[Indicators of protein, lipid, carbohydrate, purine metabolism and the level of pituitary hormones in healthy men of 22-55 years old.]

The levels of total protein, glucose, cholesterol and its fractions, triglycerides, uric acid, prolactin, thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone in the serum, blood pressure (BP), calculated body mass index (BMI) were analyzed in 60 healthy men aged 22-55 years old. To assess the age dynamics of the parameters, groups of young (up to 35 years old) and middle age (over 35 years old) were formed according to the classification adopted at the International Symposium on Age Periodization in 1965. The correlation analysis of the parameters of the biochemical parameters, level pituitary hormones, age, BMI, level of mean blood pressure was made. There are a decrease (p<0,05) of total blood protein level (within the normal range), a tendency to a decrease in prolactin levels (PRL), an increase (p<0,05) of total cholesterol, LDL cholesterol in middle age group compared with the younger group. HDL cholesterol levels did not differ in the groups; this demonstrates the constancy of antiatherogenic protection over many decades. A slight decrease in total protein in the blood and a tendency to a decrease in the level of PRL with increasing age against the background of a negative correlations between the low degree of PRL and age (r=-0,20; p=0,01), total protein and age (r=-0,35; p=0,01) confirm the participation of PRL in protein metabolism, and also indirectly indicate changes in the regulatory effect of dopamine on prolactin synthesis and secretion.

In vitro characterization of biofilms formed by Kingella kingae.

The Gram-negative bacterium Kingella kingae is part of the normal oropharyngeal mucosal flora of children <4 years old. K. kingae can enter the submucosa and cause infections of the skeletal system in children, including septic arthritis and osteomyelitis. The organism is also associated with infective endocarditis in children and adults. Although biofilm formation has been coupled with pharyngeal colonization, osteoarticular infections, and infective endocarditis, no studies have investigated biofilm formation in K. kingae. In this study we measured biofilm formation by 79 K. kingae clinical isolates using a 96-well microtiter plate crystal violet binding assay. We found that 37 of 79 strains (47%) formed biofilms. All strains that formed biofilms produced corroding colonies on agar. Biofilm formation was inhibited by proteinase K and DNase I. DNase I also caused the detachment of pre-formed K. kingae biofilm colonies. A mutant strain carrying a deletion of the pilus gene cluster pilA1pilA2fimB did not produce corroding colonies on agar, autoaggregate in broth, or form biofilms. Biofilm forming strains have higher levels of pilA1 expression. The extracellular components of biofilms contained 490 µg cm-2 of protein, 0.68 µg cm-2 of DNA, and 0.4 µg cm-2 of total carbohydrates. We concluded that biofilm formation is common among K. kingae clinical isolates, and that biofilm formation is dependent on the production of proteinaceous pili and extracellular DNA. Biofilm development may have relevance to the colonization, transmission, and pathogenesis of this bacterium. Extracellular DNA production by K. kingae may facilitate horizontal gene transfer within the oral microbial community.

Inhibition of LtxA toxicity by blocking cholesterol binding with peptides.

The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of (334) LEEYSKR(340), in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC(336) motif of LtxA (CRAC(336WT)). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of repeats-in-toxin-producing organisms.

Genotyping, local prevalence and international dissemination of ß-lactamase-producing Kingella kingae strains.

ß-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of ß-lactamase production among K. kingae isolates from different geographical origins and genetically characterized ß-lactamase-producing strains. Seven hundred and seventy-eight isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for ß-lactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, blaTEM and 16S rRNA sequencing. ß-lactamase was identified in invasive strains from Iceland (n=4/14, 28.6%), the USA (n=3/15, 20.0%) and Israel (n=2/190, 1.1%) and in carriage strains in the USA (n=5/17, 29.4%) and Israel (n=66/429, 15.4%). No French, Spanish or Canadian isolates were ß-lactamase producers. Among ß-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic ß-lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed from the four genetically unrelated Israeli ß-lactamase-producing clones. Representative strains of different genotypes produced the TEM-1 enzyme. K. kingae ß-lactamase producers exhibit a clear clonal distribution and have dissimilar invasive potential. The presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of ß-lactam resistance, and emphasizes the importance of routine testing of all K. kingae clinical isolates for ß-lactamase production.

Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells.

