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0000-0001-5552-4416
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CSIRO Queensland Bioscience Precinct
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Publisher: Elsevier BV
Date: 02-2021
Publisher: Elsevier BV
Date: 03-1992
Publisher: Springer Science and Business Media LLC
Date: 11-04-2023
DOI: 10.1007/S00484-023-02464-W
Abstract: We set out to determine the impact of moderate heat load on the plasma concentrations of a suite of hormones involved in regulating energy metabolism and feed intake. The responses of the thermally challenged (TC) feedlot steers were compared to those of feed restricted thermoneutral (FRTN) steers. Two sequential cohorts of twelve 518 ± 23 kg Black Angus steers on finisher grain ration were housed in climate-controlled rooms (CCR) for 18 days and returned to outdoor pens for 40 days. The TC group was subjected to a diurnal range of 28–35 °C for 7 days (Challenge) but held in thermoneutral conditions beforehand (PreChallenge), and in Recovery (after Challenge). The FRTN group was held in thermoneutral conditions and feed restricted throughout. Blood was collected over the three periods in CCR and two periods in outdoor pens for 40 days (PENS and Late PENS). Plasma concentrations of prolactin, thyroid stimulating hormone, insulin, leptin, adiponectin and thyroxine (T4) were determined during the five periods. Whilst the pituitary hormones were relatively stable, there were differences in plasma leptin, adiponectin and T4 between the two groups during Challenge and Recovery, and occasionally in PENS. The interaction of the plasma hormone concentrations and rumen temperature and DMI were also investigated. Whilst the positive relationship between DMI and leptin was confirmed, we found a strong negative relationship between adiponectin and rumen temperature, and a strong positive relationship between adiponectin and dry matter intake (DMI) in the TC steers only.
Publisher: FapUNIFESP (SciELO)
Date: 04-2019
DOI: 10.1590/S1984-29612019034
Abstract: Abstract Livestock infections by Trypanosoma vivax have been occurring with increasing frequency, mainly due to the presence of animals with subclinical infections and without apparent parasitaemia, making diagnosis challenging. The aim of the present study was to evaluate several techniques used for T. vivax diagnosis in order to assess the best way of using them during the course of the disease. Molecular methods demonstrated higher rates of detection than parasitological methods, detecting 33 of the 54 (61.1%) known positive s les, while the hematocrit centrifugation technique (best parasitological test) detected only 44.4%. The serological methods, IFAT and ELISA, detected seropositivity in 51 of the 54 (94.4%) and 49 of the 54 (90.7%) known positive s les, respectively. Despite being highly sensitive, the latter only demonstrates exposure to the infectious agent and does not indicate whether the infection is active. The present study was the first to use the qPCR for a South American isolate, improving disease detection and quantification. Furthermore, the analyses revealed that the patent phase of the disease may extend up to 42 days, longer than previously reported. The combination of several diagnostic techniques can lower the frequency of false negative results and contributes toward better disease control.
Publisher: Proceedings of the National Academy of Sciences
Date: 25-09-2001
Abstract: The interaction between DNA polymerases and sliding cl proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. The cl proteins also participate in a variety of dynamic and competing protein–protein interactions. However, cl -protein binding sequences have not so far been identified in the eubacteria. Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein–protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL[SD]LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding cl (the β subunit of Escherichia coli DNA polymerase III holoenzyme). Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PolC, PolB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family. The component tripeptide DLF inhibits the binding of the α (DnaE) subunit of E. coli DNA polymerase III to β at μM concentration, identifying key residues. Comparison of the eubacterial, eukaryotic, and archaeal sliding cl binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape.
Publisher: CSIRO Publishing
Date: 2020
DOI: 10.1071/AN19689
Abstract: Context Approximately 2 million sheep are exported from Australia on live export voyages annually. As voyages travel from a southern hemisphere winter to a northern hemisphere summer, production and welfare issues associated with excessive heat load may arise. Aims The aim of this study was to evaluate the responses of sheep to incremental heat load under simulated live export conditions, specifically the influence of heat load on the metabolic and inflammatory status of sheep. Methods A total of 144 Merino wethers (44.02 ± 0.32 kg) were used in a 29-day climate controlled study using two cohorts of 72 sheep (n = 2), exposed to two treatments: (1) thermoneutral, and (2) hot. Sheep in the hot treatment were exposed to heat load simulated from live export voyages from Australia to the Middle East. Blood s les were collected from all sheep (n = 144) on Day 1, then at 7-day intervals (n = 5) for the duration of each 29-day period. Blood s les were analysed to determine the cytokine, biochemistry and haematology (data not presented here) profiles. Cytokine and biochemical profiles were analysed using a repeated measures model assuming a compound symmetry covariance. The model fitted included terms for cohort and treatment (hot, thermoneutral), and a term for s le collection day (day) and a treatment × day interaction. The subject factor corresponded to the cohort × treatment combinations. Key results There were no consistent trends in plasma cytokine and biochemical profiles. Bicarbonate was the only parameter that was influenced by cohort (P = 0.0035), treatment (P = 0.0025), collection (P = 0.0001) and treatment × collection (P = 0.0025). Furthermore, interleukin-6 and glutamate dehydrogenase were the only parameters that were not influenced by cohort (P & 0.295), treatment (P = 0.2567), collection (P & 0.06) or treatment × collection (P = 0.34). Conclusions Overall, these data highlight that the metabolic and inflammatory status of sheep exposed to incremental heat load, during a simulated live export voyage from a southern hemisphere winter to a northern hemisphere summer, were not markedly altered. Implications These results provide a preliminary evaluation of the inflammatory and metabolic status of sheep on arrival in the Middle East.
