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Biomedical subjects

R E Allen

Publications and source records attributed to R E Allen.

At least 37 records · Page 2Linked to original sources
PubMed1994

Temporal expression of myogenic regulatory genes during activation, proliferation, and differentiation of rat skeletal muscle satellite cells.

The satellite cell is responsible for growth and repair of postnatal skeletal muscle. We investigated the expression of the myogenic regulatory gene (MRG) family in these cells in the stages from quiescence to fusion. Using polymerase chain reaction amplification of reverse-transcribed RNA (RT-PCR) isolated from adult rat satellite cells, we demonstrated a temporal sequence of gene activation, which is distinct from that previously observed in embryonic somatic cells. No MRG expression was detected in predominantly quiescent cells. MyoD is activated by 12 h in cell culture, prior to the first evidence of proliferation. MRF4 and myf-5 appear by 48 h and may be associated with the first division cycle. Myogenin is not detectable until 72 h after satellite cell recovery from the muscle fiber, coincidental with the first evidence of differentiation.

Animals↗
PubMed1993

Purification and characterisation of glyoxalase II from human red blood cells.

Glyoxalase II was purified from human red blood cells. The purification factor was 83,300 and the yield was 24% or 1.7 micrograms/ml red blood cells. The purified protein was a monomer with a molecular mass of 29,200 Da and an isoelectric point of 8.3. The rate of hydrolysis of S-D-lactoylglutathione to reduced glutathione and D-lactate, catalysed by glyoxalase II, followed Michaelis-Menten kinetics where the Km and kcat values were 146 +/- 9 microM and 727 +/- 16 s-1, respectively in 50 mM Tris/HCl, pH 7.4 at 37 degrees C. Other S-2-hydroxyacylglutathione derivatives were also acceptable substrates. S-p-Nitrobenzoxycarbonylglutathione was a potent competitive inhibitor of glyoxalase II with a Ki value of 1.20 +/- 0.21 microM, and the hemithioacetal formed non-enzymically from the reaction of methylglyoxal with reduced glutathione was a weak competitive inhibitor with a Ki value of 834 +/- 98 microM.

Erythrocytes↗
PubMed1993

Proliferating cell nuclear antigen (PCNA) is expressed in activated rat skeletal muscle satellite cells.

Skeletal muscle satellite cells from uninjured muscle of adult animals are generally found to be in a quiescent state, and when cultured, they remain quiescent in vitro for a period of time which is directly related to the age of the donor animal. A technique for studying the activation of satellite cells in primary cultures has been developed and employs proliferating cell nuclear antigen (PCNA) as a marker for entrance into the S phase of the cell cycle. PCNA is a protein involved in DNA replication and is maximally expressed in S phase of the cell cycle. We monitored PCNA expression in satellite cells isolated from young (3 week) and adult (9 month) rats, and our results indicate that satellite cells begin to accumulate PCNA prior to changes in cell number in both age groups. Using ELISA techniques, we demonstrated that addition of an extract of crushed muscle (CME) activated satellite cells and significantly reduced the length of the lag phase in cells from both age groups. Addition of bFGF shortened the lag phase of PCNA synthesis in satellite cells from 3-week-old rats but had no effect on the kinetics of PCNA expression in cells from 9-month-old rats. Based on our experiments, PCNA expression can be used as a marker to follow the entry of satellite cells into the cell cycle in primary mass cultures.

Animals↗
PubMed1993

A simplified method for the purification of human red blood cell glyoxalase. I. Characteristics, immunoblotting, and inhibitor studies.

Glyoxalase I (EC 4.4.1.5) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography with a modified concentration gradient of S-hexylglutathione for elution. The pure protein had a specific activity of 1830 U/mg of protein, where the overall yield was 9%. The pure protein had a molecular mass of 46,000 D, comprised of two subunits of 23,000 D each, and an isoelectric point value of 5.1. The KM value for methylglyoxal-glutathione hemithioacetal was 192 +/- 8 microM and the kcat value was 10.9 +/- 0.2 x 10(4) min-1 (N = 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had a Ki value of 0.16 +/- 0.04 microM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with a Ki value of 3.12 +/- 0.88 microM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with a Ki value of 18 +/- 8 mM. The polyclonal antibodies were raised to the purified enzyme and were found to react specifically with glyoxalase I antigen by immunoblotting. This procedure gave a protein of high purity with simple low pressure chromatographic techniques with a moderate but adequate yield for small-scale preparations.

Antibodies↗
PubMed1993

Inhibition of proliferation of human leukaemia 60 cells by methylglyoxal in vitro.

