Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase

The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) support rapid growth of a variety of Pseudomonas strains when provided as the sole source of carbon and nitrogen. All key enzymes of glutamate metabolism were detected in P. fluorescence, with glutaminase and asparaginase showing the highest specific activities. A periplasmic glutaminase/asparaginase activity (PGA) was found in all pseudomonads examined, including a number of root-colonizing biocontrol strains. The enzyme was purified and shown to be identical with the ansB gene product described previously. In addition to PGA, P. fluorescens contains a cytoplasmic asparaginase with marked specificity for Asn. PGA is strongly and specifically induced by its substrates (Asn, Gln) but also by the reaction products (Asp, Glu). In addition, PGA is subject to efficient carbon catabolite repression by glucose and by citrate cycle metabolites. A mutant of P. putida KT2440 with a disrupted ansB gene was unable to utilize Gln, whereas growth of the mutant on other amino acids was normal.     

Colocalization but differential regulation of neuronal NO synthase and nicotinic acetylcholine receptor in C2C12 myotubes

In mammalian skeletal muscle,neuronal-type nitric oxide synthase (nNOS) is found to be enriched atneuromuscular endplates. Here we demonstrate the colocalization of thenicotinic acetylcholine receptor (nAChR, stained with -bungarotoxin)and nNOS (stained with a specific antibody) in murineC₂C₁₂ myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C₂C₁₂myotubes. An antibody to the ₁-subunit of the nAChR didnot coprecipitate nNOS, and an nNOS-specific antibody did notprecipitate the ₁-subunit of the nAChR. Treatment ofmice with bacterial LPS downregulated the expression of nNOS inskeletal muscle, and treatment of C₂C₁₂ cellswith bacterial LPS and interferon- markedly decreased nNOS mRNA andprotein expression. In contrast, mRNA and protein of the nAChR (-,-, and -subunits) remained unchanged at the mRNA and proteinlevels. These data demonstrate that nNOS and the nAChR are colocalizedin murine skeletal muscle and C₂C₁₂ cells but differ in their expressional regulation.

     

Absence of the candidate male sex-determining gene dmrt1b(Y) of medaka from other fish species

Although the sex-determining genes are known in mammals, Drosophila, and C. elegans, little is known in other animals. Fishes are an attractive group of organisms for studying the evolution of sex determination because they show an amazing variety of mechanisms, ranging from environmental sex determination and different forms of hermaphroditism to classical sex chromosomal XX/XY or WZ/ZZ systems and modifications thereof. In the fish medaka, dmrt1b(Y) has recently been found to be the candidate male sex-determining gene. It is a duplicate of the autosomal dmrt1a gene, a gene acting in the sex determination/differentiation cascade of flies, worms, and mammals. Because in birds dmrt1 is located on the Z-chromosome, both findings led to the suggestion that dmrt1b(Y) is a "non-mammalian Sry" with an even more widespread distribution. However, although Sry was found to be the male sex-determining gene in the mouse and some other mammalian species, in some it is absent and has obviously been replaced by other genes that now fulfil the same function. We have asked if the same might be true of the dmrt1b(Y) gene. We find that the gene duplication generating dmrt1b(Y) occurred recently during the evolution of the genus Oryzias. The gene is absent from all other fish species studied. Therefore, it may not be the male-sex determining gene in all fishes.     

Identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses

Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.     

Splicing factor SRp30c interaction with Y-box protein-1 confers nuclear YB-1 shuttling and alternative splice site selection

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.     

Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.     

CD and NMR studies of prion protein (PrP) helix 1. Novel implications for its role in the PrPC-->PrPSc conversion process

The conversion of prion helix 1 from an alpha-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrPC of the prion protein to the pathogenic isoform PrPSc. Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrPC-->PrPSc conversion.     

Patterns and dynamics of sex-biased dispersal in a nocturnal primate, the grey mouse lemur, Microcebus murinus

We examined predictions on the proportion of dispersing natal males and females, dispersal distances, the age at dispersal and the potential for inbreeding over a 6-year period in a free-living population of grey mouse lemurs. We used monthly mark-recapture procedures to determine individual locations and interindividual distances. The analysis of seven polymorphic microsatellite markers for 213 (130 males, 83 females) individuals allowed us to estimate relatedness coefficients and kinship relationships. Closely related males ranged further from each other than closely related females and natal males were found further from their potential mothers than were females. Natal males were more likely to disperse from their birth sites than females, although male dispersal was not universal. Male breeding dispersal was detected in half of the long-term observations. Males therefore seem to be the predominant vectors for gene flow between populations and social units. Females usually stayed within one to two home range diameters of their potential mother, facilitating the evolution of cooperative behaviour by kin selection among females. Most dispersal took place before the mating season, indicating an age of less than 7 months for natal dispersal. The analysis of spatiotemporal coexistence revealed the potential for inbreeding in only 3.8% of the potential mother-son dyads, but in 21.9% of the potential father-daughter dyads and in 41.7% of other closely related male-female dyads. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour      

Choline head groups stabilize the matrix loop regions of the ATP/ADP carrier ScAAC2

ATP/ADP carriers (AACs) are essential to the cell as they exchange ATP produced in mitochondria for cytosolic ADP. Monoclonal antibodies against the isoform 2 of Saccharomyces cerevisiae AAC (ScAAC2) were used to probe the accessibility of the matrix loops 1 and 3 depending on the environment of the carrier. In mitochondrial membranes ScAAC2 was not recognized, whereas in dodecylmaltoside the antibodies bound to the carrier, suggesting that the epitopes are hidden in the native environment. Exposure of the epitopes by detergents was reversed by reconstitution of the carrier in phospholipids or by exchanging with detergents having a choline or a trimethylammonium head group. Circular dichroism spectroscopy on peptides representing the C-terminal regions of all three matrix loops showed that only phosphocholine detergents induced a structural reorganization. Since in addition phosphatidylcholine was found to be tightly associated with the purified carrier, the matrix loop regions are likely to be associated to the membrane by phosphatidylcholine.