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L Morgan

Publications and source records attributed to L Morgan.

At least 91 records · Page 5Linked to original sources
PubMed1994

Functional analysis of conserved cysteine residues in the catalytic subunit of the yeast vacuolar H(+)-ATPase.

The A subunit of the yeast vacuolar ATPase contains three highly conserved cysteines: Cys-261, Cys-284, and Cys-538. Cys-261 is located within the nucleotide-binding P-loop. Each of the conserved cysteines, and one nonconserved cysteine, Cys-254, were altered to serine by site-directed mutagenesis, and the effects on growth at pH 7.5 were determined. The Cys-254-->Ser, Cys-261-->Ser and the double mutants all grew at pH 7.5 and contained nitrate- and bafilomycin-sensitive ATPase activity. However, the ATPase activities of the Cys-261-->Ser and the double mutants were insensitive to the sulfhydryl group inhibitor, N-ethylmaleimide, demonstrating that Cys-261 is the site of inhibition by N-ethylmaleimide. Changing either Cys-284 or Cys-538 to serine prevented growth at pH 7.5. Cys-284 and Cys-538 thus appear to be essential cysteine residues which are required either for assembly or catalysis.

Base Sequence↗
PubMed1994

Analysis of nickel refinery dusts.

After characterization of bulk samples by inductively coupled plasma emission spectroscopic (ICP-ES) quantitative analysis and X-ray powder diffraction studies, single particle techniques using quantitative image analysis, scanning electron microscopy--energy dispersive analysis by X-ray, and finally laser beam ionization mass spectrometry analysis (LIMA) for surface analysis have been applied to historical nickel refinery dust samples from the nickel refining plant at Clydach in Wales. There were two historical samples of processed material from 1920 and 1929. These samples had a remarkably small particle size range, mean 3 microns and range, 0.75-24 microns. The most significant difference in elemental composition was the presence of 10% arsenic in the 1920 sample compared with 1% in the 1929 sample. The X-ray spectra revealed the presence of NiO in both. However, surprisingly, CuO was identified only in the 1929 sample. Of particular interest was the presence of a component, in the 1920 sample only, identified as the mineral orcelite, a copper-iron-nickel-arsenide-sulphide mineral, predominantly, Ni5-XAs2. Using the LIMA technique, it was found that in both samples, arsenic and arsenic derivative peaks are prominent, indicating the surface availability of arsenic compounds.

Arsenic↗
PubMed1994

Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+.

Vanadyl, (V = O)2+, is able to substitute for Mg2+ as a cofactor for ATPase activity catalyzed by the chloroplast F1-ATPase (CF1). Mg2+-dependent ATPase activity was also observed with CF1 that contained VO(2+)-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg(2+)-ATPase activity induced by VO2+ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF1 is depleted of nucleotides from all but the N1 site, a single Mg2+ remains bound at a site designated M1. Addition of VO2+ to the depleted protein gives rise to an EPR spectrum characteristic of a CF1-bound VO2+ species. The binding curve of the VO2+ complex to latent, nucleotide-depleted CF1 was determined by the integrated intensities of the -5/2 parallel peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO2+ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF1-VO2+ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated 14N nucleus. Analysis of the bound VO2+ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO2+ bound to CF1 under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.

Adenosine Triphosphate↗
PubMed1994

Direct measurement of the binding of RAS to neurofibromin using a scintillation proximity assay.

Protein-protein interactions are of major importance in many cellular processes. When no enzymic activity is involved, assays for direct binding are required. One such example is the relatively weak interaction between oncogenic Ras and the GTPase-activating protein neurofibromin (NF1). The complex between the catalytic domain of NF1 and the GTP-form of oncogenic Ras protein dissociates rapidly; hence, equilibrium binding must be quantitated. Scintillation proximity assay (SPA) technology, a radioisotopic technique that requires no separation step, was used to characterize this interaction. Leu-61 Ras complexed with [3H]GTP was generated by nucleotide exchange in the presence of a GTP-regenerating system. A SPA signal was obtained when radiolabeled Ras was mixed with NF1 fused with glutathione S-transferase (GST), anti-GST, and protein A-coated SPA beads. This signal was abolished when any of the components were omitted and also by the addition of NaCl, which potently reduces the affinity of interaction between Ras and NF1. The neutralizing anti-Ras monoclonal antibody Y13-259 and the detergent n-dodecyl maltoside, a specific inhibitor of NF1 catalytic activity, both abolished the SPA signal from the NF1/Ras assay but neither affected a control SPA signal in which a [3H]GTP.GST-Ras fusion protein was bound to protein A-coated SPA beads. This technology could be readily extended to the measurement of other protein-protein interactions and could form the basis for high-throughput screens for the discovery of novel therapeutic agents.

Escherichia coli↗
PubMed1994

The Schwann cell precursor and its fate: a study of cell death and differentiation during gliogenesis in rat embryonic nerves.

