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W S Oetting

Publications and source records attributed to W S Oetting.

46 records · Page 3Linked to original sources
PubMed1991

Non-random distribution of missense mutations within the human tyrosinase gene in type I (tyrosinase-related) oculocutaneous albinism.

Type I oculocutaneous albinism (OCA) is produced by mutations of the tyrosinase gene. We report four new missense mutations in the tyrosinase gene in patients with type IA OCA. Three of these mutations occur within exon I and the fourth mutation within exon IV. Analysis of the distribution of these four missense mutations and 12 previously reported missense mutations shows that most cluster in four areas of the gene. Two clusters involve the copper A and copper B binding sites and could disrupt the metal ion-protein interaction necessary for enzyme function. The other two clusters are in exon I and exon IV and could represent important functional domains of the enzyme. We conclude that analysis of the tyrosinase missense mutations will provide insight into the structure-function relationship of this enzyme.

Albinism, Oculocutaneous↗
PubMed1989

Production and secretion of the 21-23.5 kDa prolactin-like molecules.

We recently described the presence of a series of prolactin (PRL)-like molecules (PLMs) in the rat pituitary gland and showed that their formation was not due to artifactual proteolysis of 24 kDa PRL during extraction or to degradation of PRL in lysosomes. In this study we have found (1) in vitro translation of pituitary cell RNA to result in the production of only 24 kDa monomer isoform 2 and no PLMs, (2) that secretion of newly synthesized PLMs is differently regulated than at least a proportion of newly synthesized monomers, (3) that secretion of newly synthesized PLMs occurs after at least a 6 h delay, (4) that cysteamine (a) inhibits the release of the PLMs, (b) causes an increase in their amount versus isoform 2, and (c) causes an intracellular accumulation of pleiomorphic, immature secretory granules, and (5) that cells grown under degranulating culture conditions do not contain PLMs. These results, using normal anterior pituitary cells in primary culture, demonstrate the potential for differential release of the PLMs versus monomer PRL in vivo and are consistent with the production of the PLMs from 24 kDa monomer isoform 2 during secretory granule condensation.

Animals↗
PubMed1986

Phosphorylation of prolactin.

Rat prolactin exhibits microheterogeneity when examined in electrophoretic systems, running as three isoforms having the same molecular weight but different net charges (prolactins 1, 2, and 3 with isoform 3 being the most acidic). As there is precedent for the phosphorylation of a pituitary hormone and phosphorylation is a common cause of microheterogeneity, we examined the possibility that rat prolactin existed in differentially phosphorylated forms. The investigation included examinations of rat prolactin phosphorylation both in vitro and in vivo. For the in vitro studies, purified rat prolactin was incubated with [gamma-32P]ATP and low levels of each of five purified protein kinases. Phosphorylated rat prolactin was identified by autoradiography of silver-stained one- and two-dimensional gels. For the in vivo studies, rat anterior pituitary cells in primary culture were incubated in the presence of H3 32PO4 for 2 or 12 h, after which time the proteins were extracted from the cells, cold acetone-precipitated, or immunoprecipitated and run on two-dimensional gels. We report the in vitro phosphorylation of rat prolactin by cAMP-dependent protein kinase, casein kinase I, protease-activated kinase I, and the calcium/phospholipid-dependent kinase, that phosphorylation with these kinases results in phosphate incorporation only into isoforms 2 and 3, and the phosphorylation of prolactin in rat pituitary cells in primary culture.

Animals↗
PubMed1986

Differential isoform distribution between stored and secreted prolactin.

PRL exists within the mammotroph population in a number of different molecular forms. Three of these forms are best described as isoforms, as they have the same mol wt (24K) but differ in their net molecular charges. In this study we have examined the relative proportions of newly synthesized isoforms found in stored (intracellular) vs. secreted (extracellular) PRL. Dissociated cells from female rat anterior pituitaries were cultured for 48 h and then incubated in [35S]methionine (6 h; 37 C). Intracellular and medium proteins were then extracted and resolved by one- and two-dimensional polyacrylamide gel electrophoresis, followed by silver staining or autoradiography. Control experiments, in which biosynthetically labeled PRL was re-extracted, ensured that the isolation conditions did not in themselves promote isoform interconversion. The relative proportions of the PRL isoforms were determined by densitometric scanning of developed autoradiograms. In the cell extracts, the relative proportions were 13.6 +/- 2.1% isoform 1 (least negatively charged), 71.5 +/- 3.26% isoform 2, and 14.7 +/- 1.9% isoform 3 (most negatively charged). In the medium, the relative proportions were 60 +/- 2.89% isoform 1, 20 +/- 1.75% isoform 2, and 11 +/- 1.14% isoform 3. When the labeling was performed in the presence of 0.5 mM cysteamine (an agent we show to distinguish between newly synthesized and older stored hormone and, hence, between the previously described functional subpopulations of mammotrophs), the same ratios of newly synthesized isoforms were secreted from the cells. We conclude that secretion of the different isoforms is more complex than simple proportional release of each form, and based on the cysteamine results, this nonproportional release cannot be attributed to release of one isoform per functional subpopulation of mammotrophs.

