 = 0.83 [0.62–1.12] grade 2+ = 0.62 [0.44–0.88]) ( P = 0. 015), although no significant association with OS was present ( P = 0.605). No associations between early stomatitis or paronychia and OS or PFS were identified. Conclusion. Rash occurring early after the initiation of afatinib was significantly associated with improved OS, an indicator that rash may be a surrogate of patients likely to achieve long-term survival. Consideration of using rash as a dose adjustment target may be warranted for future prospective trials aiming to optimise outcomes with afatinib therapy.
Publisher: Mary Ann Liebert Inc
Date: 05-2019
Publisher: Springer Science and Business Media LLC
Date: 1999
Abstract: Autologous transplantation of retrovirally transduced bone marrow (BM) or neonatal blood cells was carried out on eight cats (ranging in age from 2 weeks to 12 months) with mucopolysaccharidosis type VI (MPS VI). The transducing vector contained the full-length cDNA encoding human arylsulfatase B (hASB), the enzymatic activity deficient in this lysosomal storage disorder. Following transplantation, the persistence of transduced cells and enzymatic expression were monitored for more than 2 years. Five of the cats received no myeloablative preconditioning, two cats received 370-390 cGy of total body irradiation (TBI), and one cat received 190 cGy TBI. Evidence of transduced cells, as judged by enzymatic activity and PCR detection of the provirus, was demonstrated in granulocytes, lymphocytes, or BM cells of the treated animals up to 31 months after transplantation. Radiation preconditioning was not required to achieve these results, nor were they dependent on the recipient's age. However, despite the long-term persistence of transduced cells, the levels of ASB activity in the transplanted animals was low, and no clinical improvements were detected. These data provide evidence for the long-term persistence of retrovirally transduced feline hematopoietic cells, and further documentation that engraftment of transduced cells can be achieved in the absence of myeloablation. Consistent with previous bone marrow transplantation studies, these results also suggest that to achieve clinical improvement of MPS VI, particularly in the skeletal system, high-level expression of ASB must be achieved in the treated animals and improved techniques for targeting the expressed enzyme to specific sites of pathology (e.g. chondrocytes) must be developed.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-2011
Publisher: Springer Science and Business Media LLC
Date: 03-2011
Abstract: Helicobacter pylori ( H. pylori ) is a micro-aerophilic, spiral-shaped, motile bacterium that is the principal cause of gastric and duodenal ulcers in humans and is a major risk factor for the development of gastric cancer. Despite provoking a strong innate and adaptive immune response in the host, H. pylori persists in the gastric mucosa, avoiding eradication by macrophages and other phagocytic cells, which are recruited to the site of infection. Here we have characterised the critical degradative process of phagosome maturation in primary human macrophages for five genotypically and phenotypically distinct clinical strains of H. pylori . All of the H. pylori strains examined showed some disruption to the phagosome maturation process, when compared to control E. coli . The early endosome marker EEA1 and late endosome marker Rab7 were retained on H. pylori phagosomes, while the late endosome-lysosome markers CD63, LAMP-1 and LAMP-2 were acquired in an apparently normal manner. Acquisition of EEA1 by H. pylori phagosomes appeared to occur by two distinct, strain specific modes. H. pylori strains that were negative for the cancer associated virulence factor CagA were detected in phagosomes that recruited large amounts of EEA1 relative to Rab5, compared to CagA positive strains. There were also strain specific differences in the timing of Rab7 acquisition which correlated with differences in the rate of intracellular trafficking of phagosomes and the timing of megasome formation. Megasomes were observed for all of the H. pylori strains examined. H. pylori appeared to disrupt the normal process of phagosome maturation in primary human macrophages, appearing to block endosome fission. This resulted in the formation of a hybrid phagosome-endosome-lysosome compartment, which we propose has reduced degradative capacity. Reduced killing by phagocytes is consistent with the persistence of H. pylori in the host, and would contribute to the chronic stimulation of the inflammatory immune response, which underlies H. pylori -associated disease.
