The role of adherent cells in supporting natural cytotoxicity reactions

The cytotoxic activity of natural killer (NK) cells and interferon-alpha (IFN-alpha) production by NK cells were studied. It was shown that in healthy donors adherent cells (AC) play an important role in reactions of natural cytotoxicity: they provide the main IFN production in the NK system. AC suppresses the cytotoxic activity of NK cells in patients with scleroderma and AC has a high cytotoxic activity in patients with multiple sclerosis. The role of AC in appearance and development of NK immunodeficiency in these pathologies was discussed.     

A laboratory-based survey of Campylobacter infections in Prahova County

In developing countries, as well as in many western countries, members of the genus Campylobacter are recognized as one of the most common cause of acute bacterial enteritis. Campylobacter jejuni and Campylobacter coli isolation rates have been shown to be equal to, and sometimes higher than those of other enteric pathogens. The Microbiology Laboratory of the local Public Health Authority in Prahova County conducted a one and a half-year laboratory-based survey of Campylobacter infections in patients suffering from gastrointestinal symptoms. From a total of 3284 stool samples screened, the culture-positive ones confirmed the bacterial etiology for 551 diarrhea cases. Campylobacter was found in 345 specimens, being the most frequently isolated enteropathogen. C. jejuni outnumbered C. coli species (239 vs. 106 isolates). Salmonella isolates were the second local cause of diarrhea. The highest isolation rate of Campylobacter was found in children 5 years of age (262 strains). The prevalence of campylobacteriosis declined with age. The isolation rate of Campylobacter (10.5%), the unimodal age-specific distribution of cases, as well as the identification of polymicrobial infections among the screened population were epidemiological aspects resembling reports on campylobacteriosis in developing countries. The susceptibility of Campylobacter isolates to various antimicrobial agents, including macrolides and fluoroquinolones was also assessed. Among the screened isolates, Erythromycin retained a good activity, while an increased ciprofloxacin resistance was observed. The information gathered through this local study sustains the importance of Campylobacter in the etiology of autochthonous infectious diarrhea. A development of a national surveillance program regarding the most important foodborne pathogens would be beneficial for improving prevention and controlling measures.     

<Emphasis Type="Italic">Desulfurispirillum alkaliphilum</Emphasis> gen. nov. sp. nov., a novel obligately anaerobic sulfur- and dissimilatory nitrate-reducing bacterium from a full-scale sulfide-removing bioreactor

Strain SR 1^T was isolated under anaerobic conditions using elemental sulfur as electron acceptor and acetate as carbon and energy source from the Thiopaq bioreactor in Eerbeek (The Netherlands), which is removing H₂S from biogas by oxidation to elemental sulfur under oxygen-limiting and moderately haloalkaline conditions. The bacterium is obligately anaerobic, using elemental sulfur, nitrate and fumarate as electron acceptors. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while nitrate is dissimilatory reduced to ammonium. Furthermore, in the presence of nitrate, strain SR 1^T was able to oxidize limited amounts of sulfide to elemental sulfur during anaerobic growth with acetate. The new isolate is mesophilic and belongs to moderate haloalkaliphiles, with a pH range for growth (on acetate and nitrate) from 7.5 to 10.25 (optimum 9.0), and a salt range from 0.1 to 2.5 M Na⁺ (optimum 0.4 M). According to phylogenetic analysis, SR 1^T is a member of a deep bacterial lineage, distantly related to Chrysiogenes arsenatis (Macy et al. 1996). On the basis of the phenotypic and genetic data, the novel isolate is placed into a new genus and species, Desulfurispirillum alkaliphilum (type strain SR^T = DSM 18275 = UNIQEM U250). Nucleotide sequence accession number: the GenBank/EMBL accession number of the 16S rRNA gene sequence of strain SR 1^T is DQ666683.     

Molecular and catalytic properties of a novel cytochrome c nitrite reductase from nitrate-reducing haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

A highly active cytochrome c nitrite reductase from the haloalkaliphilic sulfur-oxidizing non-ammonifying bacterium Tv. nitratireducens strain ALEN 2 (TvNiR) was isolated and purified to apparent electrophoretic homogeneity. The enzyme catalyzes reductive conversion of nitrite and hydroxylamine to ammonia without release of any intermediates, as well as reduction of sulfite to sulfide. TvNiR also possesses peroxidase activity. In solution TvNiR exists as a stable hexamer with molecular mass of about 360kDa. Each TvNiR subunit with molecular mass of 64kDa contains, as defined from spectral properties and sequence analysis, eight c-type haems. Seven of them are coordinated by the characteristic CXXCH motifs for haem c binding, while one is bonded by the unique CXXCK motif. So far, this motif coordinating the catalytic haem was found only in bacterial cytochrome c nitrite reductases (ccNiRs). All the residues essential for catalysis in the known ccNiRs were also identified in TvNiR. However, TvNiR is only distantly related to known bacterial ammonifying dissimilatory ccNiRs, sharing no more than 20% homology.     

