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Biomedical subjects

M J Dunn

Publications and source records attributed to M J Dunn.

At least 55 records · Page 3Linked to original sources
PubMed1999

Characterization of anti-heart antibodies in mice after infection with coxsackie B3 virus.

Coxsackie virus B3 (CVB3) infection results in a marked inflammatory response and the production of autoantibodies to cardiac antigens, with cardiac myosin heavy chain documented to be the most immunogenic antigen. The present study investigated the temporal appearance of anti-heart antibodies in mice after mock infection or infection with an attenuated variant of CVB3 or wildtype CVB3 by SDS-PAGE and Western blotting. Further characterization of the autoantigens was carried out using 2D electrophoresis followed by Western blotting. Mice infected with wildtype CVB3 demonstrated high levels of IgG anti-heart antibodies, reacting predominantly with myosin heavy chain but also with numerous other myocardial proteins. Significant increases in anti-myosin heavy chain, anti-actin, and anti-tropomyosin antibodies were seen in wildtype-infected mice as early as day 7 postinfection compared to those mice that were mock infected or infected with attenuated virus. Characterization of other antigens revealed novel reactivities against myosin subfragments, heat shock proteins, and desmin and its subfragments.

Actins↗
PubMed1999

The expression of heat shock protein 60 in patients with dilated cardiomyopathy.

OBJECTIVE: Heat shock proteins (hsps) have been shown to be important antigens in a number of autoimmune diseases. We have previously shown the presence of autoantibodies against hsp60 in a high proportion of patients with dilated cardiomyopathy (DCM). This study set out to investigate the expression of hsp60 in the myocardium of 30 patients with DCM and a control group of 30 normal donors. DESIGN: The expression of hsp60 was quantitated at the protein and mRNA level by Western blotting and RT-PCR, respectively, and its distribution was investigated by immunocytochemistry. RESULTS: Quantitation of hsp60 showed a 5-fold increase in the DCM hearts over that in the donor hearts. By immunocytochemistry 13 patients with DCM showed increased positive staining localised to the connective tissue. Semi-quantitative RT-PCR analysis of hsp60 showed a significantly increased amount of hsp60 at the mRNA level in the DCM hearts than in the controls. CONCLUSIONS: These results unequivocally demonstrate a raised level of expression of endogenous hsp60 in the myocardium of patients with DCM. The increased expression of hsp60 in the myocardium of patients with DCM may render such cells susceptible to react with circulating autoantibodies to hsp60 and hsp65 found in a high proportion of patients with this disease.

Adolescent↗
PubMed1999

Antibodies to endothelial cells identify myocardial damage and predict development of coronary artery disease in patients with transplanted hearts.

BACKGROUND: Transplant-induced coronary artery disease is a leading cause of graft failure in cardiac allograft recipients after the first year of transplantation, but there presently is no test to identify patients at high risk for developing the disease. Our research is focused on development of a predictive test to identify patients at high risk of developing the disease. METHODS: Sixty-eight cardiac allograft recipients transplanted and followed at Methodist Hospital between 1982 and 1996 were studied. Serial annual angiograms were used to diagnose coronary artery disease, and serial endomyocardial biopsies were used to detect cellular infiltrates and microvascular disease. Biopsy-matched serum samples were used for cardiac troponin-T determinations as measures of myocardial damage, and serum antibodies to endothelial cells were determined by using flow cytometry, enzyme-linked immunosorbent assay and immunoblotting techniques. The endothelial antibody data were evaluated statistically for associations with angiographic changes, biopsy findings and biochemical evidence of myocardial damage. FINDINGS: Antibodies to endothelial cells were identified by all three techniques, and significant associations were found for the amount of antibody identified by Western immunoblotting with histological rejection grades in biopsies, which were confirmed immunocytochemically as macrophages (p<0.01) and T lymphocytes (P = 0.03). These antibodies also associated significantly with vascular antithrombin depletion (p = 0.02), biochemical evidence of myocardial damage (p = 0.005) and subsequent development of coronary artery disease (p = 0.03). INTERPRETATION: The significant association of anti-endothelial antibodies with cellular infiltrates, depletion of vascular antithrombin and myocardial damage suggests a role for antibody in the development of transplant-induced arteriopathy. The significant association of antiendothelial antibodies with the future development of coronary artery disease further suggests that assessment of these antibodies may provide a non-invasive test to predict the development of transplant-induced coronary artery disease.