Methylglyoxal (2-oxopropanal) is the physiological substrate of the glyoxalase system. When exogenous methylglyoxal (50 microM-1 mM) was added to human leukaemia 60 (HL60) cells in culture (5 x 10(4) cells/ml), inhibition of growth and toxicity was induced. The median growth inhibitory concentration IC50 value was 238 +/- 2 microM. There was little differentiation of HL60 cells induced by methylglyoxal (a maximum of 2% differentiation with 500 microM methylglyoxal). There was no similar toxicity induced by methylglyoxal in corresponding differentiated cells, neutrophils, under the same culture conditions. Cell growth and toxicity induced by methylglyoxal (250 microM) in HL60 cells occurred in the initial 24 h of culture, after which residual surviving cells exhibited normal growth kinetics. It could also be prevented by replacing the culture medium in the initial 6 h of culture; thereafter, irreversible toxicity developed, reaching the maximum value after 24 h of culture. Growth arrest and toxicity induced by methylglyoxal increased with increasing serum composition of the medium. The mechanism of toxicity is unknown.

Antineoplastic Agents↗
PubMed1992

Inhibition of growth of human leukaemia 60 cells by S-2-hydroxyacylglutathiones and monoethyl ester derivatives.

S-2-Hydroxyacylglutathione derivatives were found to induce growth arrest and toxicity in human leukaemia 60 cells in culture. S-D-Lactoylglutathione was the most effective with a median inhibitory concentration IC50 of 82 microM (95% C.I. 65-105 microM). No similar toxicity was induced by reduced glutathione and/or the corresponding aldonic acid (500 microM) in human leukaemia 60 cells, nor by S-D-lactoylglutathione (500 microM) in mature human neutrophils under the same culture conditions. Monoethyl ester derivatives of the S-2-hydroxyacylglutathiones were prepared and also induced growth arrest and toxicity but were less effective than the corresponding unesterified compounds. S-2-Hydroxyacylglutathione derivatives also inhibited the incorporation of [3H]thymidine into DNA early in the development of toxicity: for S-D-lactoylglutathione, the median inhibitory concentration was 74 microM (95% C.I. 47-116 microM). The mechanism of the inhibition of human leukaemia cell growth by S-D-lactoylglutathione and other S-2-hydroxyacylglutathione derivatives is unknown but appears to be mediated by the inhibition of DNA synthesis.

Cell Division↗
PubMed1992

Localization of the Ca(2+)-dependent proteinases and their inhibitor in normal, fasted, and denervated rat skeletal muscle.

Immunofluorescence and immunogold localization studies show that the two Ca(2+)-dependent proteinases (mu-calpain for the micromolar Ca(2+)-requiring proteinase and m-calpain for the millimolar Ca(2+)-requiring proteinase) and their protein inhibitor (calpastatin) are located exclusively intracellularly in normal rat soleus muscle. Quantitative immunogold studies indicate that binding of antibodies to both calpains and to calpastatin is approximately two times greater at the Z-disk of myofibrils than it is at the I-band or A-band regions. Mitochondria and nuclei in muscle cells contain both calpains and calpastatin at concentrations approximately one-tenth and one-fifth, respectively, of the concentration at the Z-disk, as estimated by antibody binding. Very little calpain or calpastatin was seen in the cytoplasmic intermyofibrillar spaces, and most of the calpain and calpastatin in muscle cells is associated with intracellular structures. Immunofluorescence results suggest that concentration of m-calpain but not mu-calpain or calpastatin is, in some instances, slightly higher near the intracellular surface of the plasma membrane than elsewhere in the muscle cell. Most m-calpain, however, is distributed throughout the interior of mature rat skeletal muscle cells. Denervation, or fasting and refeeding increases the concentration of the calpains and calpastatin in the muscle cell but does not change their distribution. Some mu- and m-calpain and calpastatin is found extracellularly in denervated soleus muscle or soleus muscle from fasting rats, but the extracellular calpains and calpastatin seem to originate from "leakage" of these proteins out of the cell because serum creatine kinase levels are much higher than normal in denervated or fasting rats.

Animals↗
PubMed1992

Changes in the cytoskeleton of 3T3 fibroblasts induced by the phosphatase inhibitor, calyculin-A.

