COMPUTER PREDICTION OF BIOLOGICAL ACTIVITY OF DIMETHYL-N-(BENZOYL)AMIDOPHOSPHATE AND DIMETHYL-N-(PHENYLSULFONYL)AMIDOPHOSPHATE, EVALUATION OF THEIR CYTOTOXIC ACTIVITY AGAINST LEUKEMIC CELLS IN VITRO.

Structural analogues of ß-diketones--dimethyl-N-(benzoyl)amidophosphate (HCP) and dimethyl-N-(phenylsulfonyl)amidophosphate (HSP) were synthesized and identified by the methods of IR, 1H and 31P NMR spectroscopy. Screening of biological activity and calculation of physicochemical parameters of HCP and HSP compounds were done with the use of PASS and ACD/Labs computer programs. A wide range of biological activity of synthesized compounds, antitumor activity in particular, has been found. Calculations of the bioavailability criteria indicate that the investigated compounds have no deviations from Lipinski's rules. HCP compound is characterized by a high lipophilicity at physiological pH as compared to HSP. It was found that cytotoxic effect of the studied compounds on the leukemic L1210 cells was of time- and dose-dependent character. HCP is characterized by more pronounced and early cytotoxic effects as compared to HSP. It was shown that 2.5 mM HCP increased ROS production 3 times in the early period of incubation, and decreased cell viability by 40% after 48 h, and by 66%--after 72 h. Based on the computer calculation and undertaken research, HCP was selected for target chemical modifications and enhancement of its antitumor effect.

[Regulation of sulfates, hydrogen sulfide and heavy metals in technogenic reservoirs by sulfate-reducing bacteria].

Sulfate-reducing bacteria Desulfovibrio desulfuricans Ya-11 in the presence of sulfates and organic compounds in the medium reduce sulfates to hydrogen sulfide (dissimilatory sulfate reduction). Heavy metals in concentration over 2 mM inhibit this process. Pb2+, Zn2+, Ni2+, Co2+, Fe2+ and Cd2+ ions in concentration 1-1.5 mM display insignificant inhibiting effect on sulfate reduction process, and metals precipitate in the form of sulfides. At concentrations of heavy metals 2-3 mM one can observe a decrease of sulfates reduction intensity, and a percent of metals binding does not exceed 72%. Obtained results give reason to confirm, that sulfate-reducing bacteria play an important role in regulation of the level of sulfates, hydrogen sulfide and heavy metals in reservoirs and they may be used for purification of water environment from these compounds.

[Correction of the influence of clinostatic hypokinesis on encephalitic circulatory dynamics during the sleep in patients with hypertensive disease].

During the sleep person is in the state of antiorthostasis, this state provokes vascular cerebral abnormalities (night cerebral hypervolemia). The nature of encephalon blood supply is changing in this horizontal state, in the result there is lowering of hydrostatic blood pressure, rise of the encephalon blood supply and more difficult venous outflow. 60 patients with essential hypertension (1-2 phases) at the age of 64.2 +/- 1.8 years which were in the state of slightly raised upper segment of the head and body during night sleep were examined. By the method of the cerebral venous hemocirculation defined. Conclusion. Cerebral arterial blood flow of examined patients has increased by 25-30% during the sleep, the changes of arterial and cerebral venous hemocirculation have attended hypervolemia. Being in the state of upper (by 10-15 degrees) head of the bed decreases blood redistribution and decreases the extension of cerebral blood circulation's acute abnormalities.

[Regulation of hydrogen sulfide level by acidophobic bacteria of Thiobacillus genus in technogenic reservoirs of sulfur mining regions].

An increase of acidophobic thione bacteria quantity in Rozdil and Yavoriv reservoirs of sulfur mining regions during 2005-2009 years, which correlates with a decrease of hydrogen sulfide content in water surface layers, was shown. The ability of acidophobic bacteria of Thiobacillus genus, isolated from "Yavorivske" lake, to oxidize effectively hydrogen sulfide added into Beijerinck medium instead of thiosulfate, was discovered. It was established, that hydrogen sulfide oxidizing efficiency by Thiobacillus sp. Yav-8, Yav-11 and Yav-14 strains is the highest (78.48-84.56%) when its content in cultivation medium was increased twice: to 2584 mg/l. An increase of sulfur quantity in sodium sulfide form from to six times as compared with its standard content in sodium thiosulfate form in the Beijerinck medium does not lead to the increase of hydrogen sulfide oxidizing efficiency by cells.

[Changes in ratio of lipid components in the rat bile after applying bombesin neuropeptide].