Publisher: MDPI AG
Date: 29-11-2019
DOI: 10.3390/IJMS20236035
Abstract: Syntenin-1 is an essential multi-functional adaptor protein, which has multiple roles in membrane trafficking and exosome biogenesis, as well as scaffolding interactions with either the actin cytoskeleton or focal adhesions. However, how this functional multiplicity relates to syntenin-1 distribution in different endosome compartments or other intracellular locations and its underlying involvement in cancer pathogenesis have yet to be fully defined. To help facilitate the investigation of syntenin-1 biology, we developed two specific monoclonal antibodies (Synt-2C6 and Synt-3A11) to spatially distinct linear sequence epitopes on syntenin-1, which were each designed to be unique at the six-amino acid level. These antibodies produced very different intracellular staining patterns, with Synt-2C6 detecting endosomes and Synt-3A11 producing a fibrillar staining pattern suggesting a cytoskeletal localisation. Treatment of cells with Nocodazole altered the intracellular localisation of Synt-3A11, which was consistent with the syntenin-1 protein interacting with microtubules. In prostate tissue biopsies, Synt-3A11 defined atrophy and early-stage prostate cancer, whereas Synt-2C6 only showed minimal interaction with atrophic tissue. This highlights a critical need for site-specific antibodies and a knowledge of their reactivity to define differential protein distributions, interactions and functions, which may differ between normal and malignant cells.
Publisher: American Society for Cell Biology (ASCB)
Date: 09-2007
Abstract: The sorting of acid hydrolase precursors at the trans-Golgi network (TGN) is mediated by binding to mannose 6-phosphate receptors (MPRs) and subsequent capture of the hydrolase-MPR complexes into clathrin-coated vesicles or transport carriers (TCs) destined for delivery to endosomes. This capture depends on the function of three monomeric clathrin adaptors named GGAs. The GGAs comprise a C-terminal “ear” domain that binds a specific set of accessory proteins. Herein we show that one of these accessory proteins, p56, colocalizes and physically interacts with the three GGAs at the TGN. Moreover, overexpression of the GGAs enhances the association of p56 with the TGN, and RNA interference (RNAi)-mediated depletion of the GGAs decreases the TGN association and total levels of p56. RNAi-mediated depletion of p56 or the GGAs causes various degrees of missorting of the precursor of the acid hydrolase, cathepsin D. In the case of p56 depletion, this missorting correlates with decreased mobility of GGA-containing TCs. Transfection with an RNAi-resistant p56 construct, but not with a p56 construct lacking the GGA-ear–interacting motif, restores the mobility of the TCs. We conclude that p56 tightly cooperates with the GGAs in the sorting of cathepsin D to lysosomes, probably by enabling the movement of GGA-containing TCs.
Publisher: Wiley
Date: 28-11-2018
Abstract: The heart has high metabolic demand to maintain function. The primary source of energy supply to support correct contractile muscle function differs between a fetus and an adult. In fetal life, ATP is primarily generated by glycolysis and lactate oxidation, whereas following birth, there is a shift towards a reliance on mitochondrial metabolism and fatty acid oxidation. This change in metabolic status is an adaptation to different fuel availability, oxygenation and growth patterns. In this study, we have employed 2-photon excitation fluorescence microscopy to define the relationship between two critical metabolic cofactors nicotinamide adenine dinucleotide(P)H and flavin adenine dinucleotide, effectively utilizing a redox ratio to differentiate between the metabolic status in fetal (proliferative) and adult (quiescent/hypertrophic) hearts. Two-photon imaging was also used to visually confirm the known increase in collagen deposition in the adult heart. The changes observed were consistent with a hypertrophic growth profile and greater availability of fatty acids in the adult heart, compared to the proliferative fetal heart. Two-photon excitation fluorescence microscopy is therefore a convenient imaging technology that enables the monitoring of striated muscle architecture and the metabolic status of heart tissue. This imaging technology can potentially be employed to visualize cardiac and other muscle pathologies.
Publisher: Wiley
Date: 29-10-2012
Publisher: Springer Science and Business Media LLC
Date: 29-05-2018
DOI: 10.1038/S41598-018-24672-W
Abstract: Mitochondrial morphology is important for the function of this critical organelle and, accordingly, altered mitochondrial structure is exhibited in many pathologies. Imaging of mitochondria can therefore provide important information about disease presence and progression. However, mitochondrial imaging is currently limited by the availability of agents that have the capacity to image mitochondrial morphology in both live and fixed s les. This can be particularly problematic in clinical studies or large, multi-centre cohort studies, where tissue archiving by fixation is often more practical. We previously reported the synthesis of an iridium coordination complex [Ir( ppy ) 2 ( MeTzPyPhCN )] + where ppy is a cyclometalated 2-phenylpyridine and TzPyPhCN is the 5-(5-(4-cyanophen-1-yl)pyrid-2-yl)tetrazolate ligand and showed that this complex (herein referred to as IraZolve-Mito) has a high specificity for mitochondria in live cells. Here we demonstrate that IraZolve-Mito can also effectively stain mitochondria in both live and fixed tissue s les. The staining protocol proposed is versatile, providing a universal procedure for cell biologists and pathologists to visualise mitochondria.
