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Biomedical subjects

M Mirshahi

Publications and source records attributed to M Mirshahi.

At least 19 recordsLinked to original sources
PubMed1998

Thrombus generation after adenovirus-mediated gene transfer into atherosclerotic arteries.

Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.

Adenoviridae↗
PubMed1998

Enhanced activation of the mineralocorticoid receptor in genetically hypertensive rats.

The relative abundance and availability of the mineralocorticoid receptor (MCR) appeared to be similar in the heart, kidney and ocular tissues of the genetically hypertensive SHR and normotensive WKY rats by a number of criteria including Western blotting, immunoprecipitation, dot blot analysis, and immunohistochemistry. On the other hand, the activation of the MCR, as judged by binding to DNA cellulose, was significantly enhanced in the hearts and kidneys of 14 week-old, hypertensive, SHR rats compared to the normotensive WKY animals. The activation of the renal MCR was elevated in the SHR strain even at the age of six weeks when the tail arterial pressure was statistically identical to that of the WKY strain. Thus, precocious receptor activation may represent a primary lesion leading to hypertension in the SHR strain, thereby providing a new model to elucidate the hypertensive state.

Animals↗
PubMed1998

Cellular source of human platelet secretory phospholipase A2.

Platelets are one source of the group II extracellular form of phospholipase A2 (sPLA2) which is involved in the amplification of local and systemic inflammation. Although sPLA2 protein has been described in human platelets, its presence in human megakaryocytes has not been yet established. We demonstrated in this study that the human erythroleukaemia (HEL) cell line, which has megakaryoblastic features, constitutively expresses sPLA2. Using an anti-rhsPLA2 monoclonal antibody (mAb BA11) and dot-blot detection, we showed that HEL cells and platelets release sPLA2 into incubation medium upon stimulation by thrombin. Similar results were obtained for sPLA2 activity detected by a spectrofluorescence assay. Enzymatic activity was abolished by mAb BA11 and by protamine. In both cell types, although released, the major part of sPLA2 remained in the cell pellet, and was probably adsorbed at non-specific membrane sites. Double labelling experiments using mAb BA11 and an anti-GPIIb antiserum revealed the presence of sPLA2 in human bone-marrow megakaryocytes. The use of reverse transcription-polymerase chain reaction conjugated with hybridization analysis demonstrated the presence of mRNA encoding for sPLA2 in platelets and HEL cells. Expression of sPLA2 in platelets and megakaryocytes at both transcriptional and post-translational levels strongly argues in favour of a megakaryocytic origin of platelet sPLA2 and rules out a role for endocytosis of the enzyme from plasma by circulating platelets.

Blood Platelets↗
PubMed1997

Immunodetection of a protein related to mammalian arrestin in Euglena gracilis.

Six monoclonal antibodies directed against different epitopes of bovine retinal arrestin recognized a single polypeptide in extracts of achlorophyllous Euglena gracilis cells. This polypeptide had an apparent molecular weight slightly lower: 45kDa vs. 48 kDa, and a more basic isoelectric point compared to that of bovine visual arrestin. It was located in an insoluble cytoplasmic fraction. Immunofluorescence assays show a cytoplasmic punctuated pattern suggesting a cluster distribution or a linkage to some cell structure. The presence of arrestine-like molecules in achlorophyllous Euglena gracilis cells suggests that these proteins might be involved in a peculiar step of chemical signal transduction processes.

Amino Acid Sequence↗
PubMed1997

Immunological detection of potential signal-transduction proteins expressed during wheat somatic tissue culture.

An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein (alpha subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G alpha antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrstin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed.

Amino Acid Sequence↗
PubMed1997

Immunochemical demonstration of the mineralocorticoid receptor in ocular tissues.