Changes of lipid components ratio in the rat bile after bombesin neuropeptide infusion (1 microg per 100 g of body weight) were studied in the acute experiment using the thin-layer chromatography. The cholates-to-cholesterol, phospholipids-to-cholesterol, cholesterol-to its esters ratios and also the potent cholesterol crystallization index were calculated and analyzed. Obtained data indicate that bombesin takes influence on the colloidal parameters of bile enhancing the secretion of phospholipids, cholesterol and its esters and free fatty acids. The peptide also intensifies cholesterol etherification and improves the colloidal stability of bile. After bombesin application the potential ability of cholesterol to crystallization remains at normal rate indicating that the useful balance of micelle forming elements is unaffected, that prevents from the development of gallstone disease.

[Ethanol into acetaldehyde bioconversion by mutant strains of Hansenula polymorpha Felcao de Morais & Dália Maia].

The influence of some factors (tris-HCl buffer, pH 8.0, concentration, use of different binding agents aeration modes, genetically determined peroxisome degradation damage) on biotransformation efficiency of 0.217 M (1%) ethanol to acetaldehyde at 30 degrees C by Hansenula polymorpha 7-4A (gcrl EAO) strain cells with glucose repression block was investigated. Optimal cultivation conditions for cells were selected. Bioconversion efficiency using 1 M tris-HCl buffer, pH 8.0, was found the highest one as compared with using the buffer in concentrations from 0.1 M to 3 M. The process efficiency when using tris(hydroxymethyl)aminomethane as binding acetaldehyde agent proved much higher than when using sodium bisulfite both at aeration by air stream and incubation on shaker. Using 146 and 179 mutants cells for bioconversion with defects in alcohol oxidase inactivation during macropexophagy stimulated efficiency increase by 5.58% and 8.10%, respectively, as compared with the use of parental 7-4A strain cells.

[Sulfur-oxidizing bacteria of Yavoriv sulfur deposit reservoirs].

Eleven pure bacteria cultures, able to oxidize thiosulfate during growth at pH 7.0-9.4, were isolated from surface layers of Yavoriv sulfur deposit open pit waters. Two cultures proved to be obligate aerobes, but nine cultures performed anaerobic respiration using nitrite, N2O (5 cultures) or only nitrite (4 cultures) as terminal electron acceptors. The growth of all cultures at 22 and 28 degrees C and growth absence at 35, 42 and 55 degrees C was established. All the bacteria are obligate chemolithoauthotrophs, because after cultivation with thiosulfate in the presence ofbiotin, yeast extract, formiate, succinate, arabinose, glucose, fructose and sucrose no growth stimulation was observed, heterothropic growth of any culture was not shown. As to their morphology the cells were bacillary, cytoplasmic membrane was surrounded by three-layer cell wall typical of gram negative bacteria intracellular inclusions, nucleoid, ribosomes and polysomes were also available. On the basis of obtained physiological and morphological characteristics the isolated bacteria cultures were referred to a group of neutrophiles, representatives of genus Thiobacillus, in particular to obligate chemolithoauthotrophs.

Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis.

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

[Community-acquired pneumonia in the elderly: the peculiarities of the clinical picture, diagnosis and treatment].

The article presents the results of studying the peculiarities of the clinical picture, diagnosis and treatment for community-acquired pneumonia in the elderly in view of the interaction of combined pathology of respiratory, cardiovascular and urological systems in order to optimize medical-and-diagnostic support of the above-mentioned category of patients in conditions of the pulmonology department of versatile educational establishment. Optimization of all-round inspection of the sick with pneumonia in the advanced age at an initial stage of treatment in conditions of the pulmonology department so as to reveal the aggravation of internal bodies' combined diseases will allow making early correction of aetiotropic, pathogenetic and symptomatic combined pathology therapy available, which reduces the terms of staying at hospital without damage of diagnosis and treatment.

Structure of the human S100A12-copper complex: implications for host-parasite defence.

S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins. Together with S100A8 and S100A9, it belongs to the calgranulin subfamily, i.e. it is mainly expressed in granulocytes, although there is an increasing body of evidence of expression in keratinocytes and psoriatic lesions. As well as being linked to inflammation, allergy and neuritogenesis, S100A12 is involved in host-parasite response, as are the other two calgranulins. Recent data suggest that the function of the S100-family proteins is modulated not only by calcium, but also by other metals such as zinc and copper. Previously, the structure of human S100A12 in low-calcium and high-calcium structural forms, crystallized in space groups R3 and P2(1), respectively, has been reported. Here, the structure of S100A12 in complex with copper (space group P2(1)2(1)2; unit-cell parameters a = 70.6, b = 119.0, c = 90.2 A) refined at 2.19 A resolution is reported. Comparison of anomalous difference electron-density maps calculated with data collected with radiation of wavelengths 1.37 and 1.65 A shows that each monomer binds a single copper ion. The copper binds at an equivalent site to that at which another S100 protein, S100A7, binds zinc. The results suggest that copper binding may be essential for the functional role of S100A12 and probably the other calgranulins in the early immune response.

