Ian Harper

To 5D and Beyond: Quantitative Fluorescence Microscopy in the Postgenomic Era

Publisher: Wiley

Date: 2002

DOI: 10.1034/J.1600-0854.2002.30105.X

Abstract: Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide-field microscopes (WFM) largely depends on the type of s le, with LSMs performing best on thick s les and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large-scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.

Effects of beta-blockers and Ca²⁺ -antagonists on the response of the isolated working rat heart to adrenergic stimulants after cardioplegic arrest

Publisher: Springer Science and Business Media LLC

Date: 12-1993

DOI: 10.1007/BF00877715

Measurement of electrical potential, pH, and free calcium ion concentration in mitochondria of living cells by laser scanning confocal microscopy

Publisher: Elsevier

Date: 1995

DOI: 10.1016/0076-6879(95)60156-2

Abstract: Enteropathogenic Escherichia coli O127 is encapsulated by a protective layer of polysaccharide made of the same strain specific O-antigen as the serotype lipopolysaccharide. Seven genes encoding capsule export functions comprise the group 4 capsule (gfc) operon. Genes gfcE, etk and etp encode homologs of the group 1 capsule secretion system but the upstream gfcABCD genes encode unknown functions specific to group 4 capsule export. We have developed an expression system for the large-scale production of the outer membrane protein GfcD. Contrary to annotations, we find that GfcD is a non-acylated integral membrane protein. Circular dichroism spectroscopy, light-scattering data, and the HHomp server suggested that GfcD is a monomeric β-barrel with 26 β-strands and an internal globular domain. We identified a set of novel protein-protein interactions between GfcB, GfcC, and GfcD, both in vivo and in vitro, and quantified the binding properties with isothermal calorimetry and biolayer interferometry. GfcC and GfcB form a high-affinity heterodimer with a KD near 100 nM. This heterodimer binds to GfcD (KD = 28 μM) significantly better than either GfcB or GfcC alone. These gfc proteins may form a complex at the outer membrane for group 4 capsule secretion or for a yet unknown function.

Integrating scientific workflows and large tiled display walls: Bridging the visualization divide

Publisher: IEEE

Date: 09-2011

DOI: 10.1109/ICPPW.2011.30

Mice deficient for the chromosome 21 ortholog Itsn1 exhibit vesicle-trafficking abnormalities

Publisher: Oxford University Press (OUP)

Date: 02-08-2008

DOI: 10.1093/HMG/DDN224

Abstract: Enlarged early endosomes in the neurons of young Down syndrome (DS) and pre-Alzheimer's disease (AD) brains suggest that a disturbance in endocytosis is one of the earliest hallmarks of AD pathogenesis in both conditions. We identified a chromosome 21 gene, Intersectin-1 (ITSN1) that is up-regulated in DS brains and has a putative function in endocytosis and vesicle trafficking. To elucidate the function of ITSN1 and assess its contribution to endocytic defects associated with DS and AD, we generated Itsn1 null mice. In knockout mice we found alterations in a number of parameters associated with endocytic and vesicle trafficking events. We found a reduced number of exocytosis events in chromaffin cells and a slowing of endocytosis in neurons. Endosome size was increased in neurons and NGF levels were reduced in the septal region of the brain. Our data is the first indication that Itsn1 has a role in endocytosis in an in vivo mammalian model, and that a disruption in Itsn1 expression causes a disturbance in vesicle trafficking and endocytic function in the brain. These results imply a role for ITSN1 in the early endocytic anomalies reported in DS brains which may have ramifications for the onset of AD.

Use of granzyme B-based fluorescent protein reporters to monitor granzyme distribution and granule integrity in live cells

Publisher: Walter de Gruyter GmbH

Date: 08-2010

DOI: 10.1515/BC.2010.099

Abstract: Reporter proteins comprising granzyme B (GrB) fused to eGFP, ecliptic pHluorin or mCherry, were generated and used to study granule (lysosome) distribution and properties in COS-1 cells and natural killer cells. The reporters resembled native GrB in biosynthesis and localization, and accumulated in granules. In live cells both the eGFP and pHluorin reporters were dark in lysosomes, but fluoresced when the granule integrity or pH was perturbed by Leu-Leu methyl ester, hydrogen peroxide, naphthazarin, or sphingosine treatment. By contrast, fluorescence of the mCherry reporter was not pH-dependent. The quenching of eGFP within granules indicates that this commonly-used fluorescent protein is not appropriate as a vital intra-lysosomal marker.

