Use of the DNA-DNA hybridization method on filters for determining the toxic potency of atypical strains of Vibrio cholerae isolated from natural sources

The methods of the radioimmunoassay and the blot hybridization of restricted fragments of chromosomal DNA have been used for the characterization of V. cholerae atypical strains isolated from the natural environment. For all strains under study, the radioimmunoassay has been found to yield the most sharply defined data characterizing their atoxigenicity. The absence of the structural genes of toxin in the chromosomes of these strains has been shown by the method of blot hybridization. Some methodological simplifications of blot hybridization, having no adverse effect on the sensitivity of this method, have been tested.     

Structural characterization of aminoglycoside 4′‐O‐adenylyltransferase ANT(4′)‐IIb from Pseudomonas aeruginosa

Aminoglycosides were one of the first classes of broad‐spectrum antibacterial drugs clinically used to effectively combat infections. The rise of resistance to these drugs, mediated by enzymatic modification, has since compromised their utility as a treatment option, prompting intensive research into the molecular function of resistance enzymes. Here, we report the crystal structure of aminoglycoside nucleotidyltransferase ANT(4′)‐IIb in apo and tobramycin‐bound forms at a resolution of 1.6 and 2.15 Å, respectively. ANT(4′)‐IIb was discovered in the opportunistic pathogen Pseudomonas aeruginosa and conferred resistance to amikacin and tobramycin. Analysis of the ANT(4′)‐IIb structures revealed a two‐domain organization featuring a mixed β‐sheet and an α‐helical bundle. ANT(4′)‐IIb monomers form a dimer required for its enzymatic activity, as coordination of the aminoglycoside substrate relies on residues contributed by both monomers. Despite harbouring appreciable primary sequence diversity compared to previously characterized homologues, the ANT(4′)‐IIb structure demonstrates a surprising level of structural conservation highlighting the high plasticity of this general protein fold. Site‐directed mutagenesis of active site residues and kinetic analysis provides support for a catalytic mechanism similar to those of other nucleotidyltransferases. Using the molecular insights provided into this ANT(4′)‐IIb‐represented enzymatic group, we provide a hypothesis for the potential evolutionary origin of these aminoglycoside resistance determinants.     

Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme

Genomes of all free-living organisms encode the enzyme dUTPase (dUTP pyrophosphatase), which plays a key role in preventing uracil incorporation into DNA. In the present paper, we describe the biochemical and structural characterization of DUT1 (Saccharomyces cerevisiae dUTPase). The hydrolysis of dUTP by DUT1 was strictly dependent on a bivalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+ or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide: dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve, suggesting a positive co-operativity between the subunits. The crystal structure of DUT1 was solved at 2 ? resolution (1 ?=0.1 nm) in an apo state and in complex with the non-hydrolysable substrate α,β-imido dUTP or dUMP product. Alanine-replacement mutagenesis of the active-site residues revealed seven residues important for activity including the conserved triad Asp87/Arg137/Asp85. The Y88A mutant protein was equally active against both dUTP and UTP, indicating that this conserved tyrosine residue is responsible for discrimination against ribonucleotides. The structure of DUT1 and site-directed mutagenesis support a role of the conserved Phe142 in the interaction with the uracil base. Our work provides further insight into the molecular mechanisms of substrate selectivity and catalysis of dUTPases.     

Antibiotic susceptibility of the bacterial strains isolated at the emergency hospital]

Comparative evaluation of the incidence of the drug resistant pathogens at the patients with sepsis was performed. High ratio of the resistnt strain was shown and the most potent drugs were estimated. The investigation results demonstrates the necessity to improve diagnostic quality control. The data on drug susceptibility of the pathogens may be used for their taxonomic clarification.     