Publisher: Elsevier BV
Date: 10-1999
Publisher: Elsevier BV
Date: 10-2016
DOI: 10.1016/J.BBRC.2016.09.145
Abstract: Pompe disease is caused by a deficiency in the lysosomal enzyme α-glucosidase, and this leads to glycogen accumulation in the autolysosomes of patient cells. Glycogen storage material is exocytosed at a basal rate in cultured Pompe cells, with one study showing up to 80% is released under specific culture conditions. Critically, exocytosis induction may reduce glycogen storage in Pompe patients, providing the basis for a therapeutic strategy whereby stored glycogen is redirected to an extracellular location and subsequently degraded by circulating amylases. The focus of the current study was to identify compounds capable of inducing rapid glycogen exocytosis in cultured Pompe cells. Here, calcimycin, lysophosphatidylcholine and α-l-iduronidase each significantly increased glycogen exocytosis compared to vehicle-treated controls. The most effective compound, calcimycin, induced exocytosis through a Ca
Publisher: Elsevier BV
Date: 03-2003
DOI: 10.1016/S1096-7192(03)00007-6
Abstract: This study evaluates the immunological response following weekly 2h infusions of recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) in Mucopolysaccharidosis VI (MPS VI) cats. The results of three trials (Trial "A": 9 month duration with onset at 3-5 months of age, n = 5 and Trials "B" and "C": 6 month duration starting at birth, n = 9) were compared. No detrimental effects were noted throughout Trials B and C. Temporary hypersensitivity reactions (e.g., vomiting, diarrhoea) occurred in four cats in Trial A and were alleviated by increasing the dose of antihistamine premedication and the duration of infusion. All cats in Trial A developed antibodies to rh4S (range of final titres: 1041-134,931). All cats treated from birth showed negligible titres (range: < 50-598). In vitro inhibition of rh4S activity (up to 47%) was demonstrated with plasma from four cats with elevated titres. Significant reduction of urinary glycosaminoglycan concentration in all cats indicated the ability of rh4S to metabolize stored substrates regardless of the presence of circulating antibodies. Similarly, lysosomal storage in reticuloendothelial cells and fibroblasts of kidney interstistium, dura and skin was reduced in all cats irrespective of their antibody titre although cats with elevated titre had less beneficial effect on cardiovascular tissues (aorta smooth muscle cells, heart valve fibroblasts). Overall improvement in the disease condition (at physical, neurological, and skeletal levels) was most pronounced for cats treated from birth compared with cats treated at a later age.
Publisher: AMPCo
Date: 06-2018
DOI: 10.5694/MJA18.00324
Publisher: American Physiological Society
Date: 04-2010
DOI: 10.1152/PHYSIOL.00041.2009
Abstract: The discovery over five decades ago of the lysosome, as a degradative organelle and its dysfunction in lysosomal storage disorder patients, was both insightful and simple in concept. Here, we review some of the history and pathophysiology of lysosomal storage disorders to show how they have impacted on our knowledge of lysosomal biology. Although a significant amount of information has been accrued on the molecular genetics and biochemistry of lysosomal storage disorders, we still do not fully understand the mechanistic link between the storage material and disease pathogenesis. However, the accumulation of undegraded substrate(s) can disrupt other lysosomal degradation processes, vesicular traffic, and lysosomal biogenesis to evoke the erse pathophysiology that is evident in this complex set of disorders.
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/0885-4505(92)90028-W
Abstract: Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in alpha-L-iduronidase activity (alpha-L-iduronide iduronohydrolase, EC 3.2.1.76). Cultured fibroblasts from an MPS I patient (cell line 2827) with a severe clinical phenotype (Hurler syndrome) have been characterized using immunochemical and biochemical techniques. Using a specific immunoquantification assay, we have demonstrated that cell line 2827 had an alpha-L-iduronidase protein content (189 ng/mg of extracted cell protein) at least six times greater than the mean level found in normal control fibroblasts (30 ng/mg of extracted cell protein). This was the only MPS I cell line, from a group of 23 MPS I patients, that contained greater than 7% of the mean level of alpha-L-iduronidase protein detected in normal controls. Cell line 2827 had very low alpha-L-iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of alpha-L-iduronidase in cell line 2827 showed apparently normal levels of alpha-L-iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated alpha-L-iduronidase protein in lysosomal-enriched fractions isolated from cell line 2827, suggesting a normal cell distribution and supporting the proposed delayed processing. It is proposed that the MPS I patient described has an alpha-L-iduronidase gene mutation which affects both the active site and post-translational processing of the enzyme. This mutation must be structurally conservative because it does not result in instability either during maturation or in the lysosome.