Lanthanum probing of cell membrane permeability in the rat heart: Pathological versus artefactual alterations

Publisher: Wiley

Date: 04-1990

DOI: 10.1002/JEMT.1060140411

Abstract: The influence of fixation on membrane permeability has been examined in the isolated rat heart using lanthanum as a permeability probe. Normal and ischaemic hearts were probed at various stages during a conventional fixation programme with either ionic or colloidal lanthanum and compared with lanthanum saline administered prior to fixation. Fixation of the myocardium coincident with or followed by lanthanum probing resulted in an influx of the probe into most myocytes in normal tissue. Alterations in permeability after ischaemic episodes could not be distinguished from the artefact. However, lanthanum saline prior to fixation showed exclusion of the probe from normal tissue, while the increased permeability demonstrated after ischaemia was associated with declining myocardial performance during subsequent reperfusion. These results illustrate the need for caution in the application and evaluation of methods determining permeability in fixed tissue. Probes of differing size and charge permeated fixed tissue to varying degrees thereby implicating the formation of specific lesions during chemical fixation.

A multimode-TIRFM and microfluidic technique to examine platelet adhesion dynamics

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-538-5_3

Abstract: Fluorescence microscopy techniques have provided important insights into the structural and signalling events occurring during platelet adhesion under both static and blood flow conditions. However, due to limitations in sectioning ability and sensitivity these techniques are restricted in their capacity to precisely image the adhesion footprint of spreading platelets. In particular, investigation of platelet adhesion under hemodynamic shear stress requires an imaging platform with high spatial discrimination and sensitivity and rapid temporal resolution. This chapter describes in detail a multimode imaging approach combining total internal reflection fluorescence microscopy (TIRFM) with high speed epifluorescence and differential interference contrast (DIC) microscopy along with a novel microfluidic perfusion system developed in our laboratory to examine platelet membrane adhesion dynamics under static and flow conditions.

A revised model of platelet aggregation

Publisher: American Society for Clinical Investigation

Date: 15-03-2000

DOI: 10.1172/JCI7569

Myocardial membrane cholesterol: Effects of ischaemia

Publisher: Elsevier BV

Date: 11-1991

DOI: 10.1016/0022-2828(91)90084-Y

Abstract: Evidence has recently been presented that myocardial ischaemia is associated with a significant increased mitochondrial cholesterol content, suggesting a redistribution of cholesterol within the ischaemic cell (Rouslin et al. 1980, 1982). The aim of this study was therefore to determine the effects of different periods of ischaemia and reperfusion on the cholesterol content of myocardial mitochondria, sarcoplasmic reticulum and sarcolemma. Using the isolated perfused rat heart as experimental model, it was demonstrated that increasing periods of ischaemia (15-60 min) caused a progressive loss of cholesterol from the tissue as well as from the sarcolemma and sarcoplasmic reticulum, concomitant with a significant increase in mitochondrial cholesterol content. These compositional changes were associated with a marked increase in sarcolemmal and mitochondrial microviscosity, while that of the sarcoplasmic reticulum was reduced. To gain more insight into the mechanisms controlling intracellular cholesterol distribution, control and ischaemic hearts were perfused with either exogenous cholesterol or its precursor [U-14C]acetate as an indicator of endogenous cholesterol synthesis. Perfusion with exogenous cholesterol resulted in significant increases in the membrane cholesterol content of control hearts. However, hypoxic, low flow perfusion prevented cholesterol enrichment of the sarcolemmal and sarcoplasmic reticulum membranes, while the cholesterol content of the mitochondria was increased from 99.48 +/- 12.75 to 127.61 +/- 1.84 nmols/mg protein, indicating specific incorporation into this membrane system. Incorporation of [U-14C]acetate into cholesterol in the sarcoplasmic reticulum was increased by 120% in ischaemic conditions. However, a marked redistribution of newly synthesized cholesterol was observed within the ischaemic cell: under control conditions most of the labelled cholesterol was transferred to the sarcolemma and least to the mitochondria, while this distribution pattern was reversed in ischaemia. In view of the fact that exchange of cholesterol between membranes is affected by both phospholipid polar head-group composition and acyl chain length and saturation, it is suggested that prior ischaemia-induced membrane compositional changes might lead to intracellular cholesterol redistribution. Finally, to determine whether cholesterol loss affects sarcolemmal permeability, hearts enriched in sarcolemmal cholesterol were subjected to 15 or 30 min global ischaemia followed by reperfusion and the rate of enzyme release determined. However, enzyme release was similar in treated and untreated hearts, indicating that sarcolemmal cholesterol loss probably does not affect its permeability.

