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Biomedical subjects

M J Dunn

Publications and source records attributed to M J Dunn.

At least 163 records · Page 9Linked to original sources
PubMed1991

Endothelin receptors and coupled GTP-binding proteins in glomerular mesangial cells.

Endothelins (ETs) are a family of vasoactive peptides with profound biological actions in diverse cell systems. Among its varied actions, ET stimulates phospholipase C (PLC) in cultured mesangial cells. We investigated the presence of specific ET receptors in rat mesangial cells in culture, and studied the role of GTP-binding proteins (G proteins) in coupling PLC to the endothelin receptor. [125I]ET binding was time- and temperature-dependent, and Scatchard analysis of saturation data showed a single class of high-affinity binding sites. Heterologous displacement with two related peptides, ET-3 and sarafotoxin (SFTX), revealed the presence of two binding sites for these isopeptides. Preincubation of cells with ET-1 reduced the receptor number without affecting Kd, and this effect was not prevented by protein kinase C inhibition or downregulation. We confirmed the presence of a 41- to 43-kDa pertussis toxin substrate in rat mesangial cell membranes in an ADP ribosylation assay. ET-1 inhibits and GDP beta S enhances toxin-catalyzed transfer of ADP-ribose to this substrate. ET-1 potentiated GTP gamma S-induced phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. In addition, pertussis toxin partially inhibited ET-stimulated PI hydrolysis in intact mesangial cells. Pertussis toxin also reduced the magnitude of ET-stimulated intracellular free calcium [( Ca2+ )i]. Thus, ET-1 binds to specific receptors on rat mesangial cells and activates PLC, in part, through a pertussis toxin-sensitive G-protein.

Adenosine Diphosphate Ribose↗
PubMed1991

Demonstration by western blotting of antiheart antibodies before and after cardiac transplantation.

Western blotting has been used to detect antiheart antibodies in two groups of patients: two who required retransplantation for hyperacute rejection, and 22 consecutive patients, whose serum was tested at monthly intervals for three months following transplantation. Pretransplant and posttransplant serum samples were tested for IgM and IgG reactivity against the patients own heart and donor heart. In all patients the pretransplant lymphocytic crossmatch had been negative. In the two patients requiring retransplantation, both had multiple bands of strong IgM and IgG against their own heart prior to transplantation as well as antibodies against the donor heart. The study of 22 consecutive patients revealed that (1) the presence of strong antibody prior to transplantation is associated with unusually severe or frequent rejection episodes, (2) 20/22 patients made antiheart antibody following transplantation, but in 12 patients it was IgM only, and (3) most of the antiheart antibodies made posttransplant were not specific for the donor heart. Comparison of Western blotting with immunofluorescent detection of antibodies on frozen sections revealed that the Western blotting procedure is more sensitive and results are easier to interpret.

Adolescent↗
PubMed1991

Restricted specificity of the autoantibody response in Goodpasture's syndrome demonstrated by two-dimensional western blotting.

The autoantigen in Goodpasture's syndrome is known to be contained within the non-collagenous (NC1) domain of type IV collagen. We have examined the specificity of autoantibodies to glomerular basement membrane (GBM) using the technique of 2-D electrophoresis followed by Western blotting. Protein stains of 2-D gels of collagenase-digested human GBM revealed extensive charge and size heterogeneity. Major components were of mol. wt 24-30 kD and 43-56 kD, corresponding to monomeric and dimeric subunits of NCl. Western blotting of 2-D gels with IgG from patients with anti-GBM disease demonstrated that the most antigenic components migrated as cationic 28-kD monomers (pI 10) and similarly charged dimers, although other components were recognized less strongly. The mobility of the strongly antigenic polypeptides was different to that of the known alpha 1 and alpha 2 chains of type IV collagen. Autoantibodies from all 20 patients studied showed the same pattern of reactivity, regardless of their clinical features (in particular, the presence or absence of pulmonary haemorrhage) or HLA type. A monoclonal antibody (P1) to human GBM bound in a similar pattern, particularly recognizing the cationic components. 2-D gels of affinity-purified GBM from a P1 column showed enrichment of the 28-kD monomers, which were recognized by human autoantibodies on Western blotting. These results demonstrate that the autoimmune response in Goodpasture's syndrome is of restricted specificity, and support the suggestion that the major autoantigenic determinant is present on the novel alpha 3 chain of type IV collagen.