Publisher: Elsevier BV
Date: 09-1999
DOI: 10.1016/S0020-7519(99)00070-3
Abstract: Several peritrophins of larvae of Lucilia cuprina (sheep blowfly) have demonstrated potential as vaccine antigens, and some have been characterised and cloned. These proteins are tightly associated with the peritrophic matrix, a chitinous tube or sac lining the lumen of the gut of most insects. The peritrophins require strong denaturants for their removal from peritrophic matrix. We now report the preliminary characterisation of peritrophins of the adult stage of L. cuprina and Haematobia irritans exigua (buffalo fly). Similar SDS-PAGE profiles were obtained for proteins extracted in SDS or urea from isolated adult peritrophic matrices of both species. Radioiodination of urea-extracted peritrophins improved sensitivity, indicating numerous proteins of 15-75 kDa. Direct radioiodination of L. cuprina peritrophic matrix preferentially labelled high molecular weight complexes and proteins of 80-90 kDa. Two-dimensional gel analyses of a urea extract of adult L. cuprina peritrophic matrix revealed that most proteins were moderately acidic. Antibodies produced against SDS-extracted peritrophins, or against sonicated peritrophic matrices of these two flies were crossreactive. The sera also appeared to recognise SDS-extracted components of Triton X-100 treated and washed adult peritrophic matrix of the mosquito, Aedes vigilax (Skause). This profile altered as the peritrophic matrix matured. In concordance with extracts from the adult L. cuprina and H.i. exigua peritrophic matrices, proteins in the 50-75 kDa region were immunodominant. The vaccine potential of the peritrophins of these Diptera were examined following vaccination of cattle and rabbits with adult H.i. exigua or L. cuprina peritrophins. When the adult life stages of H.i. exigua or two mosquitoes, A. vigilax and A. aegypti (Linnaeus), were fed on the sera or blood of vaccinated hosts, there were no detrimental effects to any life cycle stages of these Diptera.
Publisher: Wiley
Date: 2012
DOI: 10.1002/BIP.22109
Abstract: The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of in idual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including s le preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges.
Publisher: Elsevier BV
Date: 12-2016
DOI: 10.1016/J.CBD.2016.04.005
Abstract: Inefficient control of temperate abalone spawning prevents pair-wise breeding and production of abalone with highly marketable traits. Traditionally, abalone farmers have used a combination of UV irradiation and application of temperature gradients to the tank water to artificially induce spawning. Proteins are known to regulate crucial processes such as respiration, muscle contraction, feeding, growth and reproduction. Spawning as a pre-requisite of abalone reproduction is likely to be regulated, in part, by endogenous proteins. A first step in elucidating the mechanisms that regulate spawning is to identify which proteins are directly involved during spawning. The present study examined protein expression following traditional spawning induction in the Haliotis laevigata female. Gonads were collected from abalone in the following physiological states: (1) spawning (2) post-spawning and (3) failed-to-spawn. Differential protein abundance was initially assessed using two-dimensional difference in-gel electrophoresis coupled with mass spectrometry for protein identification. A number of reproductive proteins such as vitellogenin, vitelline envelope zona pellucida domain 29 and prohibitin, and metabolic proteins such as thioredoxin peroxidase, superoxide dismutase and heat shock proteins were identified. Differences in protein abundance levels between physiological states were further assessed using scheduled multiple reaction monitoring mass spectrometry. Positive associations were observed between the abundance of specific proteins, such as heat shock cognate 70 and peroxiredoxin 6, and the propensity or failure to spawn in abalone. These findings have contributed to better understand both the effects of oxidative and heat stress over abalone physiology and their influence on abalone spawning.