Adult↗
PubMed1999

Class I endochitinase containing a hevein domain is the causative allergen in latex-associated avocado allergy.

BACKGROUND: In the medical literature immunoglobulin (Ig)E-mediated sensitization to avocado is rarely reported. On the other hand, more than 50% of subjects having IgE-mediated natural rubber latex allergy are sensitized to avocado fruit as demonstrated by skin-prick testing and/or specific IgE measurements and about 10-20% report hypersensitivity reactions after ingesting avocado. OBJECTIVE: The underlying pathomechanism of latex-associated avocado allergy is still unknown. The conserved hevein domain of the major latex allergen prohevein (Hev b 6.01) is a ubiquitous chitin-binding protein structure that can be found in several plant proteins and may be responsible for the observed cross-reactivity between latex and avocado fruit. METHODS: Chitin-binding avocado proteins (CBAPs) were isolated by affinity-chromatography and their IgE-binding characteristics were studied by immunoblotting using the sera from 15 avocado-sensitized latex patients. Inhibition experiments using isolated hevein and CBAPs as inhibitor solutions were performed to study the immunological cross-reactivity between both protein species and to assess the role of the CBAPs as mediators in latex-associated avocado allergy. RESULTS: In 80% of avocado-sensitized subjects (n = 15), IgE antibodies directed against a 31-kDa allergen were detected by immunoblotting. This IgE-binding protein was identified by protein sequencing to be a class I endochitinase containing a hevein domain at the N-terminus. Purified native and digested (using simulated gastric fluid) endochitinase were able to completely block all avocado-specific IgE antibodies in six out of seven avocado patients. CONCLUSIONS: Sensitization to endochitinase class I containing a hevein domain is the main underlying pathomechanism in latex-mediated avocado allergy.

Allergens↗
PubMed1999

Percutaneous trigger finger release: a comparison of a new push knife and a 19-gauge needle in a cadaveric model.

An instrument was developed for percutaneous release of the A1 pulley and this procedure was compared with percutaneous release using a 19-gauge needle in 20 fresh-frozen cadaver hands. This instrument was developed to avoid the complications of incomplete release and flexor tendon damage previously reported with percutaneous release using a 19-gauge needle. Seventy-eight fingers were used in the study. The push knife was used to release 52 fingers. The A1 pulley was completely released in 51 fingers (98%) and there were no incidents of flexor tendon damage. A 19-gauge needle was used to release 26 fingers. The A1 pulley was completely released in only 10 fingers (38%) and the flexor tendon was damaged in 19 (73%). In cadaveric hands, this instrument proved to be more effective at creating a complete release of the A1 pulley and demonstrated less disruption of the flexor tendon surface compared with current needle techniques.

Cadaver↗
PubMed1999

RGS3 inhibits G protein-mediated signaling via translocation to the membrane and binding to Galpha11.