We have characterized a cell, the Schwann cell precursor, that represents a distinct intermediate differentiation stage in the process by which Schwann cells are generated from neural crest cells. The Schwann cell precursor shows radical differences from Schwann cells which include death regulation, antigenic phenotype, pattern of cell-cell interaction, migratory behavior, and morphology. In the nerves of the rat hind limb, Schwann cells are irreversibly generated from these during a brief period, essentially embryonic days 15-17. We also provide evidence that the survival of Schwann cell precursors is regulated by neurons and identify basic fibroblast growth factor as a potential key regulator of apoptosis in Schwann cell precursors and of precursor to Schwann cell conversion. These findings have implications for our understanding of gliogenesis in the peripheral nervous system.

Animals↗
PubMed1994

Negative regulation of the P0 gene in Schwann cells: suppression of P0 mRNA and protein induction in cultured Schwann cells by FGF2 and TGF beta 1, TGF beta 2 and TGF beta 3.

During the development of peripheral nerves, Schwann cells are induced to form myelin sheaths round the larger axons. This process involves a complex series of events and the nature of the molecular signals that regulate and control myelin formation in Schwann cells is not well understood. Our previous experiments on rat Schwann cells in vitro, using serum-free defined medium, showed that a myelin-related protein phenotype could be induced in early postnatal Schwann cells in culture by elevation of intracellular cyclic AMP levels in the absence of growth factors, conditions under which the cells are not dividing. Cells with this phenotype expressed the major myelin glycoprotein P0 and expression of p75 NGF receptor, N-CAM, GFAP and A5E3 proteins was down-regulated. These changes are all characteristics associated with myelination in vivo. In contrast, when cyclic AMP levels were elevated in the presence of serum, suppression of cyclic AMP-induced differentiation resulted and DNA synthesis was induced. In this paper, we have used this model system and extended our analysis to explore the relationship between defined growth factors and suppression of myelination. We have used pure recombinant growth factors normally present in peripheral nerves, i.e. FGF1 and FGF2 and TGF beta 1, TGF beta 2, and TGF beta 3 and shown that, like serum, they can strongly suppress the forskolin-mediated induction of the P0 gene, both at the level of mRNA and protein synthesis. For both growth factor families, the suppression of P0 gene expression is dose-dependent and takes place in serum-starved cells that are mitotically quiescent. In the case of FGF2, however, even more complete suppression is obtained when the cells are simultaneously allowed to enter the cell cycle by inclusion of high concentrations of insulin in the culture medium. The present results raise the possibility that, in addition to the positive axonal signals that are usually envisaged to control the onset of myelination, growth factors present in the nerve may exert negative regulatory signals during development and thus help control the time of onset and the rate of myelination in peripheral nerves.

Animals↗
PubMed1994

Thyroid disease in the elderly. Part 1. Prevalence of undiagnosed hypothyroidism.

BACKGROUND: The purpose of this study was to examine the prevalence of previously unrecognized hypothyroidism in elderly patients. METHODS: The study was conducted in a primary care geriatrics clinic. Three hundred seventy elderly patients (287 women, 83 men) between 60 and 97 years of age were included in the study. Medical records of patients were reviewed retrospectively. Serum thyroid-stimulating hormone (TSH), free thyroxine (T4), height, weight, demographic variables, clinical signs and symptoms of hypothyroidism, history of thyroid diseases and treatment with thyroid medications, comorbidities, and current medications were obtained from the medical records. Patients who had both elevated TSH levels (5.0 to 14.9 microU/mL) and normal free T4 levels (0.7 to 2.0 ng/dL) met the criteria for "subclinical hypothyroidism." The criteria for "overt hypothyroidism" were TSH levels > or = 15 microU/mL and low free T4 levels (< 0.7 ng/dL). RESULTS: At the initial visit to the clinic, 18.1% of the patients (62 female and 5 male) had an established history of past or current thyroid disease. Another 20 women (5.4%) had a history of thyroid surgery. Of the remaining 283 patients with no history of thyroid disease, 14.6% of the women and 15.4% of the men had subclinical hypothyroidism. Overt hypothyroidism was discovered and subsequently treated in two female patients and one male patient (1.0% and 1.3%, respectively). Thyroid status was not significantly related to age group (60 to 64 years; 65 to 74; 75 to 84; 85 and older). Comorbidities typically associated with hypothyroidism were no more prevalent in hypothyroid patients than in euthyroid patients. CONCLUSIONS: We found a high prevalence of newly diagnosed subclinical hypothyroidism in both elderly male and female patients. Thyroid status was not related to age or to coexisting diseases. The clinical significance of treating subclinical hypothyroidism merits investigation.

Aged↗
PubMed1994

Thyroid disease in the elderly. Part 2. Predictability of subclinical hypothyroidism.