Publisher: Elsevier BV
Date: 07-2022
DOI: 10.1016/J.SCITOTENV.2022.154840
Abstract: Insects used to treat organic waste streams and produce valuable protein products are increasingly exposed to plastic contaminated source material assimilating plastic carbon into organic biomass, which is pervasive and hazardous to organisms. Our understanding of this increased insect-plastic interaction remains limited and needs urgent scientific attention if plastic biodegradation and production rates of quality protein are to be improved. Herein, we investigated the biochemical impact of various plastics using three insect models. Black Soldier Fly (BSF), Mealworm (MW), and Wax Moth (WM) larva were each exposed to a plastic substrate (PET, PE, PS, Expanded PE, PP, and PLA) as the primary carbon source for five days to explore any positive metabolic benefits in terms of insect performance and plastic degradation potential. Central carbon metabolism (CCM) metabolites were analyzed via a targeted tMRM liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS) method. Unique expressed pathways were observed for each insect model. When reared on PET, BSF larvae were found to have an elevated pyrimidine metabolism, while the purine metabolism pathway was strongly expressed on other plastics. BSF also exhibited a downregulated Vitamin B6 metabolism across all plastics, indicating a likely gut-symbiont breakdown. The MW and WM model insects were metabolically more active on PLA and expanded foam plastics. Further, WM exhibited an elevation in Vitamin B6 metabolism. This data suggests a positive insect-specific interaction towards certain plastic types that warrants further investigation. It is anticipated that through deeper insight into the metabolic impact and benefits afforded from certain plastics, an insect biotransformation pipeline can be established that links fit-for-purpose insect models to in idual plastic types that address our growing plastic waste issue.
Publisher: Elsevier BV
Date: 02-2002
Publisher: CSIRO Publishing
Date: 16-11-2021
DOI: 10.1071/AN21119
Abstract: Lumpy wool (dermatophilosis) develops following prolonged wetting of sheep when bacterial proliferation in wool and on skin induce an exudative dermatitis, causing a superficial skin lesion and damage to wool follicles and fibres. The incidence of dermatophilosis is strongly dependent on wet and warm weather and, hence, infection is sporadic. While older animals are less at risk than are lambs, it is unclear whether this reflects naturally acquired immune resistance or the maturation of skin and wool fibres. Dermatophilosis directly causes wool production losses and it also is a risk factor for blowfly strike, which has a substantial economic impact and increasing challenges associated with current control procedures. This review assessed research on the bacterial causes of lumpy wool, the characteristics of the resulting immune defence reactions in sheep, current control strategies, and limitations of previous attempts to control lumpy wool by sheep vaccination.
Publisher: Elsevier BV
Date: 2001
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7FD90076G
Publisher: Wiley
Date: 14-02-2011
Abstract: The hypothalamus is the central regulatory region of the brain that links the nervous system to the endocrine system via the pituitary gland. It synthesizes and secretes neuropeptide hormones, which in turn act to stimulate or inhibit the secretion of pituitary hormones. We have undertaken a detailed MS investigation of the peptides present in the bovine hypothalamus by adapting a novel heat stabilization methodology, which improved peptide discovery to direct our studies into the molecular mechanisms involved in bovine reproduction. The untreated s les contained large numbers of protein degradation products that interfered with the analysis of the neuropeptides. In the thermally stabilized s les, we were able to identify many more neuropeptides that are known to be expressed in the bovine hypothalamus. Furthermore, we have characterized a range of post-translational modifications that indicate the presence of processed intact mature neuropeptides in the stabilized tissue s les, whereas we detected many trimmed or truncated peptides resulting from post-mortem degradation in the untreated tissue s les. Altogether, using an optimized workflow, we were able to identify 140 candidate neuropeptides. We also nominate six new candidate neuropeptides derived from proSAAS, secretogranin-2 and proTRH.
Publisher: American Chemical Society (ACS)
Date: 12-11-2012
DOI: 10.1021/PR3009034
Abstract: Nontargeted metabolite fingerprinting is increasingly applied to biomedical classification. The choice of classification algorithm may have a considerable impact on outcome. In this study, employing nested cross-validation for assessing predictive performance, six binary classification algorithms in combination with different strategies for data-driven feature selection were systematically compared on five data sets of urine, serum, plasma, and milk one-dimensional fingerprints obtained by proton nuclear magnetic resonance (NMR) spectroscopy. Support Vector Machines and Random Forests combined with t-score-based feature filtering performed well on most data sets, whereas the performance of the other tested methods varied between data sets.
Publisher: Elsevier BV
Date: 11-1993
Publisher: Elsevier BV
Date: 2007
DOI: 10.1016/J.PLASMID.2006.07.005
Abstract: Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0CS00486C
Abstract: Contrary to expectations, protic hydroxyanions show a surprisingly reliable facility to dimerize or oligomerize together by hydrogen bonding.
