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Biomedical subjects

L A Graham

Publications and source records attributed to L A Graham.

8 recordsLinked to original sources
PubMed1992

Deletion of subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex specifically impairs electron transfer at the ubiquinol oxidase site (center P) in the bc1 complex.

Deletion of QCR9, the nuclear gene encoding subunit 9 of the mitochondrial cytochrome bc1 complex in Saccharomyces cerevisiae, results in inactivation of the bc1 complex and inability of the yeast to grow on non-fermentable carbon sources. The loss of bc1 complex activity is due to loss of electron transfer activity at the ubiquinol oxidase site (center P) in the complex. Electron transfer at the ubiquinone reductase site (center N), is unaffected by the loss of subunit 9, but the extent of cytochrome b reduction is diminished. This is the first instance in which a supernumerary polypeptide, lacking a redox prosthetic group, has been shown to be required for an electron transfer reaction within the cytochrome bc1 complex.

Cytochrome b Group↗
PubMed1991

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.

Amino Acid Sequence↗
PubMed1991

Rat sublingual gland as a model to study glandular mucous cell secretion.

To study the regulation of mucous cell secretion, we have developed an in vitro cell model consisting of enzymatically dispersed mucous acinar structures (cell aggregates) from rat sublingual glands. Histological and ultrastructural evidence demonstrates that the cell aggregates are highly enriched in mucous cells, retain the morphological and ultrastructural features observed in intact glands, and undergo transition to an extensive secretory state when stimulated by 10 microM carbachol. The secretory responsiveness of the cell aggregates was verified in pharmacological studies. Carbachol stimulated secretion in a dose-dependent manner with high affinity (concentration causing half-maximal response = 0.3 microM) and was completely inhibited by atropine. Secretion was also stimulated by vasoactive intestinal peptide and substance P but not by alpha- or beta-adrenergic agonists. Biochemical characterization of secretion during nonstimulated and carbachol-stimulated conditions (after preincubation in [3H]glucosamine) demonstrated that, in response to carbachol, cell aggregates synthesized and secreted mucins which were similar to mucin glycoproteins isolated from whole glands. Collectively, our results establish that the rat sublingual cell aggregate model is a viable and pharmacologically responsive cell system to study the regulation of mucous cell secretion.

Amino Acids↗
PubMed1990

Alpha-adrenergic regulation of secretion by tracheal glands.

The purpose of the present study was to begin to characterize, pharmacologically, the alpha-adrenergic regulation of glycoconjugate secretion from airway glands. Using isolated gland cells from cat trachea, we determined the binding characteristics of [3H]dihydroergocryptine ([3H]DHE), an alpha-adrenergic antagonist, with equal affinities for alpha 1- and alpha 2-adrenergic receptors. Specific binding of [3H]DHE to gland cell homogenates was saturable, of high affinity (KDapp = 4.2 nM) and inhibited with greater efficacy by epinephrine much greater than isoproterenol. Competition experiments with alpha 1- and alpha 2-adrenergic selective antagonists (prazosin and yohimbine, respectively) demonstrated high- and low-affinity sites for each antagonist, indicating the presence of both receptor subtypes. In studies of glycoconjugate secretion by cat tracheal explants, secretion was stimulated by adrenergic agonists with the rank potency: norepinephrine greater than or equal to phenylephrine greater than epinephrine much greater than clonidine. alpha-Adrenergic-stimulated secretion (epinephrine + propranolol) was inhibited by low concentrations of prazosin, but was unaffected by 100 nM yohimbine. The alpha 2-adrenergic agonists, clonidine and UK-14,304, each markedly inhibited beta-adrenergic-stimulated secretion. Collectively, these results demonstrate alpha 1-adrenergic regulation of glandular glycoconjugate secretion and suggest alpha 2-adrenergic receptors may modulate beta-adrenergic-stimulated secretion.

Animals↗
PubMed1975

Absence of facilitated glucose disposal (Staub-Traugott effect) in subjects with hypopituitarism.

Improved glucose tolerance follows glucose challenges given in rapid succession, the Staub-Traugott effect. The cause for this facilitated glucose disposal is not clear. Augmented insulin release, prior "insulinization" of cells, and suppression of a pituitary factor or free fatty acids (FFA) are previously suggested mechanisms. For information bearing on the role of the pituitary in this phenomenon, study of the Staub effect was undertaken in hypopituitary patients receiving replacement thyroid, cortisone, and sex-steroid therapy and in normal untreated controls. All subjects received three intravenous injections of glucose (0.5 g/kg) at hourly intervals. Plasma glucose, FFA, and insulin were measured. Whereas a definite Staub effect was seen in each control subject, this phenomenon was conspicuously absent in seven hypopituitary patients similarly studied. Patterns of peripheral insulin response were similar for both groups: FFA levels fell more slowly in the hypopituitary subjects. Normal pituitary function appears to be required for the Staub effect. Incremental peripheral insulin levels do not explain the effect. Subnormal suppression of free fatty acids and impaired induction of key glycolytic and glycogenic enzymes are alternative explantations for the absence of the Staub effect in hypopituitary subjects.

Aged↗