Animals↗
PubMed1985

C pigment locus mutants of the fowl produce enzymatically inactive tyrosinase-like molecules.

Three albino mutants of the fowl were tested for tyrosinase activity. Two of these mutants (c and ca) are alleles at the autosomal C locus, while the third mutant (sal) is sex-linked. Both the standard type, E, and sal are tyrosinase positive whereas the two C mutants are tyrosinase negative. Anti-chicken tyrosinase mouse serum was produced and all four genotypes were found to have cross-reacting material to this antiserum. Tyrosinase from the standard type was isolated and its location on denaturing two-dimensional gels determined. A co-migrating series of spots was found within the protein pattern of both the standard type and the tyrosinase positive albino, sal. The same pattern of spots was also observed for c and ca with no apparent change in either the pI or the molecular weight. Transmembrane blots also showed spots that reacted with anti-tyrosinase serum in all four genotypes and that migrated to the same location as that of standard tyrosinase. It is proposed that both c and ca are CRM+ mutants which produce tyrosinase-like molecules that are inactive due to a change that is electrophoretically and antigenically "silent".

Alleles↗
PubMed1985

Intracellular processing of prolactin.

PRL has been reported to exist as a number of mol wt and charge variants. We have attempted to determine the relationships among the charge and lower mol wt variants by two-dimensional gel analysis of pituitary and pituitary cell extracts. Silver-stained gels of the extracts showed three major charge isoforms of 24,000 mol wt PRL (PRLs 1, 2, and 3, with PRL 3 being the most anodic) and what appeared to be an arc of products originating in the region of PRL 3. Each spot in the arc was distinct and represented a small decrease in size (two to eight residues), with a corresponding increase in net negative charge. When primary cell cultures were labeled with [35S] methionine, the three PRL isoforms and arc products were detectable by autoradiography. Western blots of the two-dimensional gels showed the arc products to be immunologically related to PRL. Treatment of the cell cultures with hydroxynorvaline (5 mM), which inhibits processing of pre-PRL to PRL, resulted in doublet spots in the arc. Elimination of protease inhibitors or an increase in temperature during protein isolation had no effect on the relative concentrations of PRL and arc products. Inclusion of standard PRL or [125I]iodo-PRL in the extraction solution did not increase the size of the spots in the arc or produce labeled arc products, respectively. Treatment of the cell cultures with chloroquine (10(-5) M) before and during radiolabeling had no effect on the production of radiolabeled arc products. Analysis of cell culture medium showed at least some of the arc products to be secreted. We conclude that PRL or a PRL-like molecule is processed intracellularly into a number of smaller derivatives. As the arc products accumulate within the cell and are secreted, we suggest that they may be biologically important relatives of PRL.

Animals↗
PubMed1984

Mammotroph autoregulation: uptake of secreted prolactin and inhibition of secretion.

A dissociated preparation of normal adult rat pituitary cells has been used to study PRL autoregulation at the level of the mammotroph . Female rat pituitary cells previously cultured for 48 h on polylysine-coated petri dishes were washed to remove serum and accumulated PRL and then incubated in fresh medium in the absence or presence of increasing concentrations of rat PRL. Accurate balance sheets, allowing for degradation and nonspecific adsorption of PRL, showed exogenous PRL to regulate the amount of PRL released by the cells. That this regulation was partly produced by uptake of secreted PRL from the medium was demonstrated by supplementing the medium with [125I]iodo-rat PRL. Inhibition of secretion also played a role and was implied by experiments showing that ease of reversal of the inhibition was inversely proportional to the density of cell culture, which was itself proportional to the amount of PRL in the medium and the duration of autoregulation. These results indicate that normal adult rat pituitary cells in primary culture are capable of regulating the amount of PRL in their external milieu and that uptake of already secreted PRL is an important component of the regulatory mechanism.

Animals↗
PubMedDate not supplied

Molecular analysis of an extended Palestinian family from Israel with monilethrix.

We describe the molecular analysis of a large three generation Palestinian family segregating for monilethrix. Previous reports have shown that mutations in type-II hair cortex keratin genes, hHb1 and hHb6, are associated with monilethrix. Genetic linkage analysis performed on this family using markers flanking the hHb6 gene exhibited strong evidence for linkage. Sequence analysis revealed a nucleotide substitution of G --> T at nucleotide 1230 resulting in a glutamic acid to aspartic acid amino acid substitution at codon 410, identical to that reported in a French family. The family in our study provides further evidence that mutations of the hHb6 gene are responsible for monilethrix.

Amino Acid Sequence↗