Adolescent↗
PubMed1991

Regulation of cytosolic pH of cultured mesangial cells by prostaglandin F2 alpha and thromboxane A2.

Prostaglandins (PG) and thromboxane A2 (TxA2) have marked vasoactive effects on the renal glomerular microcirculation. Exposure of cultured mesangial cells to PGF2 alpha and TxA2 mimetics results in a rapid elevation of free cytosolic Ca2+ ([Ca2+]i) followed by contraction and cell proliferation. We studied whether other ionic changes mediate these effects of eicosanoids on cells of rat and human origin. Cytoplasmic pH (pHi) was monitored in cells loaded with the fluorescent, intracellularly trapped pH-sensitive probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. PGF2 alpha in rat cells and the TxA2 mimetic U-46619 in human cells induced rapid, dose-dependent cytosolic acidification followed by recovery and net alkalinization mediated by enhanced Na(+)-H+ exchange. The early acidification was also stimulated by ionomycin and Ca2(+)-mobilizing peptides, implicating a Ca2(+)-dependent mechanism. Alkalinization was abolished by removal of extracellular Na+ and by amiloride. Both components of the responses were inhibited by phorbol myristate acetate, which could mimic alkalinization, suggesting a regulatory role of protein kinase C in activation of the Na(+)-H+ exchanger by eicosanoids. Vasoconstrictor arachidonate metabolites may control glomerular cell function by a signaling mechanism centered on concurrent changes of pHi and [Ca2+]i.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

A pertussis toxin-sensitive GTP-binding protein couples endothelin to phospholipase C in rat mesangial cells.

The mechanisms of stimulation of phospholipase C (PLC) by endothelin, specifically the role of guanine nucleotide-binding proteins (GTP-binding proteins) in coupling the endothelin receptor to PLC, were investigated in rat mesangial cells. Endothelin-1 (ET) synergistically released inositol polyphosphates in the presence of the stimulatory GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in permeabilized cells. In addition, in intact cells, pertussis toxin partially inhibited the stimulation of total inositol phosphates (IPn) by ET. Pertussis toxin also reduced the peak ET-stimulated intracellular free calcium level ([Ca2+]i) in these cells, both in the presence and absence of extracellular calcium. Pertussis toxin induced ADP ribosylation of a 41- to 43-kDa protein in mesangial cell membranes, and this effect was inhibited by prior exposure to ET and augmented by the inhibitory GDP analogue, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). Thus a pertussis toxin-sensitive GTP-binding protein is involved in the activation of PLC by ET in glomerular mesangial cells.

Adenosine Diphosphate Ribose↗
PubMed1991

Regulation of prostaglandin endoperoxide synthase gene expression in cultured rat mesangial cells: induction by serum via a protein kinase-C-dependent mechanism.

Recent evidence suggests that the steady state level of prostaglandin (PG) endoperoxide synthase (PES) might regulate long term increases in PG synthesis, but the mechanism(s) regulating PES gene expression remains unclear. The present experiments investigated the regulation of PES expression in quiescent mesangial cells treated with serum. Serum induced time-dependent increases in PES steady state mRNA, protein level, and enzyme activity. Both the kinetics and cycloheximide sensitivity of PES mRNA induction suggest that PES was induced as a member of the delayed-early gene set. Beta-adrenergic-mediated cAMP accumulation was similar in Go and cycling cells, suggesting that the increase in PES expression did not reflect global up-regulation of signaling cascades in serum-treated cells. Three lines of evidence suggest that protein kinase-C (PKC) mediates serum-stimulated PES gene expression: 1) 12-tetradecanoyl phorbol 13-acetate mimicked the serum response; 2) PES gene expression by serum was attenuated in cells depleted of PKC; and 3) a PKC inhibitor, sangivamycin, blocked serum-stimulated PES gene induction. In addition, the ability of dexamethasone to block serum-induced PES enzyme activity without affecting the increase in PES mRNA points to posttranscriptional mechanisms of regulation. In summary, we conclude that in glomerular mesangial cells the PES gene is inducible, not constitutive, and that expression of this gene in Go cells is induced by serum and results in chronic elevations of PGE2. These findings are consistent with the hypothesis that differential regulation of PES gene expression might play a role in diverse cellular responses, such as mitogenesis and inflammation.