Publisher: Elsevier BV
Date: 1982
DOI: 10.3109/00313028209069036
Abstract: A monoclonal antibody, FMC 8, has been prepared after immunization with a cell line of the non-T-non-B type acute lymphoblastic leukaemia (ALL). The antigen detected by this antibody consists of a single polypeptide chain with an apparent molecular weight of 24,000 d. The antigen is absent from T cells but appears after mitogen stimulation. It is found on the majority of normal circulating B cells but is absent in most chronic lymphocytic leukaemias. All cells of the non-T-non-B type express the antigen, whereas T-ALL cells generally do not. Granulocytes, monocytes and platelets react with the antibody, but erythrocytes do not. The wide but selective distribution of the antigen renders FMC 8 potentially useful, together with other monoclonal antibodies, in the delineation of differentiation pathways and in phenotypic analysis of leukaemia and lymphoma cells.
Publisher: Elsevier BV
Date: 08-1997
DOI: 10.1016/S0925-4439(97)00036-7
Abstract: Enzyme replacement therapy (ERT) can potentially result in an immunological response to the introduced protein. The immunological response by Mucopolysaccharidosis type VI (MPS VI) cats to recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) ERT has been investigated. Plasma antibody titres to rh4S were detected in untreated MPS VI and normal control cats, but the antibody titres to rh4S were higher in ERT treated MPS VI cats. The reactivity by cats to rh4S did not appear to be just due to species cross reactivity, as plasma antibodies from normal control, MPS VI and MPS VI ERT cats reacted equally with feline and human 4-sulfatase. Normal control and MPS VI human plasma also had antibody titres to rh4S. Plasma antibodies to rh4S, from an ERT treated cat, could be temporarily removed from circulation by enzyme infusion, confirming specificity for rh4S and indicating a possible window for ERT in the absence of antibody. In enzyme distribution studies with 3H-rh4S, evidence of altered targeting, and enzyme inactivation and degradation were observed in high compared to low titre rats. In high titre rats, the observed loss of 3H-label from vacuolar organelles of the liver may represent either degradation of antibody bound 3H-rh4S for reutilisation within the liver, or antigen presentation. The development of high titre antibody may have a detrimental effect on the efficacy of ERT.
Publisher: Springer Science and Business Media LLC
Date: 22-07-2015
Publisher: MDPI AG
Date: 07-12-2022
DOI: 10.3390/V14122729
Abstract: Influenza A virus (IAV) infection during pregnancy disrupts maternal and fetal health through biological mechanisms, which are to date poorly characterised. During pregnancy, the viral clearance mechanisms from the lung are sub-optimal and involve hyperactive innate and adaptive immune responses that generate wide-spread inflammation. Pregnancy-related adaptations of the immune and the cardiovascular systems appear to result in delayed recovery post-viral infection, which in turn promotes a prolonged inflammatory phenotype, increasing disease severity, and causing maternal and fetal health problems. This has immediate and long-term consequences for the mother and fetus, with complications including acute cardiopulmonary distress syndrome in the mother that lead to perinatal complications such as intrauterine growth restriction (IUGR), and birth defects cleft lip, cleft palate, neural tube defects and congenital heart defects. In addition, an increased risk of long-term neurological disorders including schizophrenia in the offspring is reported. In this review we discuss the pathophysiology of IAV infection during pregnancy and its striking similarity to other well-established complications of pregnancy such as preecl sia. We discuss general features of vascular disease with a focus on vascular inflammation and define the “Vascular Storm” that is triggered by influenza infection during pregnancy, as a pivotal disease mechanism for short and long term cardiovascular complications.
Publisher: Elsevier BV
Date: 11-2018
Publisher: Wiley
Date: 02-2015
DOI: 10.14814/PHY2.12270
Publisher: Portland Press Ltd.
