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Biomedical subjects

L A Allen

Publications and source records attributed to L A Allen.

53 records · Page 3Linked to original sources
PubMed1989

Chinese hamster ovary cell mutants defective in peroxisome biogenesis. Comparison to Zellweger syndrome.

We have previously reported the isolation of Chinese hamster ovary (CHO) cell mutants that are defective in the biosynthesis of plasmalogens, deficient in at least two peroxisomal enzymes (dihydroxyacetonephosphate (DHAP) acyltransferase and alkyl-DHAP synthase), and in which catalase is not found within peroxisomes (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170). We now provide further evidence that three such strains are more generally defective in peroxisome biogenesis. Electron microscopic cytochemistry revealed that the mutants did not contain recognizable peroxisomes. However, immunofluorescence microscopy using an antibody directed against peroxisomal integral membrane proteins revealed the presence of peroxisomal membrane ghosts resembling those seen in cells of patients suffering from one of the human peroxisomal disorders, Zellweger syndrome. Immunoblot analyses, using antibodies specific for peroxisomal matrix proteins, demonstrated deficiencies of peroxisomal proteins in the mutant CHO cells that were similar to those in Zellweger syndrome. Fusion of a CHO mutant with fibroblasts obtained from Zellweger patients resulted in restoration of peroxisomal dihydroxyacetonephosphate acyltransferase and peroxisomal acyl-coenzyme A oxidation activities. The hybrid cells also regained the ability to synthesize plasmenylethanolamine. Moreover, normal peroxisomes were seen by immunofluorescence in the hybrid cells. These results indicate that the hybrid cells have recovered the ability to assemble peroxisomes and that, although the mutant CHO cells are biochemically and morphologically very similar to cells from patients with Zellweger syndrome, the genetic lesions are distinct. Our somatic cell mutants should be useful in identifying factors and genes involved in peroxisome biogenesis and may aid the genetic categorization of the various peroxisomal disorders.

Acyl Coenzyme A↗
PubMed1989

Cytoplasmic requirement for peroxisome biogenesis in Chinese hamster ovary cells.

Hybrids constructed by fusion of wild-type Chinese hamster ovary cells (CHO-K1) to peroxisome-deficient CHO mutants (ZR-78.1) contain normal peroxisomes, demonstrating that the mutation(s) are recessive. "Nuclear hybrids" prepared by fusion of CHO-K1 karyoplasts to mutant ZR-78.1 occasionally fail to regain intact peroxisomes (approximately 1/300 cells). These peroxisome-deficient nuclear hybrids closely resemble the original mutant cells by biochemical criteria, but their modal chromosome number is 36-38, the same as that of CHO hybrids generated from intact cells. When the peroxisome-deficient nuclear hybrids are fused to wild-type cytoplasts, a fraction of the fusion products (at least 70%) continue to propagate normal peroxisomes indefinitely. Peroxisome biogenesis cannot be reinitiated in cells of mutant ZR-78.1 by fusion to wild-type cytoplasts. Our results suggest that a wild-type nucleus by itself is necessary but not sufficient for restoration of normal peroxisome biogenesis and that a cytoplasmic component of wild-type cells, possibly a normal peroxisome, is also required.

Animals↗
PubMed1988

Glucose starvation is required for insulin stimulation of glucose uptake and metabolism in cultured microvascular endothelial cells.

In the present study we determined the uptake and disposition of glucose in serum-deprived rabbit coronary microvessel endothelial (RCME) cells. RCME cells exhibited stereospecific hexose uptake inhibited by cytochalasin B. Pretreatment of the RCME cells with potassium cyanide or 2,4-dinitrophenol inhibited 2-deoxyglucose uptake but not 3-O-methylglucose transport. A major proportion (30-60%) of the 2-deoxyglucose present in the RCME cells was not phosphorylated. These two observations suggested that the rate-limiting step in the uptake of 2-deoxyglucose was not transport but rather the phosphorylation of 2-deoxyglucose to 2-deoxyglucose 6-phosphate. When glucose-deprived cells were incubated 2 hr with [U-14C]glucose the disposition of the label was as follows: glycogen 60%, acid-soluble fraction 30%, and lipid less than 5%. In contrast glucose-fed cells exhibited lower overall glucose incorporation, and a slightly different disposition: glycogen 45%, acid-soluble fraction 50%, and lipid 5%. Glucose-deprived RCME cells also exhibited greater basal levels of 2-deoxyglucose uptake compared to glucose-fed cells. RCME cells incubated in the absence of glucose and serum for 16 hr exhibited dose-dependent insulin stimulation of hexose uptake and subsequent metabolism to macromolecules (i.e., glycogen and the acid-soluble fraction). Significant effects of insulin were observed with concentrations as low as 2 x 10(-10) M, well within the physiological range. In contrast, cells preincubated in serum-free culture medium containing 5.5 mM glucose did not exhibit insulin-enhanced hexose uptake or glucose metabolism (even at doses as high as 10(-7) M). These studies indicate that the effects of insulin on rabbit coronary microvascular endothelial cell glucose uptake and metabolism require both serum and glucose deprivation.

Animals↗
PubMed1986

Regulation of hexose transport in cultured bovine retinal microvessel endothelium by insulin.