The Development of the Digestive Glands and Enzymes in the Pitchers of ThreeNepenthesSpecies:N. alata,N. tobaica, andN. ventricosa(Nepenthaceae)

Publisher: University of Chicago Press

Date: 06-2008

DOI: 10.1086/533599

Fluorescence photobleaching analysis for the study of cellular dynamics

Publisher: Springer Science and Business Media LLC

Date: 03-2002

DOI: 10.1007/S00249-001-0202-2

Abstract: The wide availability of the confocal microscope and the emergence of green fluorescent protein (GFP) transfection technology has led to the increasing use of photobleaching studies to examine aspects of cellular dynamics in living cells. In this review, we examine the theory and practice of performing photobleaching studies using a confocal microscope. We illustrate the application of photobleaching protocols using our own measurements of fluorescently labelled red blood cells and of malaria parasite-infected erythrocytes expressing GFP fusions and examine other ex les from the literature.

Intercellular calcium communication regulates platelet aggregation and thrombus growth

Publisher: Rockefeller University Press

Date: 31-03-2003

DOI: 10.1083/JCB.200207119

Abstract: The ability of platelets to form stable adhesion contacts with other activated platelets (platelet cohesion or aggregation) at sites of vascular injury is essential for hemostasis and thrombosis. In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet–platelet adhesion contacts under the influence of flow. An examination of platelet calcium flux during platelet aggregate formation in vitro demonstrated a key role for intercellular calcium communication (ICC) in regulating the recruitment of translocating platelets into developing aggregates. We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin αIIbβ3 and the recently cloned ADP purinergic receptor P2Y12. Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth.

Mitochondrial free calcium transients during excitation-contraction coupling in rabbit cardiac myocytes

Publisher: Wiley

Date: 11-03-1996

DOI: 10.1016/0014-5793(96)00138-X

Abstract: Mitochondrial free Ca2+ may regulate mitochondrial ATP production during cardiac exercise. Here, using laser scanning confocal microscopy of adult rabbit cardiac myocytes co-loaded with Fluo-3 to measure free Ca2+ and tetramethylrhodamine methylester to identify mitochondria, we measured cytosolic and mitochondrial Ca2+ transients during the contractile cycle. In resting cells, cytosolic and mitochondrial Fluo-3 signals were similar. During electrical pacing, transients of Fluo-3 fluorescence occurred in both the cytosolic and mitochondrial compartments. Both the mitochondrial and the cytosolic transients were potentiated by isoproterenol. These experiments show directly that mitochondrial free Ca2+ rises and falls during excitation-contraction coupling in cardiac myocytes and that changes of mitochondrial Ca2+ are kinetically competent to regulate mitochondrial metabolism on a beat-to-beat basis.

Imaging Tools for Analysis of the Ureteric Tree in the Developing Mouse Kidney

Publisher: Springer New York

Date: 24-08-2013

DOI: 10.1007/978-1-60761-847-8_16

Abstract: The structure of the ureteric tree in developing mouse and rat kidneys has previously been quantified in two dimensions. While this type of analysis may provide evidence of changes in ureteric growth, these measurements are effectively inaccurate, as the ureteric tree is a three-dimensional (3D) object. Here we describe a method for measuring the ureteric tree in three dimensions. This technique involves (1) culture of the metanephric kidney at embryonic day 12 (mouse) or 14 (rat), (2) whole-mount immunofluorescence to selectively stain ureteric tree epithelium, (3) confocal microscopy to obtain a complete Z series through the ureteric tree, and (4) image analysis algorithms to binarize, skeletonize, and measure in idual branch lengths in 3D. This method has been extended to analysis of the same ureteric tree over time (4D). The results obtained provide accurate and precise quantitation of ureteric tree growth in the developing mouse or rat kidney.