Publisher: Springer International Publishing
Date: 2019
DOI: 10.1007/978-3-030-12298-0_1
Abstract: The proteome represents the total set of proteins produced by an organism or a system at a particular time or state, with proteomics being the study of the proteome. The proteome is a dynamic system wherein proteins are interconnected and serve to facilitate cellular processes in a concurrent and coordinated manner. Over the years, various biochemical and biophysical methods have been developed to elucidate the identities, structures and functions of proteins in order to understand their roles in complex biological systems. The success of proteomic approaches hinges on efficient protein extraction and s le preparation however, these preliminary steps are often considered a bottleneck in proteomic workflows. Every biological s le is unique and complex, and s le processing needs to be tailored to the nature of the protein s le due to its vulnerability towards post-collection degradation and the complexity of its non-protein constituents. S le pretreatment steps often employ buffers, solvents, salts and detergents that are not always compatible with the downstream analytical tools. This chapter will provide an overview of s le pretreatment techniques commonly used in conjunction with proteomics tools and discuss protein analysis methods. Such methods include the use of antibody-based techniques, separation sciences (e.g. chromatography, SDS-PAGE), detection methods (e.g. mass spectrometry) and structural techniques (e.g. NMR and X-ray crystallography).
Publisher: American Chemical Society (ACS)
Date: 07-03-2018
DOI: 10.1021/ACS.JPROTEOME.7B00875
Abstract: Puberty in cattle is regulated by an endocrine axis, which includes a complex milieu of neuropeptides in the hypothalamus and pituitary gland. The neuropeptidome of hypothalamic-pituitary gland tissue of pre- (PRE) and postpubertal (POST) Bos indicus-influenced heifers was characterized, followed by quantitative analysis of 51 fertility-related neuropeptides in these tissues. Comparison of peptide abundances with gene expression levels allowed assessment of post-transcriptional peptide processing. On the basis of classical cleavage, 124 mature neuropeptides from 35 precursor proteins were detected in hypothalamus and pituitary gland tissues of three PRE and three POST Brangus heifers. An additional 19 peptides (cerebellins, PEN peptides) previously reported as neuropeptides that did not follow classical cleavage were also identified. In the pre-pubertal hypothalamus, a greater ersity of neuropeptides (25.8%) was identified relative to post-pubertal heifers, while in the pituitary gland, 38.6% more neuropeptides were detected in the post-pubertal heifers. Neuro-tissues of PRE and POST heifers revealed abundance differences ( p < 0.05) in peptides from protein precursors involved in packaging and processing (e.g., the granin family and ProSAAS) or neuron stimulation (PENK, CART, POMC, cerebellins). On their own, the transcriptome data of the precursor genes could not predict the neuropeptide profile in the exact same tissues in several cases. This provides further evidence of the importance of differential processing of the neuropeptide precursors in the pituitary before and after puberty.
Publisher: American Dairy Science Association
Date: 07-2018
Abstract: Bone-derived hormones play an important role in metabolism. This study examined the hypothesis that interactions between bone and energy metabolism, particularly those involving osteocalcin, are present in dairy cattle and have feedback mechanisms over time. Associations between metabolites in blood were examined in 32 Holstein cows blocked by parity and milk yield and randomly allocated to diets containing either 0.27 mg/kg dry matter (DM) calcidiol or cholecalciferol for an anticipated intake of 3 mg/d (120,000 IU/d) at 11 kg of DM, and positive (+130 mEq/kg DM) or negative (-130 mEq/kg DM) dietary cation-anion difference (DCAD) from 252 d of gestation to calving. Blood was s led every 3 d, from 9 d prepartum to 30 d postpartum, and plasma concentrations of vitamin D
Publisher: Wiley
Date: 06-2005
DOI: 10.1080/15216540500138246
Abstract: The last 15 years of effort in understanding bacterial DNA replication and repair has identified that the donut shaped beta2 sliding cl is harnessed by very functionally different DNA polymerases throughout the lifecycle of the bacterial cell. Remarkably, the sites of binding of these polymerases, in most cases, appear to be the same shallow pocket on the beta dimer. In every case, binding of beta2 by the polymerase enhances their processivity of DNA synthesis. This binding site is also the same point of interaction between beta2 and the cl loader complex, which binds beta2, opens and places it onto the DNA strand and then vacates the site. Beta2 may also be involved in the initiation of DNA replication again via contact through this same site. While much of the research effort has focused on Escherichia coli and Bacillus subtilis, conservation of this complex system is becoming apparent in erse bacteria.
Publisher: Hindawi Limited
Date: 12-06-2017
DOI: 10.1111/ARE.13413
Publisher: Elsevier BV
Date: 02-1998
Abstract: Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA lified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.
Publisher: American Chemical Society (ACS)
Date: 23-04-2004
DOI: 10.1021/BI036229J
Abstract: The sliding cl of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding cl , a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.
Publisher: Worldwide Protein Data Bank
Date: 08-06-2011
DOI: 10.2210/PDB3QSB/PDB
Publisher: American Chemical Society (ACS)
Date: 20-02-2023
Publisher: American Society for Microbiology
Date: 06-2004
DOI: 10.1128/JB.186.11.3508-3515.2004
Abstract: In Escherichia coli , interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding cl protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.