Multiple structural states of S100A12: A key to its functional diversity.

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. Together with two other calgranulins, S100A8 and S100A9, it is mostly expressed in human granulocytes, although there is increasing evidence of expression in keratinocytes and psoriatic lesions. It is involved in host-parasite response, and linked to corneal autoimmune diseases connected with filarial parasite infestation. Interaction of S100A12 with a multiligand receptor for advanced glycation end products (RAGE) mediates inflammation. Human recombinant S100A12 was found to induce neuritogenesis of cultured hippocampal cells, similar to two other S100 proteins, S100B and S100A4. X-ray structure of S100A12 has been solved in two crystal forms: R3 and P2(1). In the R3 crystal form S100A12 is a dimer, and in the P2(1) crystal form the dimers are arranged as a hexamer. The hexameric form suggests its role in receptor oligomerisation. S100A12 binds copper at the predicted zinc/copper binding site, which is located close to the surface of the protein. We propose copper-mediated generation of reactive oxygen species by S100A12 as its function in host-parasite response.

[Biodegradation of oil hydrocarbons by Candida yeast]. / Biodegradatsiia vuhlevodniv nafty drizhdzhamy rodu Candida.

Capability of 14 yeast species to utilize oil hydrocarbons has been analyzed. All strains utilized oil hydrocarbons as a single carbon source. Four strains-destructors that are characterized by higher growth in the presence oil in cultivation medium have been chosen among them. Peroxisomes participation in utilization of oil hydrocarbons by strains-destructors has been shown. Availability of peroxisome key enzymes are characteristic of these strains grown in cultivation medium with oil. Numerous peroxisomes available in the cells of some strains grown in oil cultivation medium have been demonstrated. Utilization of a wide spectrum of oil hydrocarbons has been revealed in all four strains. Two strains are promising to be used for environment purification from oil pollution.

[Intra- and interspecies differences in the ISSR-PCR patterns of bumble bee (Hymenoptera: Bombinae)]. / Vnutri- i mezhvidovye razlichiia v ISSR-PCR kharakteristike shmelei (Hymenoptera: Bombinae).

Variation in the ISSR patterns was studied in queens of five species of bumble bee habitating on the territory of Ukraine and Belarus. Species-specific characteristics were obtained for B. sylvarum, B. lapidarius, B. pascuorum, B. terrestris, and B. hortorum with using (GTG)7A and (AGC)6G as primers. Some ISSR markers were common for the groups of species. From 73 to 100% ISSR markers were invariant within the species. Individual characteristics of B. terrestris from geographically distant populations from Kiev and Minsk Oblasts were similar. The level of intraspecific variation was different among the species. Taking into account intraspecific constancy of the major ISSR-PCR patterns and in the same time their susceptibility to individual differences we proposed these markers could be used to elucidate some complicated questions of bumble bee taxonomy.

The structure of S100A12 in a hexameric form and its proposed role in receptor signalling.

S100A12 is a member of the S100 subfamily of EF-hand calcium-binding proteins; it has been shown to be one of the ligands of the 'receptor for advanced glycation end products' (RAGE) that belongs to the immunoglobulin superfamily and is involved in diabetes, Alzheimer's disease, inflammation and tumour invasion. The structure of the dimeric form of native S100A12 from human granulocytes in the presence of calcium in space group R3 has previously been reported. Here, the structure of a second crystal form in space group P2(1) (unit-cell parameters a = 53.9, b = 100.5, c = 112.7A, beta = 94.6 degrees) solved at 2.7A resolution by molecular replacement using the R3 structure as a search model is reported. Like most S100 proteins, S100A12 is a dimer. However, in the P2(1) crystal form dimers of S100A12 are arranged in a spherical hexameric assembly with an external diameter of about 55 A stabilized by calcium ions bound between adjacent dimers. The putative target-binding sites of S100A12 are located at the outer surface of the hexamer, making it possible for the hexamer to bind several targets. It is proposed that the S100A12 hexameric assembly might interact with three extracellular domains of the receptor, bringing them together into large trimeric assemblies.

The three-dimensional structure of human S100A12.

The crystal structure of human EF-hand calcium-binding protein S100A12 in its calcium-bound form has been determined to 1.95 A resolution by molecular replacement using the structure of the S100B protein. The S100 family members are homologous to calmodulin and other related EF-hand calcium-binding proteins. Like the majority of S100 proteins, S100A12 is a dimer, with the interface between the two subunits being composed mostly of hydrophobic residues. The fold of S100A12 is similar to the other known crystal and solution structures of S100 proteins, except for the linker region between the two EF-hand motifs. Sequence and structure comparison between members of the S100 family suggests that the target-binding region in S100A12 is formed by the linker region and C-terminal residues of one subunit and the N-terminal residues of another subunit of the dimer. The N-terminal region of the target-binding site includes two glutamates that are conserved in most of the S100 sequences. The comparison also provided a better understanding of the role of the residues important for intra- and inter-subunit hydrophobic interactions. The precise role of S100A12 in cell behaviour is yet undefined, as is the case for the whole family, although it has been shown that the interaction of S100A12 with the RAGE receptor is implicated in inflammatory response.

