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Biomedical subjects

R Roy

Publications and source records attributed to R Roy.

At least 253 records · Page 14Linked to original sources
PubMed1993

Characterization of hormonally regulated and particulate-associated phospholipase A2 from bovine endothelial cells.

Hormonally regulated and particulate-associated phospholipase A2 (PLA2) was detected in endothelial cells from bovine pulmonary artery. The enzyme was solubilized and subjected to initial characterization. PLA2 activity was determined in subcellular fractions from bradykinin (BK)-stimulated and nonstimulated cells by following the release of arachidonic acid (AA) from exogenously added 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylcholine. Stimulation of cells with BK led to increased PLA2 activity in a particulate fraction (the 92,000 x g pellet of the postnuclear supernatant). The activity in the cytosolic fractions from BK-stimulated and nonstimulated cells was the same. The association of the hormonally regulated PLA2 (HR-PLA2) activity with the particulate fraction was not affected by decreasing the Ca2+ concentrations in the homogenate from 7 microM to 33 nM and therefore was not induced during homogenization by the presence of Ca2+ in the homogenate. The HR-PLA2 activity was Ca(2+)-dependent and was maximal at submicromolar concentrations of Ca2+. Incubation of the particulate fraction obtained from BK-stimulated and nonstimulated cells with 10 mM n-octyl-beta-D-glucopyranoside resulted in a differential solubilization of the HR-PLA2 activity. Its isoelectric point was determined to be 5.7. HR-PLA2 activity in the octyl glucoside extract of the particulate fraction from stimulated cells co-sedimented in sucrose gradients with the cytosolic PLA2. Their molecular mass was estimated to be 103,000 Da. The extracted enzyme from BK-stimulated cells retained its increased activity toward 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylcholine. However, its activity toward 1-palmitoyl-2-[1-14C]oleoyl-phosphatidylcholine was equal to the PLA2 activity extracted from nonstimulated cells. Treatment of the cells with 100 nM 12-O-tetradecanoylphorbol-13-acetate resulted in a 20 +/- 1.2% (mean +/- S.E., p < 0.01, n = 4) increase in the PLA2 activity in the cytosol but failed to increase PLA2 activity in the particulate fraction. In contrast, addition of 7 microM Ca2+ ionophore A23187 resulted in a 21 +/- 0.55% (mean +/- S.E., p < 0.01, n = 5) decrease in the cytosolic activity and a concomitant increase of 68 +/- 9.6% (mean +/- S.E., p < 0.05, n = 5) in the particulate-associated activity. We conclude that stimulation of endothelial cells with BK increases the activity of a Ca(2+)-sensitive high molecular weight isoform of PLA2 which is associated with the particulate fraction. Possible mechanisms of activation are discussed.

Animals↗
PubMed1993

DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor.

The human BTF2 basic transcription factor (also called TFIIH), which is similar to the delta factor in rat and factor b in yeast, is required for class II gene transcription. A strand displacement assay was used to show that highly purified preparation of BTF2 had an adenosine triphosphate-dependent DNA helicase activity, in addition to the previously characterized carboxyl-terminal domain kinase activity. Amino acid sequence analysis of the tryptic digest generated from the 89-kilodalton subunit of BTF2 indicated that this polypeptide corresponded to the ERCC-3 gene product, a presumed helicase implicated in the human DNA excision repair disorders xeroderma pigmentosum and Cockayne's syndrome. These findings suggest that transcription and nucleotide excision repair may share common factors and hence may be considered to be functionally related.

Adenosine Triphosphate↗
PubMed1993

Histopathological and immunological investigations of synthetic fibres and structures used in three prosthetic anterior cruciate ligaments: in vivo study in the rat.

Three types of prosthetic anterior cruciate ligaments were investigated by enzymatic and histological analysis of the tissue surrounding each implant and immunologically by a cytofluorometric analysis of T-cell populations in the peripheral blood of rats. Two of the prostheses had a braided construction, one made from polyester and the other from high performance polyethylene fibres. The third type also contained high performance polyethylene fibres, but had been manufactured in a knitted construction (Raschel high performance polyethylene). Five specimens from each prosthesis were implanted intraperitoneally in rats by a trocar for different periods of time up to 4 wk. A control group of rats underwent the surgery, but not the implant. No modification in peripheral T-cell populations was induced by the presence of any implant. Whilst the levels of acid phosphatase and esterase activity appeared to have increased slightly following implantation of any of the prostheses, such increases were not highly significant. Histologically, all three materials induced an intense acute inflammatory reaction at 3 d which gave way to a typical chronic response after 4 wk. The only major difference between the prostheses was that after 4 wk the polyester fibres exhibited less inflammation, and the surrounding tissue was more mature, more vascularized and more densely infiltrated with collagen than with the two high performance polyethylene implants. In conclusion, all three devices provided satisfactory biocompatibility in terms of cellular and healing response.

