Analysis of the genome of the moderate halophile Halomonas eurihalina

We have studied the genomic organization of Halomonas eurihalina, a moderately halophilic bacterium that produces an anionic exopolysaccharide with a potentially wide range of applications in industry. To estimate the genome size of strain F2-7, large restriction fragments of genomic DNA were separated by pulsed-field gel electrophoresis. According to the average size of the resolved restriction fragments, the genome size of H. eurihalina strain F2-7 was estimated to be around 2500 kb. The physical map of the chromosome for the endonuclease SwaI has been constructed. The F2-7 strain has two plasmids, pVE1 and pVE2, and in this study we have isolated three new plasmids, pVE3, pVE4, and pVE5, of 5.3, 16, and 6.5 kb, respectively, from strains H-1, H-217, and H-236 of H. eurihalina. We have characterized these plasmids and constructed their physical maps. Curing experiments produced no evidence for the involvement of these plasmids in the expression of the mucous phenotype. Received: 19 October 2001 / Accepted: 31 December 2001     

Nephrotoxicity of simultaneous exposure to mercury and uranium in comparison to individual effects of these metals in rats

Both inorganic mercury and uranium are known nephrotoxicants in mammals. In this study, the renal toxicity of a concurrent exposure to inorganic mercury and uranium was compared with the nephrotoxic effects of the individual metals in a rat model. Eight groups of rats, 10 animals per group, were subcutaneously given a single administration of mercuric chloride (HgCl₂, 0.34 mg/kg and 0.68 mg/kg), uranyl acetate dihydrate (UAD, 2.5 mg/kg and 5 mg/kg), or combinations of both compounds at the same doses. A ninth group of rats received sc injections of 0.9% saline and was designated as the control group. Necrosis of proximal tubules, which was observed in all experimental groups, was the most relevant morphologic abnormality. Marked changes, which were remarkably greater than those induced by the individual elements, were noted in some urinary parameters in the groups concurrently exposed to HgCl₂ and UAD. It could be an indicator of a synergistic interaction between mercury and uranium. In contrast, compared with the urinary levels found after individual administration of the highest doses of mercury and uranium, significant reductions in the urinary concentrations of these elements were noted following simultaneous exposure to both metals. At these doses, the reduction in the urinary metal excretion was also accompanied by significant decreases in the renal content of mercury and uranium. Whereas the results of some parameters pointed out a possible synergistic interaction between mercury and uranium, other measures hinted that an antagonistic interaction between these elements is also present.     

The N terminus of Myxococcus xanthus CarA repressor is an autonomously folding domain that mediates physical and functional interactions with both operator DNA and antirepressor protein

Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.     

Comblike complexes of bacterial poly(gamma,d-glutamic acid) and cationic surfactants

The ability of microbally produced poly(gamma,d-glutamic acid) to form stable polyelectrolyte-opposite charged surfactant complexes was investigated. A sonicated sample of polyacid with a molecular weight about 10(5) Da and a content of d enantiomer higher than 90% was used in this study. Nearly stoichiometric complexes of poly(gamma,d-glutamate) anions and alkyltrimethylammonium cations bearing linear alkyl chains with even numbers of carbon atoms from 12 up to 22 were "synthesized" by precipitation from equimolar mixtures of aqueous solutions of the two components. All complexes were found to adopt stratified supramolecular structures made of alternating layers of poly(gamma,d-glutamate) and surfactant with a periodicity increasing from 3.2 up to 4.3 nm according to the length of the alkyl side chain. No definite evidence indicative of the conformation adopted by the main chain in these complexes could be afforded. In all cases, the alkyl chains are in an extended conformation and oriented normal or nearly normal to the layer planes. Polymethylene chains with more than 16 carbon atoms were partially crystallized in the complexes in a separated paraffinic phase, whereas no crystallinity was detected for shorter lengths. The crystallized paraffinic phases were found to melt reversibly at temperatures between 40 and 70 degrees C. This process was found to happen with a concomitant expansion-contraction that amounts between 2 and 8% of the long period of the structure but without significant alteration of the layered arrangement.     

Synthesis of encoded modified oligonucleotide libraries. Part 1: orthogonal chemistry

The basis for further development of combinatorial libraries of modified oligonucleotides tagged by a codifying sequence is discussed. The chemistry involved in the orthogonal synthesis of both strands and some representative examples of building blocks are presented.     