Improved method for direct screening of true lipase-producing microorganisms with particular emphasis on alkaline conditions

A rapid and effective method for direct detection, selection and testing of microorganisms able to produce both cell-bound and extracellular true lipases is described. The method is based on formation of clearance zones on turbid solid media with emulsified olive oil around or under the colonies, cell fractions or culture supernatant of lipase-producing organisms. The method was successfully applied for the screening and isolation of microorganisms producing alkaline lipases. The article is published in the original.     

Mouse AMACO,a kidney and skin basement membrane associated molecule that mediates RGD-dependent cell attachment

The VWA domain-containing extracellular matrix protein AMACO has not been extensively characterized and its function remains unknown. It has been proposed as a potential cancer marker and carries a rare O-glucosylation and O-fucosylation on its first EGF-like domain. AMACO is a basement membrane associated protein, however its exact localization has not been determined. Here we show by immunogold electron microscopy of mouse kidney and skin that AMACO does not occur within the basement membrane but rather subjacent to the basement membrane at its stromal surface. In skin, AMACO often colocalizes with triple-helical domains of collagen VII containing anchoring fibrils as they emerge from the basal lamina. However, the immunogold patterns for AMACO and the C-terminal end of collagen VII show discrete differences, indicating that AMACO and collagen VII do not colocalize at anchoring plaques. In contrast, the localization pattern of AMACO partially overlaps with that for collagen XVIII. In addition, mouse AMACO was shown to support β1 integrin-mediated adhesion of a keratinocyte-like cell line, HaCaT, and a fibroblast cell line, Wi26, in an RGD-dependent manner, most likely using an RGD-motif near the C-terminus of AMACO. However, the loss of cell adhesion to the C-terminal part of the human AMACO, due to the unique absence of an RGD sequence in the human protein, suggests that cell adhesion is not AMACO's major function.     

Asp-to-Asn Substitution at the First Position of the DxD TOPRIM Motif of Recombinant Bacterial Topoisomerase I Is Extremely Lethal to E. coli

The TOPRIM domain found in many nucleotidyl transferases contains a DxD motif involved in magnesium ion coordination for catalysis. Medium- to high-copy-number plasmid clones of Yersinia pestis topoisomerase I (YpTOP) with Asp-to-Asn substitution at the first aspartate residue (D117N) of this motif could not be generated in Escherichia coli without second-site mutation even when expression was under the control of the tightly regulated BAD promoter and suppressed by 2% glucose in the medium. Arabinose induction of a single-copy YpTOP-D117N mutant gene integrated into the chromosome resulted in ∼ 10⁵-fold of cell killing in 2.5 h. Attempt to induce expression of the corresponding E. coli topoisomerase I mutant (EcTOP-D111N) encoded on a high-copy-number plasmid resulted in either loss of viability or reversion of the clone to wild type. High-copy-number plasmid clones of YpTOP-D119N and EcTOP-D113N with the Asn substitution at the second Asp of the TOPRIM motif could be stably maintained, but overexpression also decreased cell viability significantly. The Asp-to-Asn substitutions at these TOPRIM residues can selectively decrease Mg²⁺ binding affinity with minimal disruption of the active-site geometry, leading to trapping of the covalent complex with cleaved DNA and causing bacterial cell death. The extreme sensitivity of the first TOPRIM position suggested that this might be a useful site for binding of small molecules that could act as topoisomerase poisons.     

Microbiological monitoring of disturbed ground ecosystems of Siberia

Microbiological analysis of different elements of the forest ecosystem (soil, leaf-litter) in zones of active anthropogenic (technogenic) impact shows that microbial complexes serve as an instrument of condition diagnosis and monitoring of ground ecosystems. Fluctuations of the number of microorganisms, the scale of the ETGM number, coefficient of microbiological activity, and the reaction of introduced population of Bacillus subtilis to different doses of pollution may be considered as convenient characteristics.