BACKGROUND: The purpose of this study was to determine how well hypothyroidism could be predicted from clinical signs and symptoms in elderly patients with no known history of thyroid disease. METHODS: This retrospective study was conducted in a primary care geriatrics clinic. Two hundred eighty-three patients (205 women, 78 men), who were between the ages of 60 and 97 years of age and had no history of thyroid disease, were included in the study. Medical records of patients were reviewed for data obtained at their first visit to the clinic: serum thyroid-stimulating hormone (TSH), free thyroxine (T4), height, weight, demographic variables, clinical signs and symptoms of hypothyroidism, previous history of thyroid disease and treatment with thyroid medications, and current medications. RESULTS: Of the 283 patients with no previous history of thyroid disease, 15.4% of men and 14.6% of women had subclinical hypothyroidism (TSH levels of 5.0 to 14.9 microU/mL, and normal free T4 levels [0.7 to 2.0 ng/dL]). Overt hypothyroidism (TSH > or = 15.0 microU/mL, low free T4 < 0.7 ng/dL) was discovered and subsequently treated in one male and two female patients. There were no significant differences (P > .05) in the frequencies of any of the clinical signs and symptoms of hypothyroidism between euthyroid and hypothyroid patients. There was no significant relationship between TSH levels and the total number of hypothyroid symptoms experienced by all patients (r = -.004, P = .99). Logistic regression analyses determined that clinical signs and symptoms were poor predictors of subclinical hypothyroidism in these elderly patients. CONCLUSIONS: Thyroid status could not be predicted from clinical signs and symptoms in this sample of elderly community-dwelling patients. This finding substantiates the difficulty of diagnosing subclinical hypothyroidism in the elderly based solely on clinical features.

Aged↗
PubMed1993

Kinetics of interaction between normal and proline 12 Ras and the GTPase-activating proteins, p120-GAP and neurofibromin. The significance of the intrinsic GTPase rate in determining the transforming ability of ras.

Single turnover and equilibrium binding measurements on the interaction of Gly-12 and Pro-12 Ras.GTP with the catalytic domains of the GTPase-activating proteins, p120-GAP and neurofibromin, have been made utilizing fluorescent 2'(3')O-(N-methylanthraniloyl)-nucleotides. These have enabled the equilibrium dissociation constants (Kd) for their initial binding and the rate constants of the hydrolysis step to be measured. p120-GAP binds to both Ras proteins with a Kd of 17 microM, whereas neurofibromin binds to both Ras proteins with a Kd of 1 microM. Both p120-GAP and neurofibromin increased the rate constant of the GTP hydrolysis step of Pro-12 Ras, but the maximal activation at 30 degrees C was 120-fold and 560-fold, as compared with 70,000- and 52,000-fold, with Gly-12 Ras. The affinity with which p120-GAP and neurofibromin binds to either Gly-12 or Pro-12 Ras protein was decreased dramatically by increasing ionic strength caused by addition of NaCl. The rate constant of the cleavage step of hydrolysis catalyzed by neurofibromin increases with increasing ionic strength, whereas that catalyzed by p120-GAP appears to be unaffected. The high ionic strength within the cell might result in a much lower overall GTPase-activating protein activity than is measured under conditions of low ionic strength in vitro, with p120-GAP being more severely inhibited. The GTP hydrolysis rate of Pro-12 Ras is 2-fold faster than that of normal Ras. The low oncogenicity of Pro-12 ras is explained by a model in which the intrinsic rates of hydrolysis and exchange, as well as GTPase-activating protein- and exchange factor-stimulated rates, are determinants of the biological activity of Ras proteins in fibroblasts.

Cell Transformation, Neoplastic↗
PubMed1993

Changes in DNA synthesis rate in the Schwann cell lineage in vivo are correlated with the precursor--Schwann cell transition and myelination.

During the development of the rat sciatic nerve extensive proliferation of glial cells occurs, and there is a very substantial rearrangement of the cytoarchitecture as axons and Schwann cells assume relationships which lead to the formation of the myelinated and unmyelinated axons characteristic of adult nerve. The maturation of Schwann cells from Schwann cell precursors and the matching of Schwann cell numbers to axons is an important part of this process. We have therefore studied the proliferation of Schwann cell precursors and Schwann cells during the development of the rat sciatic nerve from embryonic day 14 to postnatal day 28 by combining bromodeoxyuridine injections of rats with double-label immunohistochemical techniques. The results reveal that DNA synthesis occurs in both Schwann cell precursors and Schwann cells throughout early nerve development. The labelling index is already substantial at embryonic day 14, but from embryonic day 17, when essentially all the glial cells have converted from precursor to Schwann cell phenotype, it rises sharply, peaking between embryonic day 19 and 20 before declining precipitously in the early postnatal period. This rapid decline in DNA synthesis coincides with the appearance of the myelin protein P0, and in individual cells DNA synthesis is incompatible with the expression of P0 protein. Nonmyelin-forming Schwann cells, which mature later in development, continue to synthesize DNA until at least postnatal day 15, but by day 28 essentially all Schwann cells in the nerve are quiescent.

Animals↗