Efficient bioconversion of ethanol to acetaldehyde using a novel mutant strain of the methylotrophic yeast Hansenula polymorpha.

We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system. The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H. polymorpha cells growing on methanol. At least three mutations were introduced in the strains. Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast. The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate. Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined. In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin. Under optimal shake-flask conditions using one of the H. polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative.

Crystallization and preliminary X-ray diffraction analysis of human calcium-binding protein S100A12.

S100A12, a member of the calgranulin family, isolated from human blood, has been crystallized by vapour diffusion in the presence of Ca(2+). Crystals belong to the space group R3 with unit-cell dimensions a = b = 99.6 c = 64.2 A. There are two monomers per asymmetric unit, with a solvent content of 57.9%. The crystals diffract to at least 2.2 A resolution and complete X-ray data have been collected to 2.5 A on a conventional laboratory source.

Structure of the intact transactivation domain of the human papillomavirus E2 protein.

Papillomaviruses cause warts and proliferative lesions in skin and other epithelia. In a minority of papillomavirus types ('high risk, including human papillomaviruses 16, 18, 31, 33, 45 and 56), further transformation of the wart lesions can produce tumours. The papillomavirus E2 protein controls primary transcription and replication of the viral genome. Both activities are governed by a approximately 200 amino-acid amino-terminal module (E2NT) which is connected to a DNA-binding carboxy-terminal module by a flexible linker. Here we describe the crystal structure of the complete E2NT module from human papillomavirus 16. The E2NT module forms a dimer both in the crystal and in solution. Amino acids that are necessary for transactivation are located at the dimer interface, indicating that the dimer structure may be important in the interactions of E2NT with viral and cellular transcription factors. We propose that dimer formation may contribute to the stabilization of DNA loops which may serve to relocate distal DNA-binding transcription factors to the site of human papillomavirus transcription initiation.

Microbial O2- and H2O2-electrode sensors for alcohol assays based on the use of permeabilized mutant yeast cells as the sensitive bioelements.

Two types of alcohol-specific microbial/electrochemical biosensors have been developed using specially constructed mutant cells of the methylotrophic yeast Hansenula polymorpha. The cells were immobilized in a calcium alginate gel, and placed between two membranes on the surface of oxygen or hydrogen peroxide-electrodes. The O2 electrode based biosensor contained mutant cells with strongly elevated alcohol oxidase activity. The peroxide electrode based biosensor consisted of catalase-defective mutant cells which produce hydrogen peroxide in the presence of alcohol. Both types of mutant cells were used in permeabilized form in order to release some components of the cellular respiration system, thus increasing the selectivity of the cellular respiration response to alcohol (cell/O2-biosensor) Permeabilization also increased sensitivity of the signal and shortened the response time (cell/H2O2-biosensor). Cell/O2 biosensors were linear up to 1.2 mM for ethanol and 0.35 mM for methanol, cell/H2O2 biosensors were linear up to 4.0 mM for ethanol, and 1.2 mM for methanol. Results were reproducible, sample pretreatment was not required, and the sensors exhibited good operational and storage stability. The use of sucrose, dulcitol or inositol during the preparation of the sensors resulted in increased stability of cells during their liophilization and storage in the dried state. Both biosensors had similar selectivity towards alcohols in the order of methanol (100%), ethanol (21%), and formaldehyde (12%). No signal was observed with glucose or glycerol as substrates.

Expression, crystallization and preliminary X-ray analysis of the E2 transactivation domain from papillomavirus type 16.

The N-terminal transactivation domain of the E2 protein from human papillomavirus type 16 has been crystallized by vapour diffusion. Crystals belong to the space group P3121 (or P3221) with unit-cell dimensions a = b = 54.3, c = 155.5 A. There is one molecule per asymmetric unit with a solvent content of 55%. Crystals diffract to at least 2.5 A resolution and complete X-ray data to 3.4 A have been collected on a conventional laboratory source. This 201 amino-acid domain of the E2 protein has been shown to interact functionally with both the HPV E1 protein and at least three cellular transcription factors, to fulfil its role in the control of viral transcription and replication. A knowledge of the structural basis of these multiple interactions should lead to a fuller understanding of the mechanism of action of this key regulator of the HPV life cycle.