Thermostable β-Glycosidase from Sulfolobus Solfataricus

The Sulfolobus solfataricus β-glycosidase (Sβgly) is a thermostable and thermophilic glycosyl-hydrolase with broad substrate specificity. The enzyme hydrolizes β-D-gluco-, fuco-, and galactosides, and a large number of /Winked glycoside dimers and oligomers, linked β1-3, β1-4, and β1-6, It is able to hydrolize oligosaccharides with up to 5 glucose residues. Furthermore, it is also able to promote transglycosylation reactions. The corresponding gene has been cloned and overexpressed both in yeast and Escherichia coli. Based on sequence and functional data, the Sβgly has been assigned to the so-called BGA family of glycosyl-hydrolases, including β-glycosidases, β-galactosidases and phosho-β-galactosidases from mesophilic and thermophilic organisms of the three domains. The Sβgly has been crystallized and the resolution of its structure is in progress. Because of its special properties, the enzymes has considerable biotechnological potential.     

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex.

The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.     

A karyotype analysis of the genus Dicentrarchus by different staining techniques

This study presents a cytogenetic analysis of the genus Dicentrarchus , represented by two species, D. labrax and D. punctatus . The karyotypes are very similar, even after staining with different techniques. Both species show 48 subtelocentric and acrocentric chromosomes, gradually decreasing in size. One pair of small size chromosomes has heteropycnotic and heteromorphtc short arms of longer size. These short arms are C- and Ag-positive, i.e. nucleolar organizer regions, NORs, are located there. Constitutive heterochromatin is also evident as a subcentromeric band on the long arms of a large chromosome pair. CMA₃-staining confirms the location and the heteromorphism of NORs. DAPI and quinacrine produce homogeneous staining of chromosomes. A review of cytogenetic studies on 'serranid' species is also presented.     

Expression of conformationally constrained adhesion peptide in an antibody CDR loop and inhibition of natural killer cell cytotoxic activity by an antibody antigenized with the RGD motif.

We report that an antibody engineered to express three Arg-Gly-Asp (RGD) repeats in the third complementarity-determining region of the heavy chain (antigenized antibody) efficiently inhibits the lysis of human erythroleukemia K-562 cells by natural killer (NK) cells. Synthetic peptides containing RGD did not inhibit. Inhibition was specific for the (RGD)3-containing loop and required simultaneous occupancy of the Fc receptor (CD16) on effector cells. The antigenized antibody inhibited other forms of cytotoxicity mediated by NK cells but not cytotoxicity mediated by major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL). A three-dimensional model of the engineered antibody loop shows the structure and physicochemical characteristics probably required for the ligand activity. The results indicate that an RGD motif is involved in the productive interaction between NK and target cells. Moreover, they show that peptide expression in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. This is a new approach towards imparting ligand properties to antibody molecules and can be used to study the biological function and specificity of short peptide motifs, including those involved in cell adhesion.     

Modulation by glycine on vascular effects of NMDA:in vivo experimental research

Summary The present study has been carried out to determine if glycine, an allosteric modulator of NMDA receptor, is involved in the vascular effect induced by the activation of the CNS NMDA receptors.Icv NMDA (from 0.01 to 1µg/rat in the 3rd ventricle) caused a significant increase in arterial blood pressure in conscious freely moving rats. Moreover, the hypertension was associated with behavioural modifications (jumping, rearing, teething and running). Glycine pretreatment (1 and 10µg/raticv), significantly increased the NMDA hypertension. Glycine alone did not cause any arterial blood pressure modification while it induced a slight sedation. HA-966 (an antagonist of the glycine site on NMDA receptor) administration (1–10µg/raticv 5 min before glycine) significantly antagonized the glycine effects on NMDA hypertension.Alone HA-966 neither modified arterial blood pressure nor antagonized NMDA hypertension. In conclusion, our investigations confirm NMDA receptor involvement in cardiovascular function and they demonstrate thatin vivo glycine positively modulates NMDA receptors.     

Liquid chromatographic assay for a butenolide endothelin antagonist (PD 156707) in plasma

A sensitive and selective liquid chromatographic assay for determining the non-peptide endothelin A receptor antagonist PD 156707 (I) in rat plasma has been developed and validated. The analyte was isolated from matrix by solid-phase extraction. Liquid chromatographic separation was achieved isocratically ona 3.2 mm I.D., ODS column with a mobile hase of acetonitrile-ammonium phosphate (50 mM, pH 3.5) (44:56, v/v). Column effluent was monitored fluorometrically. Peak-height ratios (analyte/IS) were proportional to I concentrations in rat plasma from 25 to 1000 ng/ml. Assay precision and accuracy for I, based on quality controls, was 9.5% relative standard deviation, with relative error of ±6.5%. The quantitation limit was 25 ng/ml for a 200-μl sample aliquot.     

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.     

Two independent pathways of expression lead to self-assembly of the rabbit hemorrhagic disease virus capsid protein.

The rabbit hemorrhagic disease virus capsid protein was expressed in insect cells either as an individual protein species, from a mRNA analogous to the viral subgenomic RNA, or as part of a polyprotein that included the viral 3C-like protease and the RNA polymerase. Both pathways of expression led to the assembly of viruslike particles morphologically and antigenically similar to purified virus.     

Role of fungal patchiness on vegetal detritus in the trophic interactions between two brackish detritivores,Idotea baltica and Gammarus insensibilis

We investigated patterns of resource partitioning between two brackish crustaceans (I. baltica and G. insensibilis) on trophic mosaics of fungal species colonizing vegetal detritus. Laboratory feeding experiments were carried out to assess consumption rates and diet selection of single individuals belonging to five populations. Adults of two co-occurring population of both G. insensibilis and I. baltica and one population of G. insensibilis, occurring alone in another habitat patch, were studied. Each individual were offered both fragments of Cymodocea nodosa conditioned by 8 fungal species and sterilized fragments as sole food source for 7 days. Both species preferred "conditioned" detritus but potential resource use was greater in G. insensibilis than in I. baltica. Individual niche breadth increased and phenotypic variability reduced in the allotopic sample. Trophic similarities were lower between individuals of co-occuring populations than between I. baltica and the allotopic G. insensibilis. The results suggest that co-occurrence is an important factor contributing to the enhancement of phenotypic variability and, consequently, to a trophic generalization at population level in G. insensibilis. It emphasizes the role of fungal patchiness on detritus in regulating resource partitioning between the two species.