Acid Phosphatase↗
PubMed1993

Risk factors of corneal graft failure.

PURPOSE: To measure the association between potential risk factors and corneal graft failure. Two failure outcomes are compared: those with and those without a prior immune allograft reaction. METHODS: Based on a single-center observational study design, 539 adult recipients of a corneal graft were followed for a median time of 30 months. Survival analysis was carried out. RESULTS: Eighty-two graft failures were recorded. Of 82 failures, 53 (65%) were not preceded by an immune allograft reaction. Presence of blood vessels in the recipient cornea was associated with a twofold increase in risk for both failure outcomes. Three factors increased the risk of failure without an immune reaction: prior glaucoma or uveitis (adjusted relative risk estimate = 3.1), vitreous surgery with the graft (adjusted relative risk estimate = 2.0), and a repeat graft in the study eye (adjusted relative risk estimate = 2.0). Conversely, large graft wound size (adjusted relative risk estimate = 2.0). Conversely, large graft wound size (adjusted relative risk estimate = 2.9) and human leukocyte antigen (HLA)-A, -B incompatibility (adjusted relative risk estimate = 2.2) were associated with failures that followed an immune reaction. CONCLUSION: In this study, the authors support the clinical impression that corneal graft failures with and without a prior immune reaction are distinct phenomena. Enhanced surveillance in recipients with glaucoma and early intensive treatment of allograft reactions are recommended to improve the outcome of corneal grafts.

Cataract Extraction↗
PubMed1993

ZR-75-1 breast cancer cells generate nonconjugated steroids from low density lipoprotein-incorporated lipoidal dehydroepiandrosterone.

Fatty acid esters of dehydroepiandrosterone (DHEA-FA) are present in the circulation, although no physiological function has yet been attributed to these metabolites. They are formed directly in serum and are predominantly localized in association with lipoproteins. The objective of this study was to determine the capacity of these lipoprotein-incorporated DHEA metabolites to generate nonconjugated steroids after incubation with cells in culture. A method for studying DHEA-FA using a radiolabeling technique that marks human low density lipoproteins (LDL) with tritiated DHEA-FA was elaborated. Analysis of the fatty acid composition of tritiated DHEA-FA-labeled LDL ([3H] DHEA-FA-LDL) indicated the prevalence of DHEA-linoleate/palmitoleate and DHEA-oleate. Incubation of [3H]DHEA-FA-LDL with ZR-75-1 breast cancer cells produced a time-dependent increase in labeled nonconjugated steroids in the cell culture medium, whereas the levels of tritiated DHEA-FA decreased. Lipoidal radioactivity in cells increased with time, but nonconjugated radioactivity associated with the cells showed no such increase. HPLC analysis of the culture medium indicated the presence of DHEA and androst-5-ene-3 beta,17 beta-diol. The endogenous levels of lipoidal DHEA were also determined in human plasma and its lipoprotein components to reveal that these metabolites circulate naturally in the range of 6.5 +/- 0.4 nM. Approximately 90% of this concentration was associated with the lipoprotein components, namely among the LDL and high density lipoprotein fractions. These results suggest that lipoidal DHEA may indeed act as a substrate for potent steroid formation after their entry into steroid target cells.

Breast Neoplasms↗
PubMed1993

Detection of the ryanodine receptor mutation associated with malignant hyperthermia in purebred swine populations.

A restriction endonuclease assay (REA) that reveals a C/T transition in the ryanodine receptor gene (ryr-1 locus) was proposed to test swine for malignant hyperthermia. The REA was tested in a heterologous pig population of 1,227 individuals representing Hampshire, Duroc, Landrace, and Yorkshire breeds. Of this group, 713 pigs had been classified on the basis of the halothane challenge and progeny testing into the three possible Hal genotypes (685 negatives, 24 carriers, and 4 susceptibles). The REA confirmed the four susceptibles as T/T, 17/24 carriers as C/T, and 659/685 negatives as C/C. The rest of the sample was constituted of 514 pigs that had not been selected on the basis of the halothane gene. The proportions of C/T and T/T pigs within the breeds represented were 3.2 and 1.6% for Duroc, 27.3 and 1.4% for Landrace, and 17.9 and 1.9% for Yorkshire. No T/T allelic pairs were found in the Hampshire breed, although the sample was very small. Genotypic distribution and allelic frequencies at the ryr-1, Phi, and Pgd loci were reported among the breeds.