Date: 15-12-1987
DOI: 10.1042/BJ2480755
Abstract: Initial purification of N-acetylgalactosamine-4-sulphate sulphatase from human liver homogenates containing approx. 1 mg of enzyme in 26 g of soluble proteins was achieved by a six-column chromatography procedure and yielded approx. 40 micrograms of a single major protein species. Enzyme thus prepared was used to produce N-acetylgalactosamine-4-sulphate sulphatase-specific monoclonal antibodies. The use of a monoclonal antibody linked to a solid support facilitated the purification of approx. 0.5 mg of N-acetylgalactosamine-4-sulphate sulphatase from a similar liver homogenate. Moreover the enzyme isolated contained a single protein species, shown by SDS olyacrylamide-gel electrophoresis to have an Mr of 57,000, which dissociated into subunits of Mr 43,000 and 13,000 in the presence of reducing agents. Essentially identical enzyme preparations were isolated from homogenates of human kidney and lung and from concentrated human urine. The native protein Mr of enzyme from human liver and kidney was assessed by gel-permeation chromatography to be 43,000 on Ultrogel AcA and Bio-Gel P-150. The liver N-acetylgalactosamine-4-sulphate sulphatase was shown to have pH optima of approx. 4 and 5.5 with the oligosaccharide substrate (GalNAc4S-GlcA-GalitolNAc4S) and fluorogenic substrate (methylumbelliferyl sulphate) respectively. Km values of 60 microM and 4 mM and Vmax. values of 2 and 20 mumol/min per mg were determined with the oligosaccharide and fluorogenic substrates respectively.
Publisher: Elsevier BV
Date: 03-1984
DOI: 10.1016/0161-5890(84)90075-0
Abstract: Three monoclonal antibodies (mAbs), FMC4, FMC14 and FMC15, which react with invariant sites of major histocompatibility complex (MHC) class II (Ia-like) molecules were studied in various serological assays. Sequential immunodepletion experiments show that all three epitopes are present on the same class II molecules. However, a minor subset may exist which does not express epitope 15. In competitive binding assays, using several different B-lymphoblastoid cell lines (B-LCLs), e.g. BRISTOL-8 8392, B-85, RAJI, 8866, CESS-B and LD-B, FMC4 did not block the binding of FMC14, or FMC15, and vice versa. In contrast, mutual inhibition was observed between FMC14 and FMC15. Furthermore, pairwise combinations of saturating amounts of FMC4 + FMC14 and FMC4 + FMC15 gave additive binding whilst FMC14 + FMC15 did not. These results demonstrate that epitopes 4 and 14/15 are spatially distinct 14 and 15 on the other hand appear to be spatially related. However, contrary to this partial and reciprocal inhibition was consistently observed between FMC4 and FMC14 on two other LCLs, namely DAUDI and BALM-2. Furthermore, on certain cells, FMC14 and FMC15 show markedly disparate binding. Taken together, these observations indicate that the juxtaposition of certain epitopes on class II antigens can vary according to the cell type. This demonstrates a hitherto unreported heterogeneity of antigenic determinants and of their topographical distribution on the class II molecule.
Publisher: Elsevier BV
Date: 12-2021
Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.IJPHARM.2016.09.039
Abstract: In recent years G protein-coupled receptors (GPCRs) have emerged as crucial tumorigenic factors that drive aberrant cancer growth, metastasis and angiogenesis. Consequently, a number of GPCRs are strongly expressed in cancer derived cell lines and tissue s les. Therefore a rational anti-cancer strategy is the design of nano-medicines that specifically target GPCRs to bind and internalise cytotoxic drugs into cancer cells. Herein, we report the genetic engineering of a self-assembling nanoparticle based on elastin-like polypeptide (ELP), which has been fused with gastrin releasing peptide (GRP). These nanoparticles increased intracellular calcium concentrations when added to GRP receptor positive PC-3 prostate cancer cells, demonstrating specific receptor activation. Moreover, GRP-displaying fluorescent labelled nanoparticles showed specific cell-surface interaction with PC-3 prostate cancer cells and increased endocytic uptake. These nanoparticles therefore provide a targeted molecular carrier system for evaluating the delivery of cytotoxic drugs into cancer cells.
Publisher: Frontiers Media SA
Date: 10-06-2020
Publisher: Wiley
Date: 29-09-2011
Publisher: American Chemical Society (ACS)
Date: 07-2021
Publisher: The Company of Biologists
Date: 17-05-2016
DOI: 10.1242/BIO.016642
Abstract: The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.
Publisher: Portland Press Ltd.
Date: 15-04-1995
DOI: 10.1042/BJ3070457
Abstract: The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.
No related grants have been discovered for Doug Brooks.