In microvessel endothelium obtained from young calf retinas, insulin stimulated the uptake of 2-deoxy-D-glucose in a dose-dependent manner. The maximal level of glucose uptake (50-100% over basal values) was observed at 1 ng ml-1 insulin. The effects of insulin on glucose uptake were time-dependent, with optimum stimulation of glucose transport observed after 90 min of insulin treatment. The effects of insulin were prevented by 15 min pretreatment with 2 micrograms ml-1 cycloheximide or by 2 micrograms ml-1 actinomycin D, suggesting a role for new protein synthesis in the actions of insulin on glucose transport in this cell type. These results demonstrate that cultured retinal microvessel endothelial cells exhibit an insulin-sensitive glucose transport system.

3-O-Methylglucose↗
PubMed1986

Calcium binding to cardiac sarcolemmal vesicles: potential role as a modifier of contraction.

Passive Ca binding to cardiac sarcolemmal vesicles isolated from rabbit ventricles was measured under ionic conditions similar to intracellular and extracellular media. The first of two main goals was to evaluate whether certain agents induce changes in Ca binding at the external sarcolemmal surface that might contribute to the overall effect of these agents on cardiac muscle contraction. The agents studied were ouabain, verapamil, nifedipine, Bay K 8644, caffeine, ryanodine, and milrinone over a broad range of concentrations, including concentrations at which these agents exert strong effects on cardiac contractile performance. None of these agents produced significant alterations in Ca binding, such that it is unlikely that any part of their actions can be attributed to changes in Ca binding to the external sarcolemmal surface. In contrast, when [Na] is reduced from 140 mM, sarcolemmal Ca binding increases or decreases depending on what replacement is used to avoid changes of osmolarity. Thus the possible effect of Na reduction on surface Ca must be considered in physiological experiments where extracellular [Na] is changed. The second main goal was to evaluate the effects of membrane potential, Na and Mg on Ca bound to the inner surface of the sarcolemma under ionic conditions similar to those expected intracellularly (e.g., [Ca] = 0.3-5.0 microM). Ca binding was inhibited by physiological concentrations of Na and Mg and was sensitive to membrane potential such that depolarization of a normally polarized cell would cause Ca to be released from these sarcolemmal sites. From a quantitative standpoint, it is not clear whether the effect of depolarization would be to contribute sarcolemmal Ca to the activation of the myofilaments or merely to limit the ability of the inner sarcolemmal surface to buffer the rise in intracellular [Ca] associated with contraction.

Alamethicin↗
PubMed1984

Diagnostic laparoscopy and management of patients with confirmed acute pelvic inflammatory disease.

The clinical success rate in the diagnosis of acute pelvic inflammatory disease (PID) is unacceptably low. There is an invaluable role for laparoscopy in the diagnosis, investigation and treatment of patients with suspected pelvic infection. PID at Ngwelezana Hospital is predominantly gonococcal and a combination of penicillin G and clindamycin has proved highly effective in the management of these patients.

Acute Disease↗
PubMed1983

Maternal obstetric paralysis. A case report.

A severe case of puerperal obstetric palsy is presented. Evidence in favour of the Hunerman theory is submitted. Pressure necrosis resulted in a large vesicovaginal fistula.

Adolescent↗
PubMed1983

Laparoscopic diagnosis of acute pelvic inflammatory disease.

At Ngwelezana Hospital, 103 patients admitted from the outpatient clinic with a diagnosis of pelvic infection were laparoscoped and in only 63 was the diagnosis confirmed. A disturbingly high incidence of ectopic pregnancy (8 patients) was found.

Acute Disease↗
PubMedDate not supplied

Interaction of hybridoma antibodies with normal and mutant forms of Escherichia coli recA protein.

We have prepared hybridomas which secrete antibodies against E. coli recA protein, by fusing spleen lymphocytes from immunized mice with P3X63-AG8 myeloma cells. This paper describes a preliminary survey of properties of antibody from a hybridoma population designated 156, which inhibits the strand pairing, strand assimilation, repressor cleavage, and DNA-dependent ATPase activities of recA protein. The 156 antibody consists of one or two species which have been tentatively identified as IgG2b. 156 Globulin reacts 4.6-fold more efficiently with denatured recA protein, and it reacts partially with the native tif-1, lexB30, recA44, and recA629 proteins, as well as with peptide fragments which are not common to all four proteins.

Alleles↗
PubMedDate not supplied

Somatization: a debilitating syndrome in primary care.

Somatization is a significant problem for clinical medicine. Unlike somatization disorder, which is relatively rare, abridged somatization, a less severe form of somatization, is prevalent in primary care clinics. The authors examined the clinical status and functioning of patients diagnosed with a depression or anxiety disorder comorbid with abridged somatization and compared them with patients diagnosed with a depression or anxiety disorder alone. The authors examined severity of physical functioning and psychopathology in relation to diagnostic status. Patients diagnosed with both abridged somatization and a depression or anxiety disorder were more physically impaired and more anxious than those diagnosed with a depression or anxiety disorder alone. The results suggest that abridged somatization frequently coexists with depression and anxiety and thus complicates the presentation of these disorders.

Adult↗