The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells

Publisher: Elsevier BV

Date: 12-1999

DOI: 10.1074/JBC.274.51.36241

Distinct glycoprotein Ib/V/IX and integrin αIIbβ3-dependent calcium signals cooperatively regulate platelet adhesion under flow

Publisher: Elsevier BV

Date: 2002

DOI: 10.1074/JBC.M110070200

Immunodetection of Granzyme B tissue distribution and cellular localisation

Publisher: Humana Press

Date: 21-12-2012

DOI: 10.1007/978-1-61779-527-5_17

Abstract: Investigation of Granzyme B (GrB) function and pathophysiology in both human settings and rodent models increasingly involve the use of indirect immunofluorescence imaging and fluorescence-activated cell sorting, which requires reliable GrB antibodies that do not recognise other closely related granzymes. Here, we describe the validation (using a set of recombinant granzymes, and GrB-deficient cells) and application of widely available monoclonal antibodies to specifically monitor GrB in human or mouse cells.

Erratum: Mice deficient for the chromosome 21 ortholog Itsn1 exhibit vesicle-trafficking abnormalities (Human Molecular Genetic (2008) vol. 17(21) (3281-3290) 10.1093/hmg/ddn224)

Publisher: Oxford University Press (OUP)

Date: 22-10-2009

DOI: 10.1093/HMG/DDN378

Radiation-induced changes in the ultrastructure and mechanical function of the rat heart

Publisher: Elsevier BV

Date: 12-1989

DOI: 10.1016/0167-8140(89)90044-3

Abstract: A time sequence study was performed to study the early effects of radiation on the ultrastructure of the rat heart. Wistar rats were exposed to 20 Gy electron irradiation to a field including the heart and a third of the lung. The hearts were excised at varying time intervals (1 h-180 days), and the ultrastructure of perfusion-fixed subepicardium and subendocardium studied. Changes were observed in both myocytes and interstitium at all time intervals. The most pronounced change observed in the myocyte was that of intercalated disc damage which reached a peak at 30 days post-irradiation. Mitochondrial damage, characterized by swelling and fenestration in areas of myofibrillar contracture, was focal and relatively scarce. Swelling of the capillary endothelial cells and collapse of the capillaries were marked up to 60 days. Of significance was the observation that the damage to both myocytes and interstitium receded after 60 days and the hearts exhibited an almost normal ultrastructure from 100 to 180 days post-irradiation. Mechanical function of these hearts followed a similar pattern: maximal depression was observed 60 days after irradiation. Thereafter the work performance of these hearts improved significantly, almost reaching control levels after 180 days.

Investigation of selective release of periplasmic proteins through pore size analysis and single-cell microscopy in Escherichia coli

Publisher: Elsevier BV

Date: 07-2021

DOI: 10.1016/J.BEJ.2021.108009

Sarcolemmal integrity during ischaemia and reperfusion of the isolated rat heart

Publisher: Springer Science and Business Media LLC

Date: 03-1989

DOI: 10.1007/BF01907930

Abstract: Caspase-12 functions as an antiinflammatory enzyme inhibiting caspase-1 and the NOD2/RIP2 pathways. Due to increased susceptibility to sepsis in in iduals with functional caspase-12, an early-stop mutation leading to the loss of caspase-12 has replaced the ancient genotype in Eurasia and a significant proportion of in iduals from African populations. In African-Americans, it has been shown that caspase-12 inhibits the pro-inflammatory cytokine production. We assessed whether similar mechanisms are present in African in iduals, and whether evolutionary pressures due to plague may have led to the present caspase-12 genotype population frequencies. No difference in cytokine induction through the caspase-1 and/or NOD2/RIP2 pathways was observed in two independent African populations, among in iduals with either an intact or absent caspase-12. In addition, stimulations with Yersinia pestis and two other species of Yersinia were preformed to investigate whether caspase-12 modulates the inflammatory reaction induced by Yersinia. We found that caspase-12 did not modulate cytokine production induced by Yersinia spp. Our experiments demonstrate for the first time the involvement of the NOD2/RIP2 pathway for recognition of Yersinia. However, caspase-12 does not modulate innate host defense against Y. pestis and alternative explanations for the geographical distribution of caspase-12 should be sought.

An abstract virtual instrument system for high throughput automatic microscopy

Publisher: Elsevier BV

Date: 05-2010

DOI: 10.1016/J.PROCS.2010.04.058

Virtual microscopy and analysis using scientific workflows

Publisher: IEEE

Date: 12-2009

DOI: 10.1109/E-SCIENCE.2009.41

The pH Paradox in the Pathophysiology of Reperfusion Injury to Rat Neonatal Cardiac Myocytes

