[SOD1 gene mutations in patients with amyotrophic lateral sclerosis: potential for the method of molecular].

The molecular modeling method is promising for the assessment of protein structure, being able to present an energetically beneficial protein conformation with atomic precision. This method is of great importance for studying molecular interactions and confirming pathogenic significance of the changes in the protein structure caused by particular mutations. In the present study we used molecular modeling for the assessment of mutations in the SOD1 gene in patients with amyotrophic lateral sclerosis (ALS), a severe neurodegenerative disorder characterized by the loss of the spinal and cerebral motor neurons. The product of SOD1 is a cytosolic dimeric enzyme Cu/Zn superoxide dismutase (SOD1) responsible for detoxification of the cellular superoxide radicals. We showed that all 8 revealed coding point mutations of the gene led to moderate or significant changes of the SOD1 protein energy. Mutation His49Arg increased the protein energy, and reconstruction of the respective model pointed out to spatial destabilization of the molecule and abnormal interaction with the metal ion inside the active center. The other 7 mutations (Gly17Ala, Leu85Mal, Asn87Ser, Asp91Ala, Serl06Leu, Glu134Gly, and Leul45Phe), on the contrary, led to decrease of the protein energy and increase of the spatial stability of SOD1, which is usually accompanied by increased propensity of the 'inert' mutant molecule to misfolding and cellular aggregation. Thereby, the results of in silico analysis of the SOD1 gene mutations confirm staying of ALS within the class of the so-called conformational diseases of the central nervous system, a characteristic feature of which is forming of cytotoxic insoluble protein inclusions in neurons.

[Association between the VEGF -2578С/A polymorphism and amyotrophic lateral sclerosis in a Russian population].

Genetic predisposition plays an important role in the development of amyotrophic lateral sclerosis (ALS). One of the most promising candidate genes in ALS is the vascular endothelial growth factor (VEGF) gene. In a Russian population, 192 ALS patients (103 males and 89 females), aged from 20 to 83 years (52.0±13.4), were examined. A control group comprised 128 age- and sex-matched people. All individuals studied were Slavs. Polymorphism -2578С/А (rs699947) in the VEGF gene was studied by real-time PCR. It was shown that the genotype distribution was significantly different between the ALS and control groups (χ2=11.1; р=0.004); in the ALS cohort, the 2578A/A genotype was significantly more frequent (29.7% vs. 20.3%, p=0.04). The allele distribution was also significantly different between the two groups (χ2=4.4; р=0.036). The -2578А/А genotype increased risk of ALS (OR 1.66; 95% CI 1.03-2.29), and this pattern was most obvious in the male subgroup (OR=2.18; 95% CI 1.90-2.47). It was found the association of the 'risk' 2578A/A genotype with the earlier disease onset and rapid progression. Therefore, the results obtained in the study confirm the role of the VEGF gene in the pathogenesis of ALS in a Russian population.

[Season and age related structure of acute intestinal infections morbidity in the Russian Federation].

AIM: To study seasonal and age features of etiological structure of acute intestinal infections (AII) in the territory of the RF. MATERIAL AND METHODS: A total of 7388 AII inpatients aged from 1 day to 90 years from 7 cities (Moscow, St-Petersburg, Nizhniy Novgorod, Chelyabinsk, Tyumen, Makhachkala and Khabarovsk) of the RF participated in a trial conducted from December 2001 to September 2006 The patients were examined with diagnostic tests based on polymerase chain reaction (PCR) for detection of rotaviruses of group A (RVA), noroviruses, astroviruses, adenoviruses, salmonella, termophilic campilobacteria, shigella and enteroinvasive Escherichia coli (EIEC). RESULTS: The above agents were detected in 72% children and 52% adults. In children RVA and noroviruses occurred most frequently (29.5% and 11%, respectively). The adults carried most often salmonella (9.3%), noroviruses (8.4%), RVA (7.8%) and Schigella in combination with EIEC (7.0%). CONCLUSION: Viral agents are essential or prevailing causative agents of AII at different ages. Seasonal and age-related trends of AII morbidity are characterized.

[Acute intestinal infections of virus etiology in adults].

AIM: To describe clinical symptoms of sporadic cases of noro- and astroviral infection in adults; to compare a clinical picture and a course of rotaviral infection (R VI), noroviral infection (NVI) and astroviral infection (A VI). MATERIAL AND METHODS: Reverse transcription and polymerase chain reaction (RT-PCR) method was used to examine fecal samples from 1032 patients with acute intestinal infections (All) at the age of 15 to 90 years admitted to the infectious department of the clinic of Chelyabinsk State Medical Academy in 2004-2005. Detectability and severity of some symptoms in R VI, NVI and A VI patients were analysed and clinical manifestations of viral intestinal monoinfections were compared. RESULTS: Monoinfection was diagnosed in 230 (22.3 %)fecal samples: RVI (n = 101), NVI (n = 100), A VI (n = 29). Sporadic morbidity was the highest from March to May, March to September, October to December for R VI, NVI and A VI, respectively. Gastroenteritis syndrome was prevalent in all the infections studied. Diarrhea with marked dehydration was more typical for RVI, dyspepsia and abdominal pain--for NVI. Intoxication occurred with the same frequency in all the infections. Severe RVI ran with normal body temperature in many patients over 60 years of age. CONCLUSION: Viral diarrheal diseases are frequent in adults of all ages and run with similar clinical picture. The most serious course of the infection with most severe syndromes of gastroenteritis and dehydration was typical for RVI. Etiological nature of viral intestinal infections can be established only with laboratory diagnostic methods.