We studied the presence of the mineralocorticoid receptor (MCR) in the eye with the aid of a number of immunochemical techniques. Immunoblotting with a polyclonal antibody, directed against the rat renal MCR, revealed a single band of about 102 kD in extracts prepared from whole bovine or rat retina similar to that observed in cytosol from the kidney and myocardium from these species. Isolated cells of the bovine retinal pigment epithelium (RPE) similarly exhibited a 98- to 102-kD band in Western blots developed with the aid of anti-MCR antiserum. The 98- to 102-kD band was also obtained following autoradiography of RPE cytosol irradiated in the presence of 3H-R 5020. This fluorographic pattern was abolished when RU 26752, an antagonist specific to the MCR, was allowed to compete with radiolabelled promegestone. The MCR-3H-RU 26752 complex in RPE cytosol underwent heat activation, as judged by binding to DNA cellusose, and could also be precipitated by anti-MCR IgG. In primary cultures, the proliferation of the RPE cells was inhibited by the two MCR-specific antagonists RU 26752 and ZK 91587. The loss of the MCR-specific immunofluorescence in RPE cells after only 3 passages in culture was associated with refractoriness to the inhibitory effect of both of these spironolactones. Immunohistochemistry, using MCR-specific antiserum, revealed strong fluorescence in specific areas of the rat eye. In the retina, immunopositivity was observed in Müller cells, external and internal limiting membranes, the vitreous base lining and in the pigment epithelium. Epithelial cells of the ciliary body, iris and cornea also exhibited strong MCR-specific immunofluorescence. Thus, both the epithelial and the nonepithelial compartments of the ocular tissues form interesting new targets to delineate the mechanism of action of mineralotropic hormones.

Animals↗
PubMed1996

The mineralocorticoid hormone receptor and action in the eye.

Immunoblotting with a polyclonal antibody, directed against the mineralocorticoid receptor protein purified from rat kidney in presence of the receptor-specific ligand RU 26752, labeled a single 98-102 kDa band in soluble extracts from bovine retina and from cultured bovine retinal pigment epithelial cells, identical to the receptor in several other tissues from the rat. The antibody also immunoprecipitated the receptor-3H-RU 26752 complex in bovine retinal extract. The growth of the isolated pigment epithelial cells was inhibited by RU 26752 and ZK91587, two ligands specific to the mineralocorticoid receptor. Successive passages in culture led to the disappearance of immunoreactivity in Western blots, concurrently with the refractoriness of the cells to growth inhibition by the two antagonists. On sections of the human eye, mineralocorticoid receptor-specific immunofluorescence was observed in retinal cone cells, pigment epithelium, epithelium of ciliary body, iris, cornea and lens. To our knowledge, this is the first ever demonstration of the mineralocorticoid receptor in ocular tissues.

Animals↗
PubMed1996

Fibrinogen Dusart: electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots.

Ultrastructural perturbations resulting from defects in polymerization of fibrinogen Dusart, a congenital dysfibrinogenemia with the amino acid substitution A alpha 554 arginine to cysteine, were investigated by a variety of electron microscope studies. Polymerization of this mutant fibrinogen on addition of thrombin is impaired, producing clots with decreased porosity and increased resistance to fibrinolysis, resulting in thrombotic complications in the family members with this dysfibrinogenemia. Electron microscopy of rotary-shadowed individual molecules revealed that, in contrast to control fibrinogen, most of the alpha C domains of fibrinogen or fibrin Dusart appeared to be free-swimming appendages that do not exhibit intra- or intermolecular interactions either with each other or with the central domains. The location of albumin on the alpha C domains was demonstrated by electron microscopy using anti-albumin antibodies. Electron microscopy of negatively contrasted fibrin Dusart fibers indicated that they were less ordered than control fibers and had additional mass visible. Electron microscopy of freeze-dried, unidirectionally shadowed fibers showed that they were twisted with a shorter pitch. Scanning electron microscopy revealed that intact clots were made up of thin fibers with many branch points and very small pore sizes. The viscoelastic properties of Dusart fibrin clots measured with a torsion pendulum indicated a marked increase in stiffness consistent with the structural observations.

Biomechanical Phenomena↗
PubMed1996

Evidence for receptor-mediated mineralocorticoid action in rat osteoblastic cells.