Kingella kingae is an emerging pathogen causing osteoarticular infections in pediatric patients. Electron microscopy of K. kingae clinical isolates revealed the heterogeneously-sized membranous structures blebbing from the outer membrane that were classified as outer membrane vesicles (OMVs). OMVs purified from the secreted fraction of a septic arthritis K. kingae isolate were characterized. Among several major proteins, K. kingae OMVs contained virulence factors RtxA toxin and PilC2 pilus adhesin. RtxA was also found secreted as a soluble protein in the extracellular environment indicating that the bacterium may utilize different mechanisms for the toxin delivery. OMVs were shown to be hemolytic and possess some leukotoxic activity while high leukotoxicity was detected in the non-hemolytic OMV-free component of the secreted fraction. OMVs were internalized by human osteoblasts and synovial cells. Upon interaction with OMVs, the cells produced increased levels of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL-6) suggesting that these cytokines might be involved in the signaling response of infected joint and bone tissues during natural K. kingae infection. This study is the first report of OMV production by K. kingae and demonstrates that OMVs are a complex virulence factor of the organism causing cytolytic and inflammatory effects on host cells.

Human CD18 is the functional receptor for Aggregatibacter actinomycetemcomitans leukotoxin.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is the causative organism of localized aggressive periodontitis, a rapidly progressing degenerative disease of the gingival and periodontal ligaments, and is also implicated in causing subacute infective endocarditis in humans. The bacterium produces a variety of virulence factors, including an exotoxic leukotoxin (LtxA) that is a member of the repeats-in-toxin (RTX) family of bacterial cytolysins. LtxA exhibits a unique specificity to macrophages and polymorphonuclear cells of humans and other primates. Human lymphocyte function-associated antigen 1 (LFA-1) has been implicated as the putative receptor for LtxA. Human LFA-1 comprises the CD11a and CD18 subunits. It is not clear, however, which of its subunits serves as the functional receptor that confers species-specific susceptibility to LtxA. Here we demonstrate that the human CD18 is the receptor for LtxA based on experiments performed with chimeric beta2-integrins recombinantly expressed in a cell line that is resistant to LtxA effects. In addition, we show that the cysteine-rich tandem repeats encompassing integrin-epidermal growth factor-like domains 2, 3, and 4 of the extracellular region of human CD18 are critical for conferring susceptibility to LtxA-induced biological effects.

[The epidemiological and clinicoimmunological aspects of the outburst of trichinosis in the Krymsky District of the Krasnodar Territory].

The present paper presents the epidemiological and clinicoimmunological aspects of the outburst of trichinosis in the Krymsky District of the Krasnodar Territory in January 2001. Six hundred and forty-eight persons ate infested wild boar meat, 119 (74.8%) fell ill with moderate and severe trichinosis. The authors provide the results of 3.5-year immunological studies of the patients with trichinosis and convalescents, infection-risk and healthy individuals; those of autopsies of the corpses of those who died from accidental causes unassociated with trichinosis, as well as examinations of domestic, synanthropic, and wild animals. The findings indicate that there is a focus of trichinosis with the low level of endemia in the Krymsky District of the Krasnodar Territory.

Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1.

1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of 45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates. The comparison of the Km and Vmax values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate. The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate 1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.

[Genetic control of naphthalene biodegradation by a strain of Pseudomonas sp. 8909N]. / Geneticheskii kontrol' biodegradatsii naftalina shtammom Pseudomonas sp. 8909N.

The ability of Pseudomonas sp. 8909N to grow using Naphthalene and salicylate as the sole source of carbon and energy is mediated by the presence of an 80-kb conjugative pBS1145 plasmid in this strain. Structural genes for naphthalene degradation in pBS1145 plasmid are homologous to those in the known NAH7 plasmid. Conjugational transfer of pBS1145 from the original strain is accompanied by a deletion of a plasmid DNA fragment that does not affect the Nah+Sal+ phenotype. Plasmid pBS1145 specifies a low constitutive level of catechol-2,3-dioxygenase, the key enzyme of the metha-pathway of catechol degradation. Activity of this enzyme is induced in the presence of salicylate. Enzymes of both the metha and ortho-pathway of catechol degradation (catechol-2-3-dioxygenase) were shown to operate in the process of naphthalene degradation in Pseudomonas sp. 8909N. The ability of this strain to bring about transformation of polycyclic aromatic hydrocarbon phenanthrene is also controlled by pBS1145 plasmid.