In the present study, we investigated the function and the mechanism of action of RGS3, a member of a family of proteins called regulators of G protein signaling (RGS). Polyclonal antibodies against RGS3 were produced and characterized. An 80-kDa protein was identified as RGS3 by immunoprecipitation and immunoblotting with anti-RGS3 antibodies in a human mesangial cell line (HMC) stably transfected with RGS3 cDNA. Coimmunoprecipitation experiments in RGS3-overexpressing cell lysates revealed that RGS3 bound to aluminum fluoride-activated Galpha11 and to a lesser extent to Galphai3 and that this binding was mediated by the RGS domain of RGS3. A role of RGS3 in postreceptor signaling was demonstrated by decreased calcium responses and mitogen-activated protein (MAP) kinase activity induced by endothelin-1 in HMC stably overexpressing RGS3. Moreover, depletion of endogenous RGS3 by transfection of antisense RGS3 cDNA in NIH 3T3 cells resulted in enhanced MAP kinase activation induced by endothelin-1. The study of intracellular distribution of RGS3 indicated its unique cytosolic localization. Activation of G proteins by AlF4-, NaF, or endothelin-1 resulted in redistribution of RGS3 from cytosol to the plasma membrane as determined by Western blotting of the cytosolic and particulate fractions with RGS3 antiserum as well as by immunofluorescence microscopy. Agonist-induced translocation of RGS3 occurred by a dual mechanism involving both C-terminal (RGS domain) and N-terminal regions of RGS3. Thus, coexpression of RGS3 with a constitutively active mutant of Galpha11 (Galpha11-QL) resulted in the binding of RGS3, but not of its N-terminal fragment, to the membrane fraction and in its interaction with Galpha11-QL in vitro without any stimuli. However, both full-length RGS3 and its N-terminal domain translocated to the plasma membrane upon stimulation of intact cells with endothelin-1 as assayed by immunofluorescence microscopy. The effect of endothelin-1 was also mimicked by calcium ionophore A23187, suggesting the importance of Ca2+ in the mechanism of redistribution of RGS3. These data indicate that RGS3 inhibits G protein-coupled receptor signaling by a complex mechanism involving its translocation to the membrane in addition to its established function as a GTPase-activating protein.

3T3 Cells↗
PubMed1998

A new enzyme-linked immunosorbent assay to measure anti-endothelial antibodies after cardiac transplantation demonstrates greater inhibition of antibody formation by tacrolimus compared with cyclosporine.

BACKGROUND: Chronic rejection or transplant-associated coronary artery disease (TxCAD) is the most serious complication after human cardiac transplantation. Previous studies, using Western blotting, have shown formation of antibodies against endothelial antigens of 56 and 58 kDa, which are associated with early TxCAD. These antigens were later identified as being vimentin and its breakdown products. The aims of the present study were to devise a robust assay for detection of anti-vimentin antibodies and to compare antibody formation in patients taking different immunosuppressive drugs. METHODS: 106 sequential serum samples from 19 patients taking tacrolimus and 68 sera from 12 patients taking cyclosporine were examined by enzyme-linked immunosorbent assay (ELISA) for anti-vimentin antibodies and Western blotting for reactivity against bands at 56/58 kDa. Serum samples were taken before transplantation and at 1, 3, 6, 9, and 12 months. RESULTS: The vimentin ELISA produced significantly higher numbers of positive episodes per patient (3.92+/-1.08) compared with use of Western blotting (2.54+/-0.52). Serum from patients taking tacrolimus contained significantly less antibodies measured by ELISA (15.8%) or Western blotting (6.5%) than sera from patients taking cyclosporine (46.8% for ELISA; P=0.001 and 21% by Western blotting, P=0.01). Intravascular ultrasound performed on six patients at 12 months showed a correlation between anti-vimentin antibody formation and detection of early coronary disease. CONCLUSIONS: The results demonstrate first, that differences in antibody profiles produced by different immunosuppressive drugs, and second, that detection of anti-vimentin antibodies may be a noninvasive method of detecting disease activity in transplanted vessels.

Antibodies↗
PubMed1998

Protein changes observed in pacing-induced heart failure using two-dimensional electrophoresis.

