Colocalization of somatostatin receptor sst5 and insulin in rat pancreatic beta-cells.

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is secreted by pancreatic delta-cells and inhibits the secretion of both insulin and glucagon. SRIF initiates its actions by binding to a family of six G protein-coupled receptors (sst1, -2A, -2B, -3, -4, and -5) encoded by five genes. Messenger RNA for both sst2 and sst5 have been reported in the rat pancreas, and the sst2A receptor protein has been localized to rat pancreatic alpha and pancreatic polypeptide-secreting cells in the islets as well as to pancreatic acinar cells.

Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry.

Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland.

Synthesis and biological activities of potent peptidomimetics selective for somatostatin receptor subtype 2.

A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors.

Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase.

1,3-beta-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p.

The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.

In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounced slow-growth phenotype, (ii) hypersensitivity to FK506 and cyclosporin A, (iii) a slight increase in sensitivity to echinocandin, and (iv) a significant reduction in 1,3-beta-D-glucan synthase activity in vitro.

Calcineurin-dependent growth of an FK506- and CsA-hypersensitive mutant of Saccharomyces cerevisiae.

The immunosuppressants FK506 and cyclosporin A (CsA) bound to their receptors, FKBP12 or cyclophilin, inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, preventing T cell activation or, in yeast, recovery from alpha-mating factor arrest. Vegetative growth of yeast does not require calcineurin, and in strains sensitive to FK506 or CsA, growth is inhibited by concentrations of drug much higher than those required to inhibit T cell activation or recovery from mating factor arrest. We now describe the isolation of a mutant of Saccharomyces cerevisiae which is 100-1000-fold more sensitive to the growth inhibitory properties of these drugs.

Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola.

The gene (tutA) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed. The enzyme was expressed at high levels in E. coli in the presence of isopropyl-beta-D-thiogalactopyranoside or lactose as an inducer.

Cloning of a Streptomyces clavuligerus DNA fragment encoding the cephalosporin 7 alpha-hydroxylase and its expression in Streptomyces lividans.

A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7 alpha-hydroxylase (CH) of Streptomyces clavuligerus NRRL 3585 and used to screen a DNA library from this organism. The library was constructed in the lambda GEM-11 phage system. After plaque purification and reprobing, positive recombinant phages were chosen for further analysis.

Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast.

The structurally unrelated immunosuppressants FK506 and cyclosporin A (CsA) act similarly, inhibiting a Ca(2+)-dependent signal required for interleukin-2 transcription and T-cell activation. Each drug binds to its cytosolic receptor, FKBP-12 and cyclophilin, respectively, and the drug-receptor complexes inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In yeast, calcineurin has been implicated in recovery from alpha-mating factor arrest.

Vectors for generating nested deletions and facilitating subcloning G+C-rich DNA between Escherichia coli and Streptomyces sp.

New multiple cloning sites (MCS), which facilitate the subcloning of G+C-rich DNA, were added to pUC18, M13mp18, pVE616 (a pBR322-derived insertion vector), and the low-copy-number Streptomyces vector, pIJ922. The MCS in these vectors contain sites found infrequently in Streptomyces DNA, facilitating the exchange of subclones between the vectors. The MCS added to M13mp18 and pUC18 was also designed to generate nested deletions within subcloned fragments.

Yeast FKBP-13 is a membrane-associated FK506-binding protein encoded by the nonessential gene FKB2.

The immunosuppressants FK506 and rapamycin prevent T-cell activation and also inhibit the growth of certain strains of the yeast Saccharomyces cerevisiae. It has previously been shown that yeast contains a 12-kDa cytosolic FK506-binding protein (yFKBP-12), which also possesses peptidylprolyl cis-trans isomerase activity, and that fkb1 strains lacking yFKBP-12 are resistant to rapamycin and sensitive to FK506. The absence of yFKBP-12 permitted the detection and isolation of a second FK506- and rapamycin-binding protein, which is about 13 kDa in size (yFKBP-13) and membrane-associated.

Construction of a shuttle vector consisting of the Escherichia coli plasmid pACYC177 inserted into the Streptomyces cattleya phage TG1.

The Escherichia coli plasmid, pACYC177, was inserted into the single PstI site of a deletion derivative of the Streptomyces cattleya phage, TG1. The hybrid molecule can be propagated as a phage in S. cattleya and as a plasmid in E.

Isolation and characterization of the Streptomyces cattleya temperate phage TG1.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends.

Role of glnA-linked genes in regulation of glutamine synthetase and histidase formation in Klebsiella aerogenes.

We isolated mutants of Klebsiella aerogenes with insertions of transposon Tn5 linked to the structural gene for glutamine synthetase, glnA. We found that K. aerogenes, like Escherichia coli and Salmonella typhimurium, contains a regulatory gene, glnG, which is closely linked to but distinct from glnA.

Regulation of glutamine synthetase by regulatory protein PII in Klebsiella aerogenes mutants lacking adenylyltransferase.

A mutation of Klebsiella aerogenes causing production of an altered PII regulatory protein which stimulates overadenylylation of glutamine synthetase and also prevents its derepression was combined with mutations abolishing the activity of adenylyltransferase. The results support the idea that PII plays a role in the regulation of the level of glutamine synthetase which is independent of its interaction with adenylyltransferase.

Regulation of the synthesis of glutamine synthetase by the PII protein in Klebsiella aerogenes.

Certain mutations at the glaB locus result in the failure to fully derepress glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.

Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase.

A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E.

Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase.

The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA.

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K.

Regulation of synthesis of glutamine synthetase by adenylylated glutamine synthetase.

We have examined three mutants of Klebsiella aerogenes whose genetic lesions (glnB, glnD, and glnE) are in loci unlinked to the structural gene for glutamine sythetase (glnA) and in which the control of both the level and state of adenylylation of glutamine synthetase is altered. Each mutation alters a different component of the adenylylation system of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.

Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli.

An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E. coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A.