[Antioxidant status evaluation in sportsmen using in their ration natural concentrated food products made by cryogenic technology].

The authors evaluated the level of products of lipid peroxidation and enzymatic component of antioxidant defense in 30 sportsmen (19-22 years) going in for boat racing at pre-competition period, after competition and during one month of subsequent planned training. At the same time, the authors assessed some indices of vitamin (А, Е, В1, В2) and mineral status of human organism (zinc, copper, iron). As the criteria of antioxidant defense status, catalase activity and ceruloplasmin level were detected in blood serum, content of products of lipid peroxidation was evaluated according to levels of malon dialdehyde and nitric oxide metabolites. To assess influence of food ration upon antioxidant defense in sportsmen, natural concentrated food products made by cryogenic technology were administrated to persons of the main group (n=15). The food products were administrated as powder in second course during organized diner. The food products were not given to persons in control group (n=15). First food product consisted of vegetable components and animal protein: cryopowders of rabbit meat, celery, onion, pumpkin, hips. Second food product consisted of cryopowders of red grape, topinambour, beet root and verdure of parsley. Both food products were given 10 grams during 15 days. Results showed before study 40% of sportsmen had high level of products of lipid peroxidation. It probably provided evidence about inadequacy of physical loads. These changes were detected against the background decreased content of vitamins А (10.0% persons), Е (40.0% persons), В2 (43.3% persons), В1 (100.0% persons), copper (30.0% persons) and iron (53.3 % persons). The preparation for sportive competitions led to augmentation of products of lipid peroxidation in control group. In persons of main group using the food products during pre-competition period, the increase of micronutrient saturation was noted; it probably positively influenced upon metabolic processes in human organism and prevented the augmentation of production of malon dialdehyde and nitric metabolites. The enzymatic component activity in control group did not change and was in the range of standard. Since the level of lipid peroxidation metabolites in blood serum in main group significantly decreased at the end of observation, but enzymatic component activity remained high (it provided evidence of higher antioxidant defense), the authors supposed aftereffect due to using in ration food products with elevated content of biologically active substances. Probably, sportsmen of main group more sufficiently endured planned physical loads. Obtained results provided evidence about availability of using food products made by cryogenic technology in ration of sportsmen of different kind of sport.

Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions.

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase beta (pol beta) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol beta was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mbeta16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mbeta16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol beta. These two pol beta knockdown cell lines (NRK-kcdc and Mbeta16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mbeta16tsA-wt cells or N2A cells. The levels of pol beta mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mbeta16tsA-kcdc cells with Mbeta16tsA-wt, N2A or NRK-wt cells had no effect on pol beta levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol beta levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol beta in the wt cells. The inability of Mbeta16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

Scanning near-field optical microscopy using semiconductor nanocrystals as a local fluorescence and fluorescence resonance energy transfer source.

Local fluorescence probes based on CdSe semiconductor nanocrystals were prepared and tested by recording scanning near-field optical microscopy (SNOM) images of calibration samples and fluorescence resonance energy transfer SNOM (FRET SNOM) images of acceptor dye molecules inhomogeneously deposited onto a glass substrate. Thousands of nanocrystals contribute to the signal when this probe is used as a local fluorescence source while only tens of those (the most apical) are involved in imaging for the FRET SNOM operation mode. The dip-coating method used to make the probe enables diminishing the number of active fluorescent nanocrystals easily. Prospects to realize FRET SNOM based on a single fluorescence centre using such an approach are briefly described.

MinK-related peptide 1: A beta subunit for the HCN ion channel subunit family enhances expression and speeds activation.

The HCN family of ion channel subunits underlies the currents I(f) in heart and I(h) and I(q) in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a beta subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a beta subunit for the cardiac delayed rectifier I(Kr), these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.

MEKK4 mediates differentiation in response to retinoic acid via activation of c-Jun N-terminal kinase in rat embryonal carcinoma P19 cells.