Publisher: Wiley

Date: 06-1994

DOI: 10.1111/J.1749-6632.1994.TB36714.X

Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 14-03-2016

DOI: 10.1681/ASN.2015121340

Abstract: Podocyte depletion is sufficient for the development of numerous glomerular diseases and can be absolute (loss of podocytes) or relative (reduced number of podocytes per volume of glomerulus). Commonly used methods to quantify podocyte depletion introduce bias, whereas gold standard stereologic methodologies are time consuming and impractical. We developed a novel approach for assessing podocyte depletion in whole glomeruli that combines immunofluorescence, optical clearing, confocal microscopy, and three-dimensional analysis. We validated this method in a transgenic mouse model of selective podocyte depletion, in which we determined dose-dependent alterations in several quantitative indices of podocyte depletion. This new approach provides a quantitative tool for the comprehensive and time-efficient analysis of podocyte depletion in whole glomeruli.

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

Publisher: Springer Science and Business Media LLC

Date: 31-01-2014

DOI: 10.1038/CDD.2014.7

Inhibition of Na⁺/H⁺exchange preserves viability, restores mechanical function, and prevents the pH paradox in reperfusion injury to rat neonatal myocytes

Publisher: Springer Science and Business Media LLC

Date: 1993

DOI: 10.1007/BF00795410

Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus

Publisher: Wiley

Date: 15-08-2009

DOI: 10.1002/JCB.22218

Abstract: Nuclear protein transport processes have largely been studied using in vitro semi-intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T-ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T-ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM-1-mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells.

Confocal imaging, visualization and 3‐D surface measurement of small mammalian teeth

Publisher: Wiley

Date: 11-2001

DOI: 10.1046/J.1365-2818.2001.00939.X

Abstract: The difficulties traditionally faced by functional morphologists in representing and interpreting three-dimensional objects can now be mostly overcome using available laser and computer imaging technologies. A practical method for three-dimensional imaging of small mammalian teeth using confocal microscopy is reported. Moulding and casting of the teeth were first performed, followed by confocal fluorescence imaging. Accuracy and precision of the scanned structures were tested in morphometric studies by using a new technique to measure the noise in the scan of a three-dimensional surface, and linear and angular dimensions of the scans were compared with measurements made using traditional morphological tools. It is shown that measurements can be taken with less than 4% difference from the original object. Teeth of the microchiropteran bat Chalinolobus gouldii were scanned and measured to show the potential of the techniques. Methods for visualizing the small teeth in three-dimensional space, and animating the teeth in occlusion, show the power of this approach in aiding a three-dimensional understanding of the structure and function of teeth and other three-dimensional structures.

Preconditioning with a single short episode of global ischemia in the isolated working rat heart: Effect on structure, mechanical function, and energy metabolism for various durations of sustained global ischemia

Publisher: Springer Science and Business Media LLC

Date: 02-1995

DOI: 10.1007/BF00877750

Abstract: Potential long-lasting adverse effects of child maltreatment have been widely reported, although little is known about the distinctive long-term impact of differing types of maltreatment. Our objective for this special article is to integrate findings from the Mater-University of Queensland Study of Pregnancy, a longitudinal prenatal cohort study spanning 2 decades. We compare and contrast the associations of specific types of maltreatment with long-term cognitive, psychological, addiction, sexual health, and physical health outcomes assessed in up to 5200 offspring at 14 and/or 21 years of age. Overall, psychological maltreatment (emotional abuse and/or neglect) was associated with the greatest number of adverse outcomes in almost all areas of assessment. Sexual abuse was associated with early sexual debut and youth pregnancy, attention problems, posttraumatic stress disorder symptoms, and depression, although associations were not specific for sexual abuse. Physical abuse was associated with externalizing behavior problems, delinquency, and drug abuse. Neglect, but not emotional abuse, was associated with having multiple sexual partners, cannabis abuse and/or dependence, and experiencing visual hallucinations. Emotional abuse, but not neglect, revealed increased odds for psychosis, injecting-drug use, experiencing harassment later in life, pregnancy miscarriage, and reporting asthma symptoms. Significant cognitive delays and educational failure were seen for both abuse and neglect during adolescence and adulthood. In conclusion, child maltreatment, particularly emotional abuse and neglect, is associated with a wide range of long-term adverse health and developmental outcomes. A renewed focus on prevention and early intervention strategies, especially related to psychological maltreatment, will be required to address these challenges in the future.