Rapid ventricular pacing in dogs results in a low output cardiomyopathic state which is similar to idiopathic dilated cardiomyopathy in man. However, the pathophysiological mechanisms which cause this failure following pacing are unknown. Five dogs underwent rapid ventricular pacing. Hearts were stimulated at 245 beats per min (bpm) for four weeks and then reduced to 190 bpm to stabilize the failure. Six unoperated dogs were used as controls. This paper compares the two-dimensional gel electrophoresis (2-DE) protein patterns of left ventricular samples from the paced myocardium with the control dogs. Changes in protein expression were analyzed qualitatively and semi-quantitatively. In the paced dog samples 69 protein spots were significantly altered of which 42 were decreased and 27 were elevated. One qualitative change was observed: elongation factor Tu was present only the control hearts. Of these proteins, 20 have been identified by a combination of N-terminal protein microsequencing, peptide mass profiling by mass spectrometry, amino acid compositional analysis, and by comparison with databases of canine and human ventricular proteins. Ten of these are associated with mitochondria and energy production, including: pyruvate dehydrogenase E1 component, isocitrate dehydrogenase subunit alpha, HSP60 and HSP70, creatine kinase M and fatty acid binding protein. The cytoskeletal protein desmin was detected in reduced quantities and a spot corresponding to a fragment of desmin was increased. These results indicate that the development of heart failure in the paced dog involves alterations in mitochondrial energy production, the cytoskeleton and calcium activation.

Animals↗
PubMed1998

Cardiac protein abnormalities in dilated cardiomyopathy detected by two-dimensional polyacrylamide gel electrophoresis.

The aim of the investigation was to determine whether there are specific global quantitative and qualitative changes in protein expression in heart tissue from patients with dilated cardiomyopathy (DCM) compared with ischaemic heart disease and undiseased tissue. Two-dimensional (2-D) polyacrylamide gel electrophoresis and computer analysis was used to study protein alteration in DCM biopsy material (n=28) compared with donor heart biopsy samples (n=9) and explanted hearts from individuals suffering from ischaemic heart disease (IHD; n = 21). A total of 88 proteins displayed decreased abundance in DCM versus IHD material while five proteins had elevated levels in the DCM group (p<0.01). The most prominent changes occurred in the contractile protein myosin light chain 2 and in a group of proteins identified as desmin. These changes do not appear to be artefactual degradation events occurring during sample processing. These proteins are not apparent in electrophoretic separations of vascular tissue or cultured endothelial cells, mesothelial cells or cardiac fibroblasts, which are clearly distinguishable from the 2-D protein patterns of whole heart and of isolated cardiac myocytes and do not appear to reflect variations in the cellular composition of biopsy samples. The different protein patterns observed in cardiomyopathy showed no obvious relationship with New York Heart Association (NYHA) functional class or haemodynamic parameters. The study has demonstrated significant alterations in quantitative protein expression in the DCM heart which would have serious implications for myocyte function. These changes might be explained by altered protease activity in DCM which could exacerbate contractile dysfunction in the failing heart.

Adult↗
PubMed1998

Multiple parameter cross-species protein identification using MultiIdent--a world-wide web accessible tool.

Recent increases in the number of genome sequencing projects means that the amount of protein sequence in databases is increasing at an astonishing pace. In proteome studies, this is facilitating the identification of proteins from molecularly well-defined organisms. However, in studies of proteins from the majority of organisms, proteins must be identified by comparing analytical data to sequences in databases from other species. This process is known as cross-species protein identification. Here we present a new program, MultiIdent, which uses multiple protein parameters such as amino acid composition, peptide masses, sequence tags, estimated protein pI and mass, to achieve cross-species protein identification. The program is structured so that protein amino acid composition, which is highly conserved across species boundaries, first generates a set of candidate proteins. These proteins are then queried with other protein parameters such as sequence tags and peptide masses. A final list of database entries which considers all analytical parameters is presented, ranked by an integrated score. We illustrate the power of the approach with the identification of a set of standard proteins, and the identification of proteins from dog heart separated by two-dimensional gel electrophoresis. The MultiIdent program is available on the world-wide web at: http://www.expasy.ch/sprot/multiident.h tml.