Differentiation of P19 embryonal carcinoma cells in response to the morphogen retinoic acid is regulated by Galpha(12/13) and is associated with activation of c-Jun N-terminal kinase. The role of MEKK1 and MEKK4 upstream of the c-Jun N-terminal kinase was investigated in P19 cells. P19 clones stably expressing constitutively active and dominant negative mutants of MEKK1 and MEKK4 were created and characterized. Expression of the constitutively active form of either MEKK1 or MEKK4 mimicked the action of retinoic acid, inducing these embryonal carcinoma cells to primitive endoderm. Expression of the dominant negative form of MEKK1 had no influence on the ability of retinoic acid to induce either JNK activation or primitive endoderm formation in P19 stem cells. Expression of the dominant negative form of MEKK4, in contrast, effectively blocks both morphogen-induced activation of JNK and cellular differentiation. These data identify MEKK4 as upstream of c-Jun N-terminal kinase in the pathway mediating differentiation of P19 stem cells to primitive endoderm.

Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars.

Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.

[Role of migration processes in oncologic epidemiology of Siberia and Far East]. / Rol' migratsionnykh protsessov v onkoépidemiologii Sibiri i Dal'nego Vostoka.

The paper presents the analysis of the cancer death rates among the population of a Norilsk industrial area and compares the rate and structure among newcomers and natives. The persons who long live in this area more frequently die of cancer diseases than migrants. They have high mortality rates for most sites (the stomach, esophagus, bowel, liver, pancreas). The migrants die younger and chiefly of tumors of respiratory organs, the skin, brain, and female genitals. The lower mortality rates in the newcomers are attributable to their departure to their former residence places.

[Chromatomass spectrometric determination of free fatty acids in blood serum]. / Khromatomass-spektrometricheskoe opredelenie svobodnykh zhirnykh kislotv syvorotke krovi.

Complete analysis of blood serum free fatty acids was carried out after methylation of the acids in the mixture containing methanol-acetyl chloride 50:1 and subsequent evaluation of their methyl esters using a gas chromatography analyser. Standard mixtures of free fatty acids and their methyl esters were analyzed before study of blood serum free fatty acids; composition of fish-liver oil (Far Eastern mackerel), containing definite fatty acids was also studied. Combination of gas chromatography and mass spectrometry enabled to identify all the free fatty acids and to detect their main characteristics (time of retention, typical mass spectrum).

NaF and mononucleotides as inhibitors of 3'-5'-exonuclease activity and stimulators of polymerase activity of E. coli DNA polymerase I Klenow fragment.

It has been shown that, in the absence of dATP in the poly(dT).oligo(dA) template-primer complex, the rate of primer cleavage by the E. coli DNA polymerase I Klenow fragment equals 4% of polymerization rate, while in the presence of dATP it equals as much as 50-60%. NaF and NMP taken separately inhibit exonuclease cleavage of oligo(dA) both with and without dATP. The addition of NaF (5-10 mM) or NMP (5-20 mM) increases the absolute increment of polymerization rate 5-9-fold relative to the absolute decrement of the rate of nuclease hydrolysis of primer. This proves the assumption that not more than 10-20% of primer molecules, interacting with the exonuclease center of polymerase, are cleaved by the enzyme. Presumably, NaF and nucleotides disturb the coupling of the 3'-end of oligonucleotide primer to the exonuclease center of the enzyme. As the primers mostly form complexes with the polymerizing center, the reaction of polymerization is activated.

Interaction of dNTP, pyrophosphate and their analogs with the dNTP-binding sites of E. coli DNA polymerase I Klenow fragment and human DNA polymerase alpha.

The 3',5'-exonuclease center of the Klenow fragment of E. coli DNA polymerase I (FK) was selectively blocked by NaF. The latter was shown to forbid the binding of nucleotides and their analogs to the enzyme exonuclease center. In the presence of poly(dT).r(pA)10 template.primer complex and NaF, we observed AMP, ADP, ATP, PPi and dATP to be competitive inhibitors of the FK-catalyzed DNA polymerization. The interactions of the nucleotides with FK and human DNA polymerase alpha were compared to reveal similarity of binding to the DNA polymerizing