GLUTARADEHYDE‐INDUCED PERMEABILIZATION OF CELL MEMBRANES

Publisher: Wiley

Date: 02-1986

DOI: 10.1111/J.1365-2818.1986.TB02709.X

Abstract: Once released, norepinephrine is removed from cardiac synapses via reuptake into sympathetic nerves, whereas transmitter ATP is catabolized by ecto-NTP diphosphohydrolase 1 (E-NTPDase1)/CD39, an ecto-ATPase. Because ATP is known to modulate neurotransmitter release at prejunctional sites, we questioned whether this action may be ultimately controlled by the expression of E-NTPDase1/CD39 at sympathetic nerve terminals. Accordingly, we silenced E-NTPDase1/CD39 expression in nerve growth factor-differentiated PC12 cells, a cellular model of sympathetic neuron, in which dopamine is the predominant catecholamine. We report that E-NTPDase1/CD39 deletion markedly increases depolarization-induced exocytosis of ATP and dopamine and increases ATP-induced dopamine release. Moreover, overexpression of E-NTPDase1/CD39 resulted in enhanced removal of exogenous ATP, a marked decrease in exocytosis of ATP and dopamine, and a large decrease in ATP-induced dopamine release. Administration of a recombinant form of E-NTPDase1/CD39 reproduced the effects of E-NTPDase1/CD39 overexpression. Exposure of PC12 cells to simulated ischemia elicited a release of ATP and dopamine that was markedly increased in E-NTPDase1/CD39-silenced cells and decreased in E-NTPDase1/CD39-overexpressing cells. Therefore, transmitter ATP acts in an autocrine manner to promote its own release and that of dopamine, an action that is controlled by the level of E-NTPDase1/CD39 expression. Because ATP availability greatly increases in myocardial ischemia, recombinant E-NTPDase1/CD39 therapeutically used may offer a novel approach to reduce cardiac dysfunctions caused by excessive catecholamine release.

Melatonin protects against ischaemic-reperfusion myocardial damage

Publisher: Elsevier BV

Date: 02-2001

DOI: 10.1006/JMCC.2000.1306

Electron dense artefactual deposits in tissue sections: The role of ethanol, uranyl acetate and phosphate buffer

Publisher: Informa UK Limited

Date: 1990

DOI: 10.3109/10520299009105614

Abstract: The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.

A live cell micro-imaging technique to examine platelet calcium signaling dynamics under blood flow

Publisher: Springer New York

Date: 17-11-2012

DOI: 10.1007/978-1-61779-307-3_6

Abstract: The platelet is a specialized adhesive cell that plays a key role in thrombus formation under both physiological and pathological blood flow conditions. Platelet adhesion and activation are dynamic processes associated with rapid morphological and functional changes, with the earliest signaling events occurring over a subsecond time-scale. The relatively small size of platelets combined with the dynamic nature of platelet adhesion under blood flow means that the investigation of platelet signaling events requires techniques with both high spatial discrimination and rapid temporal resolution. Unraveling the complex signaling processes governing platelet adhesive function under conditions of hemodynamic shear stress has been a longstanding goal in platelet research and has been greatly influenced by the development and application of microimaging-based techniques. Advances in the area of epi-fluorescence and confocal-based platelet calcium (Ca(2+)) imaging have facilitated the in vitro and in vivo elucidation of the early signaling events regulating platelet adhesion and activation. These studies have identified distinct Ca(2+) signaling mechanisms that serve to dynamically regulate activation of the major platelet integrin α(IIb)β(3) and associated adhesion and aggregation processes under flow. This chapter describes in detail a ratiometric calcium imaging protocol and associated troubleshooting procedures developed in our laboratory to examine live platelet Ca(2+) signaling dynamics. This technique provides a method for high-resolution imaging of the Ca(2+) dynamics underpinning platelet adhesion and thrombus formation under conditions of pathophysiological shear stress.

Monash University

Location: Australia

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Stellenbosch University

Location: South Africa

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University Of Natal

Location: South Africa

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South African Medical Research Council

Location: South Africa

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Linkage Projects - Grant ID: LP0882735

Start Date: 07-2008

End Date: 07-2011

Amount: $570,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100165

Start Date: 2010

End Date: 12-2010

Amount: $250,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0561229

Start Date: 03-2005

End Date: 08-2007

Amount: $518,427.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100037

Start Date: 02-2012

End Date: 12-2012

Amount: $350,000.00

Funder: Australian Research Council

Research Networks - Grant ID: RN0460002

Start Date: 08-2004

End Date: 08-2009

Amount: $2,000,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0668435

Start Date: 2006

End Date: 03-2007

Amount: $1,300,000.00

Funder: Australian Research Council