An efficient enzymatic preparation of 4(S)-hydroxy-1 (R)-acetoxy-cyclopent-2-ene by using new yeast isolate

Summary The Enzymatic enantioselective hydrolysis of prochiral 1,4-cyclopent-2-ene diacetate (3) was carried out using yeast and fungal cultures from inhouse culture collection. Of all the cultures tested, the yeast sp. NCIM 3574 gave 4 (S)-hydroxy-1 (R)- acetoxy-cyclopent-2-ene (4b) in high optical yields (99% ee).     

Separation of a complete set of basic, diastereomeric pentapeptides by reversed-phase ion-pair chromatography

The resolving power of liquid chromatography systems containing 1-pentane sulfonate, previously used to analyze basic hydrophilic peptides related to substrates of cAMP-dependent protein kinase, has been studied with respect to diastereomeric peptides. A set of peptides comprising RRASV and its five diastereomers containing one D-amino acid were used as model compounds. Complete resolution of all the peptides could be accomplished both with ethanol and acetonitrile as organic modifiers. The separation of the various peptides within the set turned out to be only modestly changed under the conditions investigated. These new results clearly demonstrate the potential of the chromatography systems studied.     

Plasma selenium-dependent glutathione peroxidase. Cell of origin and secretion

Human plasma glutathione peroxidase (GSHPx) has been shown to be a glycosylated selenoprotein distinct enzymatically, structurally, and antigenically from known cellular glutathione peroxidases. The extracellular location of the enzyme and the fact that it is glycosylated suggested that it is a secreted protein. Utilizing mutually non-cross-reactive antibodies to human cellular and plasma GSHPx, we conducted a search to determine the tissue of origin for plasma GSHPx. The cells screened were endothelial cells because they are the main source of extracellular superoxide dismutase, HL-60 cells (myeloid cell line) because they are the main source of extracellular H2O2, and Hep G2 cells (hepatic cell line) because they are the source of many plasma proteins. Human umbilical vein endothelial cells were metabolically labeled with either [35S]methionine or [75Se]selenious acid, and HL-60 cells and Hep G2 cells were metabolically labeled with [75Se]selenious acid. Proteins were immunopurified from the labeled cells and their media with either anti-red blood cell (RBC) GSHPx IgG or with anti-plasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, only the cellular form of the enzyme was precipitated from all the cells tested but not from their media. When anti-plasma GSHPx IgG was applied to the cells and their media, a selenoprotein was precipitated only from the media of Hep G2 cells. When Hep G2 cells were incubated in the presence of the carboxylic ionophore monensin, an intracellular selenoprotein could be detected using anti-plasma GSHPx IgG. The precipitation of the cellular form from all three cell types was partially inhibited by preincubation of the anti-RBC GSHPx IgG with purified RBC GSHPx while the precipitation of the selenoprotein from the medium of Hep G2 cells by anti-plasma GSHPx IgG was prevented by preincubation of the antibody with purified plasma GSHPx. We suggest that plasma GSHPx is synthesized by and secreted from hepatic cells. This is, to the best of our knowledge, the only known selenoprotein with a defined function that has been shown to be synthesized for secretion by mammalian cells.     

Daily fluctuations of titratable acidity, content of organic acids (malate and citrate) and soluble sugars of varieties and wild relatives of Ananas comosus L. growing under natural tropical conditions

The genus Ananas has its centre of origin in northern South America. In this area, several varieties of Ananas comosus are widely cultivated, and a number of wild species are found growing under variable conditions of light intensity, soil fertility and water availability. Here we report detailed daily courses of titratable acidity, and malate, citrate and free-sugars content of several cultivated varieties of A. comosus and of A. ananassoides, a closely related species growing on granitic rock-outcrops in southern Venezuela. Day-night oscillations of both malate and citrate were detected in plants growing under full sun, but malate was by far the most important organic anion associated with CAM performance in ail populations sampled. Fructose was the dominant compound in the neutral fraction, but only sucrose showed a consistent inverse relation with the cycle of titratable acidity. The diel oscillations of free sugars measured were not always enough to account for the amount of organic anions accumulated during the night. Plants cultivated under shady conditions always showed a lower night-time increase in titratable acidity and organic acids, and also smaller oscillations in the amount of free sugars than sun exposed plants. In all populations growing under full sun, osmolality increased during the night, but it was not always possible to explain these changes on the basis of variations in molar concentrations of organic acids and sugars. Besides, no diel variations in the cations K⁺, Ca²⁺ and Mg²⁺ were detected. K⁺ was always the dominant cation (K/Ca ratios ～ 19), while Mg²⁺ was always higher than Ca²⁺ (Mg/Ca ～ 2).     

