Effect of Erythropoietin on the interaction of Concanavalin A with rat erythrocytes

Effect of Erythropoietin (Ep) on the interaction of Concanavalin A (Con A) with rat erythrocytes was studied using ¹²⁵I-labelled Con A. Binding of Con A to erythrocytes was dependent on time and cell concentration. Starvation caused an elevation of the lectin binding capacity of red cells which again came down towards the normal level on Ep administration to starved rats. Binding of Con A to erythrocytes decreased linearly with increasing concentration of Ep. Specificity of binding was confirmed by inhibition studies with -methyl-D-mannopyranoside (Me Man) Cells from the starved rats compared to those from normal and Ep treated animals were less prone to inhibition by this sugar analog. Positive cooperative binding of Con A to rat erythrocyte was observed at low concentration of Con A but was absent at higher lectin concentrations. Starvation caused an increase in the number of binding sites per cell which returned to normal level after Ep treatment. Under identical conditions, binding affinities were not much changed in these cells. Cells from the starved animals were more susceptible to agglutination compared to those from normal and Ep-treated rats. Microviscosity and cholesterol/phospholipid ratio of red cell membrane decreased in the starved animals which retraced its way back towards the normal level after Ep treatment.     

Analysis of codon usage in genes for nitrogen fixation from phylogenetically diverse diazotrophs

Summary A cluster analysis based on codon usage in genes for biological nitrogen fixation (nif genes) grouped diazotrophs into three distinct classes: anaerobes, cyanobacteria, and aerobes. In thenif genes ofKlebsiella pneumoniae there was no evidence for selection pressure in favor of highly translatable codons. However, in the nitrogen regulatory operonglnAntrBntrC of enteric bacteria the stoichiometrically high level of glutamine synthetase may be facilitated by the presence of efficiently translatable codons inglnA. Thenif genes of the cyanobacteriumAnabaena showed codon selection in favor of translational efficiency. Computation of codon adaptation indices for expression in heterologous systems indicated that the reading frames most suitable for expression ofnif genes inEscherichia coli, Bacillus subtilis, andSaccharomyces cerevisiae were present in azotobacters, clostridia, and cyanobacteria, respectively. In codon-usage-based cluster analysis, type 3 nitrogenase genes ofAzotobacter vinelandii grouped along with type 1 and type 2 genes. This is in contrast to the nucleotide sequence-based multiple alignment in which type 3 nitrogenase genes ofA. vinelandii have been reported to cluster with entirely unrelated diazotrophs such as methanogens and clostridia. This may be indicative of lateral transfer ofnif genes among widely divergent taxons. The chromosomal- and plasmid-locatednif genes of rhizobia also cluster separately in nucleotide sequence-based analysis but showed similar codon usage. These analyses suggested that the phylogeny ofnif genes drawn on the basis of nucleotide sequence homology was not masked by the taxon-specific pressure on codon usage.     

The effect of zwitterion buffers on the freezability of Boer goat semen

Twenty semen samples with mass activity greater than +3 were collected from six healthy, mature Boer goat bucks. Each ejaculate was divided into four equal parts and extended at 37 degrees C in Tris, Test, Tes and Bes buffers containing egg yolk and glycerol. Semen was placed into medium size French strawsand after 2 hours of equilibration at 5 degrees C, frozen in the vapour phase and stored in liquid nitrogen for 7 days at -196 degrees C. Progressive motility, the number of live spermatozoa and glutamic oxaloacetic transaminase (GOT) release were studied after the initial extension, after equilibration and after 15 minutes and 7 days of freezing of semen. Semen samples when extended with Tris yolk glycerol showed significantly (P<0.01) higher progressive motility and live spermatozoa than when extended with the other zwitterion buffer-based extenders. The change of extenders did not influence the release of GOT at various stages of freezing of semen (P>0.05).     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.     