[Changes in Acholeplasma laidlawii plasma membrane functions during development of resistance to ciprofloxacin and tetracycline]. / Izmenenie funktsional'nogo sostoianiia plazmaticheskoi membrany Acholeplasma laidlawii pri formirovanii rezistentnosti k tsiprofloksatsinu i tetratsiklinu.

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin and tetracycline was investigated. It was shown that one of the important steps of resistance development in acholeplasms is a complex of adaptation metabolic reactions providing limited antibiotic accumulation by the cells. In the case of ciprofloxacin resistance metabolism changes concerning transport systems took place before mutations in target genes. Development of tetracycline resistance of mollecutes after incubation in the medium with enhancing antibiotic concentrations and not connected with the presence of tet(M) determinant was demonstrated for the first time.

[Role of mutations in the A-subunit of Acholeplasma laidlawii PG-8B topoisomerase IV in formation of resistance to fluoroquinolones]. / Rol' mutatsii v A-sub''edinitse topoizomerazy IV Acholeplasma laidlawii PG-8B v formirovanii razistentnosti k ftorkhinolonam.

Nucleotide sequence of Acholeplasma laidlawii genome site PG-8B (1000 n.p.), containing topoisomerase IV subunit genes (parE and parC), has been determined. Sequenced genome site contains a gene fragment coding for the C-terminal region of ParE and gene fragment coding for N-terminal region of ParC. Topoisomerase IV subunite genes in A. laidlawii genome are situated near each other and overlapping by 4 nucleotides. Selection in liquid nutrient medium with ascending antibiotic concentrations resulted in derivation of A. laidlawii PG-8B cells resistant to ciprofloxacin, a fluoroquinolone. The resistant clones contain a mutation in the parC QRDR region determining fluoroquinolone resistance: Ser(91) (corresponding to Ser(80) in Escherichia coli ParC) replacement) for Leu.

[The participation of the transport-barrier functions of the plasma membrane in the development of fluoroquinolone (ciprofloxacin) resistance in Acholeplasma laidlawii]. / Uchastie transportno-bar'ernykh funktsii plazmaticheskoi membrany v razvitii rezistentnosti k ftorkhinolonam (tsiprofloksatsinu) u Acholeplasma laidlawii.

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin was investigated. It was shown that ethidium bromide used as fluoroquinolone analogue in plasmatic membrane efflux pump was accumulated in ciprofloxacin-resistant cells in much less amount. It was estimated that ethidium bromide efflux depended on temperature, glucose and transmembrane electro-chemical proton potential. Inhibitors of efflux systems--reserpine and verapamil enhanced the ethidium bromide accumulation much more intensively in ciprofloxacin resistant cells. The results of investigation allowed to consider the existence of active efflux system for toxic agents in acholeplasma; in the case of ciprofloxacin-resistant strain these systems are inducible.

[Changes in the properties of the plasma membrane of Acholeplasma laidlawii under the action of tetracycline]. / Izmenenie svoistv plazmaticheskoi membrany Acholeplasma laidlawii pri deistvii tetratsiklina.

The role of physico-chemical rearrangements in the cell plasmalemma of Acholeplasma laidlawii in the development of resistance to tetracycline was investigated. The cells of A.laidlawii were shown to be tolerant to tetracycline and to preserve a rather high titre of the cells even at a concentration of the antibiotic in the inoculation medium much higher than the MIC. The results of the investigation of the structural rearrangements in the plasmatic membrane of the cells grown in the presence of tetracycline revealed changes in the lipid flow in the surface layer and an increase in the cholesterol and phospholipid contents. The size of the changes depended on the time of tetracycline addition and the phase of the culture growth.

[Formation of M. hominis and A. laidlawii resistance to fluoroquinolones]. / O formirovanii rezistentnosti k ftorkhinolonam u M. hominis i A. laidlawii.

Mycoplasma hominis and Acholeplasma laidlawii cultures resistant to antibacterial fluoroquinolone drugs ciprofloxacin (Cpf), ofloxacin (Ofl), and lomefloxacin (Lmf) were prepared by selection in liquid nutrient medium with ascending concentrations of Cpf. Resistant mycoplasma clones contained point mutations in the gyrase. A gene region determining quinolone resistance (QRDR gyrA): M. hominis contained C-->T transition resulting in substitution of Ser(83) for Leu and A. laidlawii G-->A resulting in substitution of Asp (91) for Asn. The phenomena of mutation formation during mycoplasma culturing in the presence of fluoroquinolones is studied. In the presence of Cpf in culture medium in concentrations of up to 10 micrograms/ml (for M. hominis) and 1 microgram/ml (for A. laidlawii) the mycoplasma populations contained cells with both altered and wild genotype. Culturing in the presence of higher Cpf concentrations resulted in elimination of cells nonmutant for QRDR gyrA. Besides in vitro studies, we analyzed clinical strains of M. hominis in the presence of different Cpf concentrations. M. hominis clones resistant to Cpf varying in genotypes were detected. These data permit a conclusion that the mechanism of fluoroquinolone resistance formation in mycoplasma includes several stages.