Amino Acid Sequence↗
PubMed1998

Sub-antihypertensive doses of ramipril normalize sarcoplasmic reticulum calcium ATPase expression and function following cardiac hypertrophy in rats.

We examined the hypothesis that the angiotensin converting enzyme inhibitor ramipril at sub-antihypertensive concentrations could improve sarcoplasmic reticulum (SR) CaATPase expression and function in compensated hypertrophied rat hearts. Five weeks after abdominal aortic constriction, rats received a daily dose (50 micrograms/kg/day) of ramipril or vehicle for 4 weeks. Cardiac angiotensin-converting enzyme (ACE) activity increased with cardiac hypertrophy (CH) but returned to normal following ramipril treatment. SR CaATPase protein levels and activity decreased with CH (P < 0.05) and were normalized following ramipril treatment (P < 0.05 for protein and activity). No change in phospholamban (PLB) protein levels could be demonstrated between any of the groups. In contrast, ramipril treatment specifically increased control SR CaATPase and PLB mRNA levels by > 60% (P < 0.01) and > 30%, respectively. In the hypertrophied group, SR CaATPase increased by 35% (P < 0.05 n = 6) after ramipril treatment. Calsequestrin mRNA levels were unaffected by ramipril administration. In conclusion, ramipril normalizes SR CaATPase protein expression and function in pressure-overloaded and compensated CH. The effects of ramipril are however multifaceted, affecting RNA and protein expression differentially.

Animals↗
PubMed1998

Relative induction of heat shock protein in coronary endothelial cells and cardiomyocytes: implications for myocardial protection.

OBJECTIVES: Induction of the 70 kd heat shock protein in the heart is known to exert a protective effect against postischemic mechanical and endothelial dysfunction. However, the exact site of induction and the mechanisms involved remain unknown. The aim of this study was to investigate the relative capacity of endothelial and myocardial cells to express the 70 kd heat shock protein in response to heat stress, as well as their significance. METHODS: (1) Postischemic recovery of cardiac mechanical and endothelial function was studied in isolated rat hearts with and without endothelial denudation with saponin. (2) Semiquantitative determination of induction of 70 kd heat shock protein by Western immunoblotting was performed in the whole cardiac homogenate, in isolated cardiac myocytes, and in coronary endothelial cells. (3) Immunocytochemistry was used to visualize the distribution of induction of 70 kd heat shock protein in both cell types. RESULTS: Postischemic recovery (percent preischemic value +/- standard error of the mean) of cardiac output in hearts from heat-stressed animals was significantly improved (66.7 +/- 6.9 vs 44.5 +/- 4.5 in the control group, p < 0.01). In heat-stressed hearts treated with saponin no improvement in the recovery of cardiac output was noted (44.7 +/- 6.9 in heat-stressed hearts vs 38.0 +/- 4.0 in heat-stressed, saponin-treated hearts, p = not significant). Endothelial function (as assessed by the vasodilatory response to the endothelium-dependent vasodilator 5-hydroxytryptamine) improved from 31.0 +/- 5.2 in the control group to 65.8 +/- 7.1 in heat-stressed hearts (p < 0.02 vs control) and dropped to -1.9 +/- 3.8 in heat-stressed hearts treated with saponin. Immunocytochemistry showed that only sections of hearts from heat-treated rats showed a strong specific reaction with heat shock protein antibody. The positive staining was seen in endothelial cells. Induction of 70 kd heat shock protein content in the whole cardiac homogenate from heat-stressed rats as measured by Western immunoblotting was 5.2 +/- 1.9 (vs 0.0 in non-heat-stressed rats, p < 0.0001) and dropped to 0.0 in heat-stressed hearts treated with saponin. The tentative amount of 70 kd heat shock protein was 18.1 +/- 7.8 in isolated endothelial cells from heat-stressed hearts and 2.3 +/- 2.3 in isolated cardiac myocytes (p < 0.01 vs endothelial cells). CONCLUSIONS: Coronary endothelial cells are the main site of induction of 70 kd heat shock protein in the heart and appear to contribute to the protective effects of heat stress on the recovery of mechanical and endothelial function.