Publisher: Wiley
Date: 15-04-1996
DOI: 10.1111/J.1432-1033.1996.0414K.X
Abstract: The angiotensin-converting enzymes (ACE) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. A carboxydipeptidase similar to mammalian ACE has now been identified in the adult stage of the haematophagous fly, Haematobia irritans exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin-affinity chromatography and ion-exchange chromatography and migrated as a doublet of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (HieACE) is not membrane bound. The amino acid sequence of an internal peptide from HieACE and a conserved amino acid region present in all mammalian ACE were used to design degenerate oligonucleotide primers suitable for PCR. A DNA fragment lified from adult buffalo fly cDNA was used to identify a cDNA clone that encoded the enzyme. The cDNA sequence encodes a carboxydipeptidase with 41-42% amino acid identity to the mammalian testicular ACE. The active-site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatically, HieACE is very similar to its mammalian counterparts, with comparable Km and V(max) values for the synthetic substrate, benzoylglycylglycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin-8. In the adult fly, HieACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this enzyme is induced in the maturing male reproductive system, which suggests conservation of ACE function in these species.
Publisher: Elsevier BV
Date: 10-2001
DOI: 10.1016/S0965-1748(01)00051-0
Abstract: The ersity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an ex le of concerted evolution of a trypsin gene in Chrysomya bezziana.
Publisher: Wiley
Date: 15-11-2000
DOI: 10.1046/J.1365-3024.2000.00335.X
Abstract: Chrysomya bezziana is an endemic pest of livestock or a threat to livestock production in large areas of Africa, the Middle East, southern and south-east Asia and Australia. Its control is difficult. The feasibility of vaccinating against this pest has now been explored. In-vitro and in-vivo assays have been established. Using these assays, it has been shown that first instar larvae, third instar peritrophic membrane and cardia are all sources of material able to induce immunological reactions in sheep which lead to significant reductions in larval growth. In-vitro assays following vaccination with peritrophic membrane also show larval mortality. Taken together, these effects lead to an 82% reduction in the weight of recovered larvae in vitro and 45% reduction in vivo. Preliminary evidence suggests that the mechanism of protection may be complex.
Publisher: Elsevier BV
Date: 02-1999
DOI: 10.1016/S0965-1748(98)00123-4
Abstract: The peritrophic matrix (or peritrophic membrane) lines the gut of most insects at one or more stages of the life cycle. It has important roles in the facilitation of the digestive processes in the gut and the protection of the insect from invasion by microorganisms and parasites. The traditional view of the peritrophic matrix as a relatively insert sieve, composed largely of proteins and glycosaminoglycans embedded in a chitinous matrix, is under revision as more is learned about the molecular characteristics of the peritrophic matrix proteins. This review summarizes emerging knowledge of the main protein constituents of the peritrophic matrix. The availability of the first sequences of integral peritrophic matrix proteins has coincided with the explosion of information in sequence databases. It is therefore possible to examine common structural themes in this family of proteins as well as in proteins of unknown location and function from a variety of other insects, nematodes and viruses. The review concludes with speculation about the biological functions of the proteins in this matrix.
Publisher: Wiley
Date: 28-02-2020
DOI: 10.1002/RCM.8723
Abstract: Cytokines are cell regulatory molecules of high importance as indicators for homeostasis and pathology in many species. The current method to measure cytokines in body fluids is reagent dependent, requiring highly specific paired antibodies. A liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS)-based approach was developed to simultaneously establish the limits of detection (LODs) and quantification (LOQs) for recombinant cytokines IL-1β, IL-6, IFNγ and TNFα as pure standards and in bovine sera. All experimental LC/MRM-MS data are available at CSIRO Data Access Portal repository under identifier 0.25919/5de8a0232a862. The present method enabled LODs and LOQs as low as 1.05 and 1.12 fmol/μL in the experiment comprised of pure standards. Comparable results were obtained in the experiment where digested cytokines were mixed with pre-digested sera proteins. The intrinsic matrix effects were evident when intact cytokines were co-digested within undiluted and undigested sera decreasing the ability to detect and quantify cytokines by 10,000-fold compared with pure standards and pre-digested sera. The developed LC/MRM-MS method provided insights into the difficulties in detecting the target peptides when embedded in complex matrices. Nonetheless, the method may potentially be readily applied in biomarker-focused research interrogating fluids of lesser complexity such as synovial fluid, cerebrospinal fluid and tissue culture media.