Aldosterone significantly enhanced the proliferation of osteoblastic cells from rat calvaria, and this effect was inhibited by RU 26752 and ZK 91587, two antagonists specific to the mineralocorticoid receptor (MCR). In addition, aldosterone inhibited the activity of alkaline phosphatase, a marker of the osteoblastic phenotype, and this effect was also reversed by RU 26752. Cytoplasmic staining of MCR was observed in rat calvaria osteoblasts incubated with a specific polyclonal antiserum raised against rat kidney MCR. This anti-MCR immunoglobulin G immunoprecipitated and macroaggregated the MCR-[3H]RU 26752 complex in osteoblastic cytosol. A single 98-kDa band was observed when osteoblastic cytosol was analyzed by Western blotting with anti-MCR serum. The 98-kDa band was also obtained after autoradiography of irradiated osteoblastic cytosol-[3H]R 5020 complex, and this was abolished in the presence of RU 26752. A p26MR probe, specific to COOH-terminal end of MCR, hybridized with the predicted product after amplification of total cell RNA by polymerase chain reaction technique. Furthermore, hybridization of poly(A)+ mRNA from at calvaria osteoblastic cells with the p26MR probe revealed a major band of approximately 4.2 kb. Collectively, our studies demonstrate the existence of a functional MCR in rat calvaria osteoblasts.

Aldosterone↗
PubMed1995

Expression of elastase and fibrin in venous leg ulcer biopsies: a pilot study of pentoxifylline versus placebo.

The pathogenesis of venous leg ulcers is based on the leakage of fibrinogen leading to a pericapillary fibrin cuff and plugging of capillaries by white blood cells. On the basis of a previous work, we had assumed that the key event in the pathogenesis of venous leg ulcers is related to inflammation generated by activated white blood cells that accumulate under unrelieved blood pressure, because in ulcer biopsies we had detected the presence of tumor necrosis factor-alpha (TNF-alpha) in intracapillary monocytes, elastase in the polymorphonuclear leukocytes near the vessels, and a pericapillary undegraded fibrin cuff causing a diffusion barrier to oxygen. This concept was developed because TNF-alpha synthesized by activated monocytes is responsible for many deleterious effects. It has a potent mitogenic effect on fibroblasts, leading to new collagen deposition and angiogenesis, it induces an increase in collagenase production, it acts through upregulation of an intracellular adhesion molecule (ICAM-1), leading to leukocyte sequestration and consequently a release of toxic metabolites by the polymorphonuclear cells, an early step in chronic inflammation, it activates the coagulation pathway via a marked increase in monocyte-associated tissue factor (TF) procoagulant activity, and it inhibits fibrinolysis by promoting the release of PAI-1, contributing to undegraded fibrin deposition. Therefore, we were interested in evaluating, in patients with venous leg ulcers, the effect of pentoxifylline administered at 1,200 mg daily (versus placebo) for 2-months, as this drug induces a decrease in TNF-alpha synthesis and also blocks its activity. This pilot assay was performed in blind. Evolution of several parameters in ulcer biopsies are analyzed: TNF-alpha, intact fibrin, fibrin degradation products, ICAM-1, TF, and elastase. Pentoxifylline administration induced a decrease of local elastase and of fibrin deposit. These results support the hypothesis that accumulation of activated leukocytes is the key event in venous leg ulcers.

Antibodies, Monoclonal↗
PubMed1995

Human anti-streptokinase antibodies induce platelet aggregation in an Fc receptor (CD32) dependent manner.

Exposure to streptokinase (SK) elicits anti-SK antibodies (Abs), which inhibit fibrinolysis and induce platelet aggregation. The mechanism of the latter is not fully understood, although it seems to involve platelet binding by a plasminogen streptokinase and anti-SK ternary complex. Anti-SK Abs were purified by affinity chromatography from serum of patients having received SK for acute myocardial infarction (AMI), and were shown to be of the IgG type. Their effects were studied with (i) human platelets in citrated plasma in the presence of SK or acetylated plasminogen-SK activator complex (APSAC), and (ii) in washed platelets, resuspended in Tyrode buffer after lowering the ionic strength, in the presence of APSAC (which provides both SK and plasminogen). An antibody concentration-response curve was obtained, showing a plateau in the presence of 0.1 mg/ml IgG. By increasing the concentration of APSAC, we obtained a unimodal response curve, the optimal concentration of APSAC being 0.05 U/ml. Aggregation was suppressed by chelating calcium with EDTA, blocking fibrinogen binding by the synthetic peptide Arg-Gly-Asp-Ser (RGDS), and raising intraplatelet cAMP with Iloprost (a prostacyclin analogue). Aggregation required the interaction of the anti-SK Ab Fc domain with the platelet Fc-gamma receptor type II, also known as CD32, since: (i) it was blocked by the monoclonal antibody IV-3 directed against CD32, (ii) it did not occur with F(ab)'2 fragments, which block the response to the intact IgG. The clinical relevance of these platelet-activating anti-SK antibodies remains to be determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Evidence from in vivo manipulations of lipid composition in mutants that the delta 3-trans-hexadecenoic acid-containing phosphatidylglycerol is involved in the biogenesis of the light-harvesting chlorophyll a/b-protein complex of Chlamydomonas reinhardtii.