Proteomics-based identification of novel Candida albicans antigens for diagnosis of systemic candidiasis in patients with underlying hematological malignancies

Systemic candidiasis remains a major cause of disease and death, particularly among patients suffering from hematological malignancies. In an attempt to contribute to the discovery of useful biomarkers for its diagnosis and therapeutic monitoring, we embarked on a mapping of Candida albicans immunogenic proteins specifically recognized by antibodies produced during the natural course of systemic Candida infection in this high-risk population. About 85 immunoreactive protein species were detected with systemic candidiasis patients' serum specimens by using immunoproteomics (i.e., two-dimensional electrophoresis followed by Western blotting), and identified through a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), de novo peptide sequencing using nano-electrospray ionization-ion trap (ESI-IT) MS, and genomic database searches. This proteomic approach has led to the characterization of 42 different housekeeping enzymes as C. albicans antigens. Their biological significance is also discussed. Furthermore, this study is the first to report that 26 of them exhibit antigenic properties in C. albicans, and 35 of them become targets of the human antibody response to systemic candidiasis. Our findings suggest that the production of antibodies to C. albicans phosphoglycerate kinase and alcohol dehydrogenase during systemic candidiasis could be associated with a differentiation of the human immune response. We also highlight the relationship between changes in maintenance of circulating levels of specific anti-Candida antibodies and patients' outcome. Some of these variations, especially the rise of high anti-enolase antibody concentrations, appear to be related to recovery from systemic candidiasis in these patients, which might serve as markers for predicting their outcome. This approach could therefore provide new challenges for diagnosis and clinical follow-up of these fungal infections, and even for antifungal drug or vaccine design.     

Protective effect of Montilla-Moriles appellation red wine on oxidative stress induced by streptozotocin in the rat

This study evaluated the protective effect of Montilla-Moriles appellation red wine (Cordoba, Spain) on oxidative stress, course and intensity of symptoms in experimental diabetes induced by the injection of streptozotocin in male Wistar rats. The rats were injected with a single dose of streptozotocin (60 mg/kg i.p.) and given water and red wine separately. After 4 weeks of treatment, blood samples were obtained to determine sugar and fructosamine concentrations in blood plasma, serum insulin concentration, and percentage of glycosylated hemoglobin in blood. The kidney, liver, and pancreas were removed to determine lipid peroxidation levels, reduced glutathione content, and antioxidative enzyme activity. A significant increase of glucose concentration in urine was found in the rats after injecting the streptozotocin. The administration of red wine before streptozotocin elevated reduced glutathione content and antioxidative enzyme activity, while lowering the lipid peroxidation level. Moreover, the red wine induced decreased levels of glycemia, plasma fructosamine and percentage of glycosylated hemoglobin, while increasing levels of insulin. These data suggest that red wine has a protective effect against oxidative stress and diabetes induced by streptozotocin.     

Crystallographic and thermodynamic analysis of the binding of S-octylglutathione to the Tyr 7 to Phe mutant of glutathione S-transferase from Schistosoma japonicum

Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained. These new structural data combined with fluorescence spectroscopy and thermodynamic data, obtained by means of isothermal titration calorimetry, allow for detailed characterization of the ligand-binding process. The binding of S-octyl-GSH to SjGST(Y7F) is enthalpically and entropically driven at temperatures below 30 degrees C. The stoichiometry of the binding is one molecule of S-octyl-GSH per mutant dimer, whereas shorter alkyl derivatives bind with a stoichiometry of two molecules per mutant dimer. The SjGST(Y7F).GSH structure showed no major structural differences compared to the wild-type enzyme. In contrast, the structure of SjGST(Y7F).S-octyl-GSH showed asymmetric binding of S-octyl-GSH. This lack of symmetry is reflected in the lower symmetry space group of the SjGST(Y7F).S-octyl-GSH crystals (P6(3)) compared to that of the SjGST(Y7F).GSH crystals (P6(3)22). Moreover, the binding of S-octyl-GSH to the A subunit is accompanied by conformational changes that may be responsible for the lack of binding to the B subunit.     

The plot thickens: New perspectives of primary cell wall modification

Recent studies have further confirmed the ubiquity of cell wall restructuring during plant growth and development, and have emphasized the fact that our understanding of the breadth of molecular processes that mediate wall modification is still rudimentary. In the past few years, both enzymatic and non-enzymatic agents that apparently contribute to wall disassembly have been identified, and it is likely that additional mechanisms will continue to be revealed. These discoveries are being propelled by the development of new biochemical and biophysical assays, by database mining in the wake of the explosion of plant sequence information from genome sequencing and expressed sequence tags, and by a variety of strategies used to catalog the cell wall proteome. The daunting question of how these mechanistically diverse and complex processes are coordinated remains unresolved.     

Binding of calmodulin to the carboxy-terminal region of p21 induces nuclear accumulation via inhibition of protein kinase C-mediated phosphorylation of Ser153

Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.