Publisher: Springer Science and Business Media LLC
Date: 14-08-2022
DOI: 10.1007/S00484-022-02349-4
Abstract: Responses to heat stress in ruminants reflect the integration of local climatic conditions, environment roduction system and the animal's homeostatic and homeorhetic capacities. Thus, the goal of ameliorating heat stress requires experimental settings that, within limits, closely resemble the target production system and cohort. We investigated the blood biochemical changes of two sequential cohorts of twelve 518 ± 23 kg grain fed Black Angus steers. Each cohort consisted of two treatments of 6 head/group: a thermally challenged (TC) treatment and a feed restricted thermoneutral (FRTN) treatment. Both groups were housed in climate controlled rooms for 19 days, with the TC group experiencing three distinct periods: PreChallenge, Challenge and Recovery. PreChallenge and Recovery delivered thermoneutral conditions, while Challenge consisted of 7 days of moderate diurnal heat load. The FRTN group was maintained in thermoneutral conditions at all times. Both groups were then relocated to outdoor pens for a further 40 days to detect any enduring change to metabolism as a consequence of the treatments. We compared blood biochemical responses of the treatments and inferred likely metabolic changes. Relative to the FRTN group, the TC animals experienced limited supply of triglycerides, cholesterol and glutamine during moderate heat load, suggesting constraints to energy metabolism. Lower blood urea during Recovery and in outdoor pens implied a requirement to capture N rather than allow its excretion. Altered liver enzyme profiles indicated a higher level of hepatic stress in the TC group. By the completion of feedlot finishing, the groups were not separable on most measures.
Publisher: Elsevier BV
Date: 04-2021
Publisher: Elsevier BV
Date: 08-1995
Abstract: Four cDNA clones (GST-1, -7, -47, and -51) encoding isoenzymes of the detoxification enzyme glutathione S-transferase (GST) have previously been identified and characterised from Fasciola hepatica. In the present study, antisera were generated to synthetic peptides of regions unique to each of the four GST proteins predicted by the cDNAs. The antisera were characterised, and two were found to distinguish GST-1 from GST-7, GST-47, and GST-51 as a group. These two antisera were used to localise different GSTs in adult and newly excysted juvenile F. hepatica. The antiserum to GST-1 was specific and localised GST-1 to the parenchyma of adult fluke but not to the lamellae of the intestinal caeca. The antiserum to a GST-51 peptide, which cross-reacted with GST-7 and GST-47 but not GST-1, localised the other GSTs not only to the parenchyma but also to the intestinal lamellae of adult fluke. This appears to be the first evidence of tissue-specific expression of GST isoenzymes in trematodes. In contrast to adult fluke, immunolocalisation of the GSTs in juvenile F. hepatica revealed the binding of both the GST-1 and GST-51 antisera to the parenchymal cytoplasm, to cytoplasmic extensions of the parenchyma cells in the subtegumental area, as well as the excretory ducts. No labeling was observed in the intestinal epithelium of the juvenile fluke. These results demonstrate that adult F. hepatica, in contrast to juvenile flukes, contain a GST, which is not GST-1, associated with the lamellae of the gut and suggest that GSTs in adult fluke may play a role in the absorptive function of the adult gut.
Publisher: Oxford University Press (OUP)
Date: 04-2016
Publisher: Oxford University Press (OUP)
Date: 04-2016
Publisher: Elsevier BV
Date: 05-1997
DOI: 10.1016/S0965-1748(97)00020-9
Abstract: HieACE, a soluble 70 kDa protein related to the angiotensin-converting enzyme (ACE) has recently been identified, characterized and cloned from the adult buffalo fly (Haematobia irritans exigua). HieACE is enzymatically similar to the mammalian ACEs and its predicted amino acid sequence has 42% identity with the mammalian testicular ACEs. In adult H.i. exigua, HieACE expression is restricted to the compound ganglion and posterior midgut, and the maturing male reproductive system. Western blot analysis was used to investigate the expression of HieACE and its homologues in the larvae of H.i. exigua, Drosophila melanogaster, the sheep blowfly (Lucilia cuprina), the Old World screwworm fly (Chrysomya bezziana) and a secondary strike fly, Chrysomya rufifacies. Dipteran ACE homologues of 65-70 kDa were detected in all the larval instars investigated. Most of the immunoreactive proteins were concentrated in the soluble fraction. The first and second larval instars of L. cuprina and C. bezziana appeared to express two ACE homologues. These larvae were also found to secrete (or excrete) the ACE homologue in larval cultures. The presence of ACE-like enzymes in these larvae was confirmed by the measurement of carboxydipeptidase activity that was inhibited by the specific ACE inhibitor, captopril. The tissue distributions of the ACE homologues in the third instar larvae of H.i. exigua and L. cuprina were examined. As in adult H.i. exigua, HieACE was detected in the larval ganglion, but in contrast to the restricted distribution in the adult stage midgut, HieACE was found throughout the digestive system, and in the salivary glands of H.i. exigua larvae. The expression pattern in the gut of L. cuprina larvae was similar despite the differences in diet and habitat. The most striking difference from the adult stage H.i. exigua was the expression of HieACE and its L. cuprina homologues in the hindgut and Malpighian tubules of these larvae. These results suggest that the role(s) played by the dipteran ACE-like enzymes differ between the adult and larval stages.