Nonhyperbolic kinetics of Na,K-ATPase--a new viewpoint]

The kinetics of the 130 kDa monomer obtained by treatment of duck salt gland Na,K-ATPase with C12E8 was compared with that of the membrane-bound enzyme. The shapes of the substrate-velocity curves for the membrane-bound and solubilized forms were quite different: a hyperbolic one for the monomeric Na,K-ATPase and a nonhyperbolic one for the native enzyme. A reaction scheme for ATP hydrolysis based on a comparative analysis of kinetic properties of these two forms is proposed. Experimental evidence in favour of this hypothesis is presented.     

Effect of left atrial pressure on bronchial vascular hemodynamics

We studied the bronchial vascular response to downstream pressure elevation by increasing left atrial pressure (Pla) and mean airway pressure (Paw) with positive end-expiratory pressure (PEEP). In seven pentobarbital-anesthetized ventilated sheep, we cannulated and perfused the bronchial branch of the bronchoesophageal artery. Steady-state bronchial artery pressure- (Pba) flow (Qba) relationships were obtained as Pla was increased by inflating a balloon catheter in the left atrium. Bronchial vascular resistance (BVR), determined by the inverse slope of the Pba-Qba relationship, increased significantly from 3.2 +/- 0.3 (SE) mmHg.ml-1.min-1 at a Pla of 2.9 +/- 0.7 mmHg to 5.1 +/- 0.5 mmHg.ml-1.min-1 at a Pla of 20.1 +/- 2.0 mmHg (P = 0.0007). Under control Qba (23.3 +/- 1.2 ml/min), these changes in BVR represent a 3.6 +/- 0.7-mmHg increase in Pba per mmHg increase in Pla. The zero-flow pressure increased 1.3 +/- 0.2 mmHg/mmHg increase in Pla. After infusion of papaverine, a smooth muscle paralytic agent, directly into the bronchial artery, BVR decreased significantly to 1.3 +/- 0.7 mmHg.ml-1.min-1 (P = 0.0004). Under these dilated conditions, BVR was unaltered by increases in Pla. After papaverine administration, Pba increased 0.9 +/- 0.1 and 1.2 +/- 0.1 mmHg/mmHg increase in Pla during control and zero-flow conditions, respectively. Thus the effect of Pla elevation on BVR appears to be dependent on active smooth muscle responses. Paw elevation had similar effects on Pba. Under control Qba, Pba increased 2.2 +/- 0.4 mmHg/mmHg increase in Paw.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repression of lipopolysaccharide biosynthesis in Escherichia coli by an antisense RNA of Acetobacter methanolicus phage Acm1

Lysogenic Acetobacter methanolicus strains carrying the prophage Acm1 were found to be unable to synthesize both the capsutar polysaccharide (CPS) and the O-specific side-chain of lipopolysaccharide (LPS) and to represent rough variants of the host bacterium. A 262 bp DNA fragment of phage Acm1, obviously required for interference with LPS biosynthesis, was cloned and expressed in Escherichia coli Independently of the O-type, transformation of various E. coli strains with the recombinant DNA resulted in a suppression of biosynthesis of the O-specific chains. The DNA fragment of phage Acm1 contained three very short open reading frames of 21, 24, and 36 bp. However, attempts to express phage-encoded peptides were not successful. Instead, the Acm1-derived DNA fragment was shown to code for the synthesis of a trans-acting RNA molecule of 97 nucleotides, designated lbi (L PS b iosynthesis-i nterfering) RNA. This RNA contains sequence complementarity to E. coli target RNA sequences and appears to have the ability to form intracellularly RNA hybrid duplexes with mRNA. The data presented in this study support the hypothesis that the phenotypic effect of conversion to rough-type LPS is accompanied by the expression of an antisense RNA of phage Acm1.     

Rapid determination of members of the family Enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen.

An immunological method for the detection of members of the family Enterobacteriaceae in drinking water was developed. The method was based on a sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody immunoglobulin G2a 898 against enterobacterial common antigen. The enterobacterial common antigen sandwich ELISA combined with selective preenrichment culture could be performed in only 24 h. Six hundred sixty-eight water samples from a variety of German public water supplies were screened to verify the effectiveness of the new method. Ninety-eight percent of the results obtained by the immunological method could be confirmed by conventional microbiological methods. The immunological method proved to be considerably faster and more specific and sensitive than the standard method specified by the German drinking water regulations.