Animals↗
PubMed1998

Different activation of mitogen-activated protein kinases in experimental proliferative glomerulonephritis.

Mitogen-activated protein (MAP) kinases are critical for cell signaling goals such as cellular proliferation and induction of apoptosis. We examined whether MAP kinases, as a point of convergence for multiple extracellular stimuli, are activated in proliferative glomerulonephritis (GN) in vivo. Accelerated crescentic anti-glomerular basement membrane (GBM) GN was induced in rats preimmunized with rabbit IgG by administration of rabbit anti-rat GBM serum. Whole cortical tissue and isolated glomeruli were then subjected to kinase activity assays and Western blot analysis. Cortical activity of the archetypal MAP kinase, extracellular signal-regulated kinase (ERK), was increased significantly one, three, and seven days after induction of GN. In contrast, activation of MAP kinases with antiproliferative actions, stress-activated protein kinase, and p38 MAP kinase was detectable only in the early stages of proliferative GN (days one and three), implying that different MAP kinases serve distinct roles in the pathogenesis of GN.

Animals↗
PubMed1998

Donor ACE gene polymorphism: a genetic risk factor for accelerated coronary sclerosis following cardiac transplantation.

AIMS: To investigate the role of angiotensin converting enzyme (ACE) (I/D) gene polymorphism in the development of coronary sclerosis after cardiac transplantation. METHODS AND RESULTS: Eighty cardiac transplant recipients (44 transplant associated coronary artery disease; 36 non-transplant associated coronary artery disease) and their donors were genotyped by polymerase chain reaction. The allele frequencies of the recipients in the transplant associated coronary artery disease and non-transplant associated coronary artery disease groups (I = 0.47 and 0.48, D = 0.53 and 0.52, respectively) did not differ significantly between the groups. However, there was a negative association between the frequency of the I allele in the donor and the development of transplant associated coronary artery disease. The D allele in the donor population of the non-transplant associated coronary artery disease group had a significantly (P < 0.01) lower frequency (0.35) than either the transplant associated coronary artery disease group (0.53) or that of the general population (0.57). Other factors analysed were recipient family history, cholesterol levels, age, sex and body mass index, donor age and acute rejection, of which the significant (P < 0.05) factors were acute rejection and sex of the recipient. CONCLUSION: These results suggest that the ACE genotype of the donor organ may be an additional risk factor for the development of coronary artery disease following cardiac transplantation and that tissue rather than circulating ACE could be implicated in the pathogenesis of this disease.

Acute Disease↗
PubMed1997

Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase.

Endothelin-1 (ET-1) induces cell proliferation and differentiation through multiple G-protein-linked signaling systems, including p21ras activation. Whereas p21ras activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21ras activation induced by engagement of G-protein-coupled receptors remains to be fully elucidated. In the present study we show that ET-1 induces a biphasic activation of p21ras in rat glomerular mesangial cells. The first peak of activation of p21ras, at 2-5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21ras results in activation of the extracellular signal-regulated kinase (ERK) cascade. We demonstrate that ET-1 signaling elicits a negative feedback mechanism, modulating p21ras activity through ERK-dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21ras and ERK deactivation, Sos1 reverts to the non-phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21ras at 30 min does not lead to ERK activation, correlating with intensive, ET-1-induced expression of MAP kinase phosphatase-1, but does result in increased p21ras-associated phosphatidylinositol 3-kinase activity. Our data provide evidence that ET-1-induced biphasic p21ras activation causes sequential stimulation of divergent downstream signaling pathways.

Adaptor Proteins, Signal Transducing↗