Mechanistic Studies on Metal-pyrophosphate Hydrolysis. Structure of Tetraammine(chloroaquo)cobait(III) dichloride ([CO(NH3)4ClH20]2+•-2Cl−), an Acid Hydrolysis Product of Co(NH3)4HP2O7 and Co(NH3)4PO4

Abstract

The octahedral complex tetraammine(chloroaquo)cobalt(III) dichloride is shown to be the HCl hydrolysis product of both P¹,^2-bidentate tetraammine(pyrophosphato)cobalt(III) [CO(NH₃)₄HP₂0₇ or CoPP] and bidentate tetraammine(phosphato)cobalt(III) [Co(NH₃)₄P0₄or CoP]. The complex crystallizes in the orthorhombic space group Pna2¹ with cell dimensions α=13.033(2)Å, b=6.710(1) Å, and c=10.318(2)Å; the crystal structure was refined to a final disagreement index of 0.033. The average of the four Co-N distances is 1.944±6Å. The Co-Cl distance is 2.257(2)Å and the Co-O(W) distance is 1.971(4)Å. Both protons of the coordinated water molecule are engaged in strong hydrogen bonds to the two nonbonded chloride counterions with 0(W)-C1 distances of 3.087(6)Å and 3.123(6)Å. Each nonbonded chloride is engaged in seven hydrogen bonding interactions resulting from the high ratio of hydrogen bond donors to acceptors in the CoP structure. Cobalt bisphosphate (CoP₂) is the final enzyme hydrolysis product when CoPP is used as substrate in the yeast inorganic pyrophosphatase reaction. The bridge oxygen atom is the site of initial CoPP cleavage both, for HCl catalyzed hydrolysis as well as for enzyme catalyzed hydrolysis.     

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.     

Combination of ascorbate and alpha-tocopherol as a preventive therapy against structural and functional defects of erythrocytes in visceral leishmaniasis

The redox unbalance in erythrocytes has been found to contribute significantly in the development of anemia in visceral leishmaniasis (VL). The present study revealed enhanced production of reactive oxygen species (ROS) and gradual depletion of alpha-tocopherol and ascorbate in the erythrocytes of infected animals. The response of erythrocytes to chronic treatment with antioxidants was studied in hamsters during leishmanial infection. Treatment with a combination of alpha-tocopherol and ascorbate proved to be the most effective preventive for the proteolytic degradation of erythrocyte membrane. Erythrocytes from infected animals were thermally more sensitive compared to the control ones. Combination of both antioxidants was most successful in resisting heat induced structural defects in the cells. Cross-linking of membrane proteins subsequent to oxidative damage in the red cells was accompanied by the formation of high molecular weight protein band at the top of the resolving gel in the presence of the cross-linking agent dimethyladepimidate (DMA). Marked inhibition of cross-linking was observed with combination of both antioxidants. Treatment with alpha-tocopherol and ascorbate together could withstand osmotic lysis of erythrocytes in the infected animals very efficiently. Decreased hemoglobin (Hb) level was successfully replenished and was coupled with significant increase in the life span of red cells after treating the animals with both antioxidants. Results indicate better efficacy of the combination therapy with alpha-tocopherol and ascorbate in protecting the erythrocytes from structural and functional damages during leishmanial infection.     

The EMBL Nucleotide Sequence Database: major new developments

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) incorporates, organizes and distributes nucleotide sequences from all available public sources. The database is located and maintained at the European Bioinformatics Institute (EBI) near Cambridge, UK. In an international collaboration with DDBJ (Japan) and GenBank (USA), data are exchanged amongst the collaborating databases on a daily basis to achieve optimal synchronization. Webin is the preferred web-based submission system for individual submitters, while automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via FTP, Email and World Wide Web interfaces. EBI's Sequence Retrieval System (SRS) integrates and links the main nucleotide and protein databases plus many other specialized molecular biology databases. For sequence similarity searching, a variety of tools (e.g. Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT. All resources can be accessed via the EBI home page at http://www.ebi.ac.uk.