Structural and biochemical characterization of a novel Mn2+-dependent phosphodiesterase encoded by the yfcE gene

Escherichia coli YfcE belongs to a conserved protein family within the calcineurin-like phosphoesterase superfamily (Pfam00149) that is widely distributed in bacteria and archaea. Superfamily members are metallophosphatases that include monoesterases and diesterases involved in a variety of cellular functions. YfcE exhibited catalytic activity against bis-p-nitrophenyl phosphate, a general substrate for phosphodiesterases, and had an absolute requirement for Mn2+. However, no activity was observed with phosphodiesters and over 50 naturally occurring phosphomonoesters. The crystal structure of the YfcE phosphodiesterase has been determined to 2.25 A resolution. YfcE has a beta-sandwich architecture similar to metallophosphatases of common ancestral origin. Unlike its more complex homologs that have added structural elements for regulation and substrate recognition, the relatively small 184-amino-acid protein has retained its ancestral simplicity. The tetrameric protein carries two zinc ions per active site from the E. coli extract that reflect the conserved di-Mn2+ active site geometry. A cocrystallized sulfate inhibitor mimics the binding of phosphate moeities in known ligand/phosphatase complexes. Thus, YfcE has a similar active site and biochemical mechanism as well-characterized superfamily members, while the YfcE phosphodiester-containing substrate is unique.     

General RNA‐binding proteins have a function in poly(A)‐binding protein‐dependent translation

The interaction between the poly(A)‐binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA‐binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB‐1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB‐1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP‐dependent after the addition of YB‐1. In this system, eIF4E binding to the cap structure is inhibited by YB‐1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB‐1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB‐1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.     

Hierarchy of alpha fetoprotein (AFP)-specific T cell responses in subjects with AFP-positive hepatocellular cancer

We identified a series of immunodominant and subdominant epitopes from alpha fetoprotein (AFP), restricted by HLA-A*0201, which are recognized by the human T cell repertoire. The four immunodominant epitopes have been tested for immunogenicity in vivo, in HLA-A*0201+AFP+ advanced stage hepatocellular cancer (HCC) patients, and have activated and expanded AFP-specific IFN-gamma-producing T cells in these patients, despite high serum levels of this self Ag. Here, we have examined the frequency, function, and avidity of the T cells specific for subdominant epitopes from AFP. We find that T cells specific for several of these epitopes are of similar or higher avidity than those specific for immunodominant epitopes. We then tested the peripheral blood of subjects ex vivo with different levels of serum AFP for the hierarchy of response to epitopes from this Ag and find that HCC patients have detectable frequencies of circulating IFN-gamma-producing AFP-specific CD8+ T cells to both immunodominant and subdominant epitopes. We find the immunodominant and subdominant peptide-specific T cells to be differentially expanded with different modes of Ag presentation. Whereas spontaneous and AFP protein-stimulated responses show evidence for immunodominance, AdVhAFP-transduced dendritic cell-stimulated responses were broader and not skewed. Importantly, these data identify subdominant epitopes from AFP that can activate high-avidity T cells, and that can be detected and expanded in HCC subjects. These subdominant epitope-specific T cells can also recognize tumor cells and may be important therapeutically.     

Biosynthesis of chlorophyll P-680 of photosystem II in greening leaves of plants adapted to heat shock

In etiolated pea and maize leaves illuminated after incubation at 38 degreesC, a new dark reaction was shown manifested in the bathochromic shift of spectral bands and accompanied by esterification of the product of protochlorophyllide photochemical reduction--Chld 684/676: Chld 684/676 --> Chl 688/680. After completion of the reaction a rapid (20-30 sec) quenching of the fluorescence of the reaction product (Chl 688/680) was observed. The reaction Chld 684/676 --> Chl 688/680 is inhibited under anaerobic conditions and in the presence of cyanide; the reaction accompanied by Chl 688/680 fluorescence quenching is not observed in pea mutants with impaired function of photosystem II reaction centers. The spectral properties of the formed Chl form with the absorption maximum at 680 nm, fluorescence quenching, and simultaneous synthesis of pheophytin suggest that the reaction is connected with the chlorophyll of photosystem II reaction center--P-680.