Animals↗
PubMed1991

The regulation and role of prostaglandin biosynthesis in cultured bovine glomerular endothelial cells.

The experiments described in this article were designed to investigate the regulation of, and functional response to, prostaglandin (PG) synthesis in cultured bovine glomerular endothelial cells. Glomerular endothelial cells in culture synthesized PGE2 much greater than PGF2 alpha greater than thromboxane A2 greater than PGI2 in the presence of 30 microM arachidonate. Basal levels of PGE2 synthesis in these cells were one sixth less than that of bovine mesangial cells. PGE2 synthesis was time dependent and required the continuous presence of serum. Moreover, PGE2 synthesis was regulated by phospholipase A2 as shown by ionomycin-stimulated PGE2 accumulation as well as by bradykinin- and thrombin-stimulated PGE2 accumulation. Even though acidic fibroblast growth factor and heparin were required to support glomerular endothelial cell growth in culture, these agents downregulated PGE2 synthesis. PG endoperoxide synthase activity, as shown by arachidonate-stimulated PGE2 accumulation, also regulated PGE2 synthesis. PGE2 and PGF2 alpha, as well as thromboxane A2 and PGI2 mimetics (1 microM) evoked mitogenesis in quiescent glomerular endothelial cells. PGE2 and PGF2 alpha were most potent, and threshold doses of these PG were 10 nM. These data suggest that glomerular endothelial cells are similar to other microvascular endothelial cells in their regulation of PG synthesis and that, under physiological conditions, the proliferation of glomerular endothelial cells might be regulated by PG in an autocrine or paracrine fashion.

Animals↗
PubMed1991

The effects of endothelin on cytosolic calcium in cultured human and rat glomerular mesangial cells.

Isopeptides of the endothelin gene family, including sarafotoxin, caused a concentration-dependent increase in intracellular-free calcium (cytosolic calcium) in cultured human and rat glomerular mesangial cells. The calcium increments had two components, a spike increment resulting from mobilization of intracellular calcium and a sustained calcium increment as a result of endothelin activation of calcium influx. ET-dependent calcium influx occurred through dihydropyridine-insensitive calcium channels which were blocked by NiCl and allowed entry of Mn2+. In human mesangial cells, ET stimulated periodic oscillations of cytosolic Ca2+ that reflect synchronous signalling in localized populations. Calcium signalling evoked by the ET isopeptides showed cross-desensitization among the four ET-peptides tested. Although protein kinase C activation acutely reduced ET-induced calcium signalling, the desensitization by ET isopeptides was independent of protein kinase C. If ET was added to cells incubated with an inhibitor of the endoplasmic reticulum calcium ATPase, it became apparent that ET activated a calcium efflux pathway which reduced cytosolic calcium. These data, therefore, demonstrate that calcium signalling is a common response in both human and rat cultured mesangial cells incubated with different ET isopeptides.

Animals↗
PubMed1991

Endothelin binding and receptor down regulation in rat glomerular mesangial cells.

Endothelin (ET) exerts various biological actions in mesangial cells, including stimulation of proliferation, contraction and phospholipase C activation. We investigated the presence of specific ET receptors on cultured rat mesangial cells, incubating the cells in the presence of [125I]ET-1 both at 22 and 4 degrees C. ET binding was time- and temperature-dependent and achieved equilibrium at 2 hr at 22 degrees C and at 5 hr at 4 degrees C. Scatchard analyses of equilibrium saturation curves with [125I]ET-1 and homologous competition curves revealed the presence of a single class of high-affinity binding sites (Kd = 31.4 +/- 7.08 pM). Heterologous competition experiments with ET-3 and sarafotoxin, however, indicated the presence of two binding sites for ET-related peptides in mesangial cells with a Kd for ET-3 of 41.5 +/- 19.2 and of 374 +/- 38.5 nM. Nifedipine and arginine-vasopressin failed to compete for ET binding sites. Preincubation of the cells with 1 nM ET-1 caused a dramatic decrease in ET binding capacity (from 0.5-0.02 fmol/100,000 cells) without affecting the Kd for the receptors (38 pM). ET receptor down regulation was not prevented by protein kinase C inhibition with H-7 and sangiovamycin, or after down regulation of protein kinase C induced by 24-hr preincubation with phorbol myristate acetate. ET receptor down regulation also exerts functional effects, as we found a decrease in intracellular-free calcium response to ET-1 after long-term preincubation with the same agonist. Our results are consistent with the presence of two binding sites for ET in rat mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Comparison of mastectomy and lumpectomy for medial carcinoma of the breast.