The phosphatidylglycerol containing the unusual delta 3-trans hexadecenoic fatty acid is specifically found in photosynthetic membranes of eukaryotic organisms. Its involvement in the biogenesis and the structure of the light-harvesting chlorophyll a/b-protein complex has been evidenced by in vivo targeting this lipid to photosynthetic membranes of Chlamydomonas reinhardtii mutants lacking this lipid. In the mf1 and mf2 mutants, this deficiency results in (a) the absence of the oligomeric light-harvesting complex of photosystem 2; (b) an extensive destacking of thylakoid membranes; (c) a very low 77-K fluorescence emission in the photosystem-2 region. We show in this paper that these deficiencies result from modifications in the pigment and polypeptide compositions of the photosystem-2 light-harvesting complex; it contains less chlorophyll b and some of its constitutive polypeptides are absent or reduced in amount, while immunologically related polypeptides of lower molecular mass accumulate. The direct involvement of the lack of trans-C16: 1-phosphatidylglycerol in these deficiencies is evidenced by the partial restoration of normal characteristics of the light-harvesting complex (pigment and polypeptide composition, oligomerization) after liposome-mediated, in vivo incorporation of this lipid into the photosynthetic membranes of the mf2 mutant. Trans-C16:1-phosphatidylglycerol, therefore, is involved in the biogenesis of the photosystem-2 light-harvesting chlorophyll a/b-protein complex through a mechanism that may prevent degradation processes. Its contribution to the structural conformation of neosynthesized monomers and to their organization into stable oligomeric form is discussed.

Animals↗
PubMed1994

Properties of the mineralocorticoid receptor immunopurified from bovine kidney.

The mineralocorticoid receptor (MCR) from bovine kidney was purified on an affinity column containing covalently linked polyclonal IgG raised in the rabbit against rat kidney protein purified in the presence of RU 26752 that is specific to the MCR. The immuno-affinity eluate was excluded as a single peak during gel permeation chromatography and could be resolved as a single band of approximately 98 kDa by western blot and gel electrophoresis. Immunohistochemistry revealed MCR-specific staining in both the cortical and glomerular regions of bovine kidney. Interestingly, the purified MCR could not be activated in the presence of the specific ligand RU 26752 whereas binding to DNA-cellulose increased by 100% when crude cytosol was left at room temperature for 45 min. The binding of calcium to the MCR resulted in an increase in the fluorescence signal that could be partially reversed by EDTA. By a calcium-specific fluorescence dye technique, 1.13 nM of ionized Ca2+ was bound per 0.01 nM MCR. The binding of ATP32 to the immunopurified receptor was observed following chromatography on P-10 columns. The fluorescence signal of etheno-ATP was maximally attenuated by the receptor at 1/1 stoichiometry of the ATP-MCR complex. Asparagine-linked complex chain N-glycosylation of the purified MCR was also observed. Analysis by far-UV circular dichroism spectra showed that MCR contains 33% alpha helices and 30% beta sheets, compatible with a relatively flat conformation of the native protein. These data provide experimental proof for the predicted computer simulation regarding the structural features of the steroid receptor superfamily and suggest crosstalk between several protein families.

Adenosine Triphosphate↗
PubMed1994

Nicotiana tabacum contains a putative mineralocorticoid receptor.