Addition of the protein phosphatase inhibitor, calyculin-A, to 3T3 fibroblasts causes a marked change in cell morphology. Initially the cells become rounded, develop surface blebs and then detach from the substratum. In the detached cells an unusual ball-like structure is observed. This study focuses on the cytoskeleton during these calyculin-A-induced morphological changes. Stress fibres disappear as the cells begin to round and aggregates of actin are formed towards the apical surface of the cell. These aggregates condense, in the detached cells, to form the ball structure of approximately 3 microns diameter. Between the ball and the nucleus are cables of intermediate filaments that appear to be attached to the surface of the ball and to the nuclear lamina. Using a procedure designed for the isolation of nuclei the nucleus-ball complex can be obtained. Analysis of the nucleus-ball preparation by immunofluorescence and electron microscopy demonstrate that the ball contains actin and that intermediate filaments are located between the ball and the nucleus. In this preparation, the intermediate filaments also appear to attach to the surfaces of the ball and the nucleus. Electrophoretic analysis of the nucleus-ball preparation indicates that, in addition to actin, a major component of the ball is myosin. It is suggested that the formation of the ball is caused by an actin-myosin-based contractile process, initiated by the phosphorylation of myosin. The aggregation of the actomyosin draws together the intermediate filaments into the area between the ball and nucleus. This hypothesis requires that vimentin binds both to the nucleus and to some component of the ball.

3T3 Cells↗
PubMed1992

Chemiluminescence assay of mucosal reactive oxygen metabolites in inflammatory bowel disease.

Previous studies suggesting increased reactive oxygen metabolite (ROM) production in inflammatory bowel disease have been restricted to peripheral blood and isolated intestinal phagocytes. In the current study, chemiluminescence and the effect of various scavengers, enzymes, and enzyme inhibitors were used to show that ROMs account for the increased production of oxidants by colorectal mucosal biopsy specimens in inflammatory bowel disease. Luminol-amplified chemiluminescence was increased in active ulcerative colitis [macroscopic grade 1: 25 photons.mg-1.min.10(-3) (median), 8-47 (95% confidence intervals), n = 40; grade 2: 89, 65-156, n = 30; grade 3: 247, 133-562, n = 13] and Crohn's disease [mild: 9, 3-84, n = 6; severe: 105, 25-789 (range), n = 5] compared with normal-looking mucosa (ulcerative colitis: 0.8, 0.4-1.4, n = 22, P less than 0.01; Crohn's disease: 0.8, 0.1-2, n = 6, P less than 0.05) and controls (0.6, 0.04-1.4, n = 52, P less than 0.01). In ulcerative colitis, luminol chemiluminescence correlated with microscopic inflammation (Spearman's p = 0.74, P = 0.0001) and was decreased by sodium azide (-89%, P less than 0.05), taurine (-31%, P less than 0.05), catalase (-23%, P less than 0.05), and dimethyl sulfoxide (-29%, P less than 0.05). Superoxide dismutase and oxypurinol decreased lucigenin chemiluminescence in ulcerative colitis by -63% (P less than 0.05) and -27% (P less than 0.05), respectively. Luminol chemiluminescence correlated with lucigenin chemiluminescence (Spearman's rho = 0.72, P = 0.003). These results suggest that neutrophil-derived oxidants (superoxide, hydrogen peroxide, hydroxyl radical, and hypochlorite) are generated in colorectal mucosa in active inflammatory bowel disease and support the hypothesis that production of such metabolites by neutrophils is of major pathogenetic importance.

Acridines↗
PubMed1991

Calyculin-A increases the level of protein phosphorylation and changes the shape of 3T3 fibroblasts.

Calyculin-A, an inhibitor of type 1 and 2A phosphatases, was applied extracellularly to 3T3 fibroblasts. At 0.1 microM, calyculin-A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phosphorylation following treatment with calyculin-A was observed in vimentin, the 20-kD myosin light chain, and an unidentified 440-kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells. Calyculin-A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin-A (0.1 microM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin-A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin-A an unusual "ball-like" structure was observed with transmission electron microscopy. This unique structure was 2-3 microM in diameter and was located close to the nucleus. The use of calyculin-A adds further support to the idea that cell shape is controlled, at least in part, by concerted actions of a kinase-phosphatase couple.

Animals↗
PubMed1991

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Growth factor regulation of bovine satellite cell growth in vitro.

This study examined the effects of basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) on the proliferation and differentiation of primary bovine satellite cells (BSC) in vitro. Individually, these three factors had the following effects on satellite cells: bFGF stimulated proliferation (P less than .01) but inhibited differentiation (P less than .05); IGF-I had no effect on proliferation but stimulated differentiation (P less than .01); and TGF-beta inhibited both proliferation and differentiation (P less than .01). When combined, the following effects were observed: maximum stimulation of proliferation (P less than .01) occurred in the presence of bFGF and IGF-I and differentiation was not stimulated; TGF-beta and bFGF continued to inhibit differentiation (P less than .01), but in the presence of bFGF, TGF-beta stimulated proliferation (P less than .01). No stimulation was observed in the presence of TGF-beta and IGF-I. Bovine satellite cells respond to these three growth factors that have been shown to regulate the activity of other myogenic cells, and in most instances, the responses among cells from various species are similar. These experiments indicate that the interactions of growth factors may be critical in regulating bovine satellite cell activity.

Animals↗