Deactivation ofCandida rugosa lipase

Summary Deactivation ofCandida rugosa lipase was found to be complex. Hydrophobic interaction induced by iso-octane influenced the initial phase of deactivation, and increased the turn-over rate of the intermediate in the transition phase. After urea-treatment the structure of the last phase was not further influenced by thermal treatment, whereas that of initial phase was more sensitive to temperature change. Charge interaction was important in maintaining the structure during the deactivation, and especially anion charge might be a major factor.     

Cleavage of territrems by alkaline hydrogen peroxide: Isolation and characterization of the aromatic moiety of territrems

The chemical reaction of cleavaging territrem B to give 3,4,5-trimethoxy benzoic acid by alkaline hydrogen peroxide was investigated. The method was applied for confirmation of the chemical structure of the aromatic moiety of territrem A, A’, B, and B’. The physicochemical properties of the aromatic cleavage product of territrem Aindicated the structure as 3,4-methylendioxy, 5-methoxy benzoic acid (or 4-methoxy, 6-carboxy, 1, 3-benzodioxole). The experiment also gave the evidences that territrem A and A’, on the other hand territrem B and B’ have the identical aromatic moieties on their structures.     

Human immunodeficiency virus neutralizing antibodies recognize several conserved domains on the envelope glycoproteins.

Serum neutralizing antibodies against the human immunodeficiency virus were frequently detected in infected individuals, and low or absent serum neutralizing titers correlated with poor prognosis. Multiple diverse human immunodeficiency virus isolates were found to exhibit similar susceptibility to neutralization by a panel of human seropositive sera, suggesting that neutralizing antibodies are largely directed against conserved viral domains. Furthermore, utilizing antisera raised against a library of synthetic env peptides, four regions which are important in the neutralization process have been identified within both human immunodeficiency virus envelope glycoproteins (gp41 and gp120). Three of these are in conserved domains and should be considered for inclusion in a candidate vaccine.     

The pancreatic glucagon and C-peptide secretion during hyperinsulinemia in euglycemic glucose clamp with or without somatostatin infusion in normal man

6 normal subjects received two times of 2 hr euglycemic glucose clamp studies (insulin infusion rate 40 mU/M2/min) one with and the other without somatostatin (SRIF) infusion (500 microgram/hr). Serum C-peptide and glucagon levels were measured during clamp to study the sensitivity of pancreatic alpha and beta cells to the suppressive effects of exogenous hyperinsulinemia during normoglycemia in normal subjects and to find whether SRIF had any modulative effects on endocrine pancreas secretion at the status of hyperinsulinemia. The results showed that in normal man the degree of suppression of pancreatic glucagon secretion by hyperinsulinemia (approximately 100 uU/ml) during euglycemic glucose clamp without SRIF infusion was less than that of C-peptide with mean value of 62 +/- 4% of basal glucagon remained at the end of clamp study; while only about 30 +/- 2% of basal C-peptide concentrations remained. But during SRIF infused glucose clamp studies (SRIF was infused from 60 to 120 min), 32 +/- 2% of mean basal C-peptide concentrations and 38 +/- 6% of mean basal glucagon concentrations left at the end of 2 hr clamp studies when serum insulin level was about 100 uU/ml. For the glucose infusion rate (M value), it was significantly greater in our normal subjects in response to insulin + SRIF as compared to insulin alone (12.0 + 0.9 vs 8.8 +/- 1.4; P less than 0.01). We concluded: during hyperinsulinemia (100 uU/ml), the sensitivity of pancreatic alpha cells to insulin seems less than that of beta cells in normal man at normoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tn4556, a 6.8-kilobase-pair transposable element of Streptomyces fradiae.

A 6.8-kilobase-pair (kbp) transposable element (Tn4556) was found in a neomycin-producing strain of Streptomyces fradiae. This element was first observed in two 30.3-kbp plasmids (pUC1123 and pUC1124) which arose when a thiostrepton resistance gene (1 kbp) was ligated with the BclI-2 fragment (22.5 kbp) that contains the origin of replication of phage SF1. The Tn4556 segment was deleted when these plasmids were transduced into another S. fradiae host with phage SF1. These deletion plasmids (pUC1210 and pUC1211) had copy numbers of less than 1 per chromosome and were unstable. In contrast, pUC1123 and pUC1124, with copy numbers of 12 to 15 per chromosome, respectively, were relatively stable. When pUC1210 and pUC1211 were reintroduced into S. fradiae by protoplast transformation, the Tn4556 element transposed again to the plasmids at numerous new locations in either of two orientations. A copy of Tn4556 was found in the S. fradiae chromosome by hybridization studies. It appears that Tn4556 originated from the chromosome, transposed into unstable pUC1210 and pUC1211, and made stable plasmids. A temperature-sensitive hybrid plasmid carrying a viomycin resistance derivative of Tn4556 (pMT660::Tn4556::vph) was constructed. When Streptomyces lividans UC8390 containing the hybrid plasmid was grown at 39 degrees C, Tn4556::vph (Tn4560) transposed to random positions in the host chromosome.     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.     

Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.