Alleles↗
PubMed1993

Rapid selection of donor myoblast clones for muscular dystrophy therapy using cell surface expression of NCAM.

This study describes an easy 3 step-procedure to prepare rapidly and at low cost, pure myoblast cell cultures from a normal muscle biopsy. Following collagenase and trypsin treatment of the tissue (step 1), dissociated cells were cloned at a density of 10 cells/ml in MCDB 120 medium (0.2 ml/well). Clones that grew were then tested for NCAM cell surface expression by cytofluorometric analysis (CFA) using Coulter CD56-PE monoclonal antibodies (step 2). Only those clones with more than 98% strongly labelled positive cells were expanded (step 3) for further trials in cell transfer therapy for dystrophic patients. Visualization of the pattern of NCAM expression was performed by immunoperoxidase assay, while the potential ability to form myotubes was confirmed by the observation of their formation within a period of 1 to 2 weeks. The 65% of the CD56+ clones in CFA were the same clones that proved to be myogenic with positive immunoperoxidase assay and myotube formation. This method avoids the fastidious and costly approach of cell sorting (whenever available), avoids contamination hazards due to many manipulations of the clones. Moreover this approach leads to a pure myoblast population free of any contaminating fibroblast which could contribute to connective tissue implement already deleterious in dystrophic patients.

Antibodies, Monoclonal↗
PubMed1992

Jung et al. reply.

Explore the source record for details and available documents.

Journal Article↗
PubMed1992

Human myoblast transplantation: preliminary results of 4 cases.

Myoblasts from immunocompatible donors have been transplanted into the muscles (tibialis anterior, biceps brachii, and/or extensor carpi radialis longus) of 4 Duchenne patients in the advanced stages of the disease. Although no immunosuppressive treatment was used, none of the patients showed any clinical signs of rejection such as fever, redness, and inflammation. One patient transiently produced antibodies against the donor myoblasts as determined by cytofluorometric analysis. This patient and 2 others were shown to form antibodies against their donor's myotubes. Muscle biopsies of the injected tibialis anterior of 4 patients revealed that 80%, 75%, 25%, and 0% of the muscle fibers, respectively, showed some degree of dystrophin immunostaining. The contralateral noninjected muscles of the latter 3 patients did not contain any dystrophin positive fibers, while that of the first patient showed dystrophin expression in 16% of the fibers examined. Myoblasts were also injected into the extensor carpi radialis longus or the biceps brachii of these patients. A few months subsequent to injection, one patient was shown to have a 143% increase of strength during static wrist extension. This result must be interpreted with caution because a double-blind strength-measuring protocol was not used. Furthermore, we have noted that this change slowly decayed over time. The strength of 2 other patients was increased less remarkably (41% and 51%), while the strength of the fourth patient was unchanged.

Adolescent↗
PubMed1992

Administration of pregnenolone and dehydroepiandrosterone to guinea pigs and rats causes the accumulation of fatty acid esters of pregnenolone and dehydroepiandrosterone in plasma lipoproteins.

Steroids were administered continuously to guinea pigs and rats using subcutaneously applied silastic tubing implants, and the effects on circulating steroid and steroid conjugate levels were monitored. Using implants filled with pregnenolone, we observed that pregnenolone had a marked effect on increasing the levels of its fatty acid-esterified derivative, while dehydroepiandrosterone-releasing implants produced a rise in circulating nonconjugated dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, androstenedione, testosterone, and lipoidal derivatives of both dehydroepiandrosterone and androst-5-ene-3 beta,17 beta-diol. Implants filled with androstenedione produced a 20-fold increase in plasma androstenedione levels relative to untreated controls and a corresponding five-fold increase over control testosterone levels. No fatty acid-esterified derivative of testosterone could be detected within the plasma. Lipoproteins were isolated from both rats and guinea pigs treated with implants filled with pregnenolone or dehydroepiandrosterone. The steroid and steroid fatty acid esters present in each fraction were analyzed, revealing that approximately 75% of all the fatty acid esters of pregnenolone recovered in the lipoproteins was localized within the high-density lipoprotein (HDL) fraction of both guinea pig and rat plasma. Similarly, lipoidal dehydroepiandrosterone was found associated predominantly with the low-density lipoprotein and HDL fractions in the guinea pig, while in the rat this steroid conjugate was exclusively within the HDL fraction. High-density lipoprotein-incorporated tritiated pregnenolone fatty acid esters and dehydroepiandrosterone fatty acid esters were injected into castrated male guinea pigs to study the fate of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