Total protein from N. tabacum cell cytosol, partially purified by ammonium sulphate precipitation, contains a 52 kDa protein that tests positive for mineralocorticoid receptor (MCR)-like activity by Western blots, immuneprecipitation and photochemical cross-linking. Binding of RU 26752, a ligand specific to the MCR, suggests 100 fmol steroid bound per mg of tobacco protein; this is equivalent to about 6000 sites per cell assuming a 1/1 stoichiometry. Northern blots of total RNA from isolated tobacco cells, hybridized with a probe specific to mammalian MCR, revealed 2.8 kb and 2.2 kb transcripts, whereas the poly A mRNA was resolved as a prominent message of 1500 bp, preceded by a band of 2200 bp. This appears to be the first ever demonstration where a vascular plant is endowed with a pygmy protein similar to the much larger steroid hormone receptors in the animal world.

Blotting, Western↗
PubMed1994

Recombinant hirudin: a specific thrombin inhibiting anticoagulant for hemodialysis.

The first experience with hirudin as an alternative anticoagulant for heparin in hemodialysis is reported. Recombinant hirudin (HBW 023) was administered in 20 patients as a bolus before dialysis with low flux polysulfone dialyzers (PS400), the dosage being adapted stepwise from patient to patient by 0.02 mg/kg to the occurrence of clotting or bleeding. Four different administration schedules were studied. The first three schedules (0.02 mg/kg, N = 1; 0.04 mg/kg, N = 1; 0.06 mg/kg, N = 4) were discontinued because of clotting. The 0.08 mg/kg schedule was maintained without clotting event in 14 patients. Bleeding was not observed. Plasma hirudin averaged 503.9 +/- 214.0 and 527.7 +/- 217.1 ng/ml after two and four hours of dialysis, and decreased during an interdialytic interval of 44 hours to 223.2 +/- 86.2 ng/ml. Modified antithrombin III (P < 0.05) and activated partial thromboplastin times were lower (P < 0.01) under hirudin compared to heparin; these coagulation parameters were closer to normal during hirudin treatment. The patients developing clotting could be distinguished from those without clotting by the registration of the activated clotting times (9.2 +/- 3.0 vs. 18.7 +/- 3.2 min after 2 hr, P < 0.01; 8.1 +/- 3.0 vs. 16.2 +/- 3.8 min after 4 hr of dialysis, P < 0.05); cut-off value below which clotting is to be expected was 12 min). It is concluded that administration of hirudin as a bolus before the start of dialysis, at a dosage of 0.08 mg/kg, is not complicated by clotting or by bleeding. Coagulation tendency can optimally be monitored by the registration of the activated clotting time.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Light-induced changes in S-antigen (arrestin) localization in retinal photoreceptors: differences between rods and cones and defective process in RCS rat retinal dystrophy.

The subcellular localization of S-antigen (arrestin), a protein regulating phototransduction in retinal rods, was studied by immunocytochemistry using monoclonal antibodies on sections of Swiss mouse, Lewis, Brown Norway (BN), Royal College of Surgeons (RCS) rdy-p+ (dystrophic) and RCS rdy(+)-p (non-dystrophic) rat retinas. In normal retinas, the topography of S-antigen immunoreactivity in photoreceptor cells varied according to the lighting environment of the animals. In dark-adapted eyes, outer segments did not display any S-antigen immunoreactivity while the inner segments, cell bodies and synaptic terminals were strongly labeled. A few minutes after light exposure, there was an inversion of the pattern of labeling: the label increased in the outer segment but was strongly reduced in the other compartments. After 1 h of light, S-antigen immunoreactivity remained only in outer segments and in a few synaptic terminals. We show that the kinetics of this change is slower in cone than in rod cells, and thus allows the transient visualization of the scarce cone photoreceptors. On the 17th day after birth, photoreceptor cells are well differentiated in all rat strains, including RCS rdy-p+ rats. At this time, the S-antigen shift phenomenon occurred in the non-dystrophic strains, but was not observed in rdy-p+ rats: after light exposure, the intracellular distribution of S-antigen remained the same as in the dark. We suggest that an abnormality in the mechanisms of intracellular protein transport could be a characteristic of this genetic disease.

Amino Acid Sequence↗