Publisher: Springer Science and Business Media LLC
Date: 07-2005
DOI: 10.1007/S10858-005-7946-4
Abstract: Cell-free protein synthesis systems provide facile access to proteins in a nascent state that enables formation of soluble, native protein-protein complexes even if one of the protein components is prone to self-aggregation and precipitation. Combined with selective isotope-labeling, this allows the rapid analysis of protein-protein interactions with few 15N-HSQC spectra. The concept is demonstrated with binary and ternary complexes between the chi, psi and gamma subunits of Escherichia coli DNA polymerase III: nascent, selectively 15N-labeled psi produced in the presence of chi resulted in a soluble, correctly folded chi-psi complex, whereas psi alone precipitated irrespective of whether gamma was present or not. The 15N-HSQC spectra showed that the N-terminal segment of psi is mobile in the chi-psi complex, yet important for its binding to gamma. The s le preparation was greatly enhanced by an autoinduction strategy, where the T7 RNA polymerase needed for transcription of a gene in a T7-promoter vector was produced in situ.
Publisher: Wiley
Date: 21-05-2001
DOI: 10.1002/ARCH.1038
Abstract: The midgut of most insects is lined with a peritrophic matrix, which is thought to facilitate digestion and protect the midgut digestive epithelial cells from abrasive damage and invasion by ingested micro-organisms. The type 2 peritrophic matrix is synthesised by a complex and highly specialised organ called the cardia typically located at the junction of the cuticle-lined foregut and midgut. Although the complex anatomy of this small organ has been described, virtually nothing is known of the molecular processes that lead to the assembly of the type 2 peritrophic matrix in the cardia. As a step towards understanding the synthesis of the peritrophic matrix, the synthesis and secretion of the intrinsic peritrophic matrix protein, peritrophin-15 has been followed in the cardia of Lucilia cuprina larvae using immuno-gold localisations. The protein is synthesised by cardia epithelial cells, which have abundant rough endoplasmic reticulum, Golgi, and vesicles indicative of a general secretory function. Peritrophin-15 is packaged into secretory vesicles probably produced from Golgi and transported to the cytoplasmic face of the apical plasma membrane. The vesicles fuse with the plasma membrane at the base of the microvilli and release peritrophin-15 into the inter-microvilli spaces. The protein then becomes associated with the nascent peritrophic matrix, which lies along the tips of the epithelial cell microvilli. It is proposed that peritrophin-15 binds to the ends of chitin fibrils present in the nascent peritrophic matrix, thereby protecting the fibril from the action of exochitinases.
Publisher: Elsevier BV
Date: 03-2023
Publisher: Elsevier BV
Date: 02-1995
DOI: 10.1016/0166-6851(94)00205-2
Abstract: Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses. The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE. Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. All four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with EPNP. rGST47 and 51 showed activity with the other four substrates. rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles.
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.JPBA.2012.04.028
Abstract: Thousands of metabolites are excreted in urine, and potentially can be detected in NMR spectra. Currently, NMR spectral information for about one thousand metabolites has been deposited in publicly available sources, limiting the identification of chemical compounds that are potential biomarkers for clinical and subclinical applications. This study reports the identification of crotonyl glycine, one of the key metabolites detected by ¹H NMR as excreted in the urine of sheep after 48 h road transport and during the subsequent 72 h recovery period. This metabolite was important in separating the metabolic responses as expressed in the urine from animals undergoing shorter road transport treatments. At the time of the metabonomic analysis, the NMR signals from this metabolite were designated as unassigned as no match was found in public databases or the literature. Selected sheep urine s les containing the metabolite were resolved by reversed phase HPLC reducing the s le complexity. Subsequent ¹H NMR spectra of the collected fractions revealed that the unknown metabolite was present in a single HPLC fraction. High-resolution 1D and 2D ¹H NMR spectra of this fraction followed by mass determination of the parent ion and its fragments by nanoESI-TOF-MS/MS revealed the identity of the compound as crotonyl glycine (N-but-(E)-2-enoyl glycine). The HPLC fraction was subsequently spiked with synthetic crotonyl glycine which confirmed identification.
Publisher: American Society for Microbiology
Date: 08-2006
DOI: 10.1128/JB.00231-06
Abstract: The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication ( oriV ). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli β sliding cl (β 2 ). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial cl -binding consensus motif. T1 forms a stable complex with β 2 and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and β 2 and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1 (P. D. Kim, T. M. Rosche, and W. Firshein, Plasmid 43:214-222, 2000). The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the β-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent overinitiation in E. coli through its interaction with the initiator protein, DnaA, and β 2 . Our results support a model in which T1 toxicity is caused by T1 binding to β 2 , especially when T1 is overexpressed, preventing β 2 from interacting with host replication proteins such as Hda during the early events of chromosome replication.