The recurrence and five year disease-free survival rates of 86 patients with carcinoma of the breast located in the medial quadrants who had either lumpectomy and radiotherapy or mastectomy were compared. Patients who had lumpectomy and mastectomy were comparable with respect to variables reflecting extent of the disease. One of 54 patients who had lumpectomy had a local recurrence and five had a recurrence regionally or at distant sites. In the group of 32 patients who had a mastectomy, one patient had a local recurrence and an additional four patients had a recurrence at other sites. The five year study disease-free survival rate was 83 per cent for the lumpectomy group and 78 per cent for the mastectomy group. These data suggest that, for medial quadrant lesions, there may be a disease-free survival advantage associated with lumpectomy and radiotherapy over mastectomy. Further study of this treatment modality for this subset of patients seems indicated.

Breast Neoplasms↗
PubMed1991

Aortic counterpulsation for up to 28 days with autologous latissimus dorsi in sheep.

This article reports the development and assessment of an entirely autologous extraaortic counterpulsation system using skeletal muscle (latissimus dorsi). The technique has been performed and assessed in 16 sheep to quantify the effectiveness of counterpulsation over periods up to 28 days and to optimize the stimulation regimens for muscle contraction and fiber-type transformation. Hemodynamic changes have been quantified by calculation of the endocardial viability ratio. This has shown an increase of between 12% and 89% for 28 days. The wide variety of increase observed has been related to the development of an optimum flap configuration. The technique of surface impedance monitoring of flap blood flow has allowed the start of electrical stimulation after 48 hours with the introduction of hemodynamic benefit (1:4 mode) during the process of fiber-type transformation (in situ training). Extraaortic counterpulsation with autologous latissimus dorsi has been shown to be effective and safe for as long as 28 days. It has not been associated with any thromboembolic or infective complications, which we attribute to the exclusion of any foreign material in the design.

Animals↗
PubMed1990

Endothelin isopeptides evoke Ca2+ signaling and oscillations of cytosolic free [Ca2+] in human mesangial cells.

Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+] ([Ca2+]i) in human glomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [Ca2+]i constitutes a common pathway of signal transduction for the ET peptide family.

Calcium↗
PubMed1990

Nature of the mononuclear infiltrate and the mechanism of muscle damage in juvenile dermatomyositis and Duchenne muscular dystrophy.

Normal skeletal muscle does not express class I MHC antigens. In contrast, these antigens are strongly expressed at the periphery of muscle fibres in patients with juvenile dermatomyositis (JDM) and Duchenne muscular dystrophy (DMD). Interferons can induce the expression of class I antigens, but in this study interferon-gamma could not be detected in JDM muscle biopsy specimens using four different immunocytochemical techniques. However, an infiltrate of mononuclear cells capable of synthesising interferons was present in biopsies from JDM and DMD patients. The predominant cell types detected in both diseases were macrophages and T lymphocytes, these two cell types comprising more than 80% of the infiltrating mononuclear cells. A striking predominance of CD4+ helper/inducer T cells was observed. The majority of these cells expressed class II MHC antigens and were, therefore, considered to be activated. Additional evidence for the functional activity of CD4+ T cells was derived from the finding that it was this population of cells from JDM biopsies which proliferated in response to interleukin-2 in vitro. T cell subsets in peripheral blood were also investigated in JDM and DMD patients. Only in the case of JDM were any significant differences from normal observed, where a significant reduction in the number of peripheral blood CD8+ T cells resulted in an elevation of CD4+/CD/8+ ratios. The role of CD4+ T cells and aberrant class I MHC antigen expression in mediating muscle damage in JDM and DMD is discussed.

CD4-Positive T-Lymphocytes↗