Publisher: Elsevier BV
Date: 02-1992
DOI: 10.1016/0014-4894(92)90142-W
Abstract: Glutathione S-transferases (GSTs) from Fasciola hepatica have been purified by glutathione affinity chromatography. Two closely migrating species of Mr 26,000 and 26,500 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several species resolved by two-dimensional gel analysis, indicating substantial heterogeneity among the GSTs. N-terminal amino acid sequencing revealed one core sequence containing three polymorphisms, whereas the sequence of GST peptides implied a minimum of three different GSTs. The amino acid sequence data assigned the F. hepatica GSTs to the mu class of GSTs with high similarities to these proteins in other helminths and mammals. The native GSTs of F. hepatica appeared to behave as dimers as determined by molecular sieving chromatography. The observation that the GSTs of F. hepatica are heterogeneous in sequence and behave as dimers in the native state suggest that these isoenzymes may exhibit considerable functional heterogeneity which may be of importance to the parasite. Immunocytochemical studies suggest that the main source of GST in F. hepatica are the parenchymal cells and peripheral tissues of the parasite. Some extracellular GST is associated with the lamellae of the intestinal epithelium. The identification of an intestinal GST is unique among trematodes studied to date.
Publisher: American Chemical Society (ACS)
Date: 31-01-2011
DOI: 10.1021/PR100862T
Abstract: The physical, endocrine, and metabolic responses of livestock to road transport have been evaluated by conventional hematological and biochemistry parameters for more than 20 years. However, these measures are relatively insensitive to subtle metabolic adaptations. We applied NMR-based metabonomics to assess system-wide metabolic responses as expressed in urine and serum of a large cohort of animals (n = 80) subjected to 12 and 48 h road transport. The profiling of (1)H NMR spectra revealed that the transported animals experienced altered gut and energy metabolism, muscle catabolism, and possibly a renal response. The animals transported for 48 h exhibited a deeper metabolic response to the transport event and a complex and expanded metabolic trajectory over the 72 h recovery period. Intriguingly, excretion of acyl glycines and a dicarboxylic acid was observed after transport and during recovery, implicating peroxisomal fatty acid oxidation as a metabolic response to transport-induced stress.
Publisher: Elsevier BV
Date: 03-1994
Abstract: There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infection. A cysteine proteinase complex identified in the regurgitant of adult F. hepatica was examined in this context. The thiol-cathepsin-related proteinases of M(r) 28,000 were purified and tested in vaccine trials of sheep infected with liver fluke. Ten animals were immunised with the purified proteinases and developed antibodies to the cysteine proteinases prior to challenge with F. hepatica metacercariae. Infection appeared to cause a boost in antibody response by Week 4 into infection, and antibody levels were generally sustained throughout infection. The cysteine proteinases are not novel antigens, since low-level antibody titres were also detected in nonimmunised controls by late infection. On completion of the trial, there was no difference in worm burden between the two groups. However, faecal egg counts and therefore worm fecundity were significantly decreased.
Publisher: International Union of Crystallography (IUCr)
Date: 27-06-2003
DOI: 10.1107/S0907444903009958
Abstract: The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding cl that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 A [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 A at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 A resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.
Publisher: Elsevier BV
Date: 11-2009
DOI: 10.1016/J.MOLBIOPARA.2009.07.001
Abstract: Two different classes of small nematode specific lipid-binding proteins, the nematode polyprotein allergens/antigens (NPAs) and the fatty acid- and retinol-binding (FAR) proteins, are secreted by helminth parasites. Until now, there was no evidence of the expression or secretion of these two families of proteins in Haemonchus contortus. In this study, we applied proteomic and bioinformatic tools in an iterative manner to reveal the expression and complexity of these proteins in the excretory/secretory products (ESP) of adult H. contortus at the protein and gene levels. Initial examination of the mass spectra of ESP fractions against standard databases returned nine peptides mapping to Ostertagia ostertagi NPA and FAR sequences. Searches of the H. contortus EST and genomic contig databases with the O. ostertagi and Caenorhabditis elegans homologues retrieved erse sequences encoding H. contortus NPA and FAR proteins. H. contortus sequences were then integrated into a customized database and a new search of the mass spectra achieved a 10-fold improvement in coverage of the predicted H. contortus NPAs. The final analyses of the mass spectra achieved 49-60% coverage of H. contortus NPAs and 7-47% coverage of H. contortus FARs. Moreover, the ersity in structures of the encoding genes was revealed by assembling the genomic sequence data with predicted protein sequences confirmed by the peptide evidence. We predict there are at least one Hc-NPA gene and six Hc-FAR genes in H. contortus, and life stage gene expression of Hc-FAR-1 to -6 revealed unique transcription patterns for each of these genes.
Publisher: Elsevier BV
Date: 08-2014
DOI: 10.1016/J.JPROT.2014.06.001
Abstract: Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases.
Publisher: Elsevier BV
Date: 05-2000
DOI: 10.1016/S0020-7519(00)00055-2
Abstract: Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.
Location: Australia
No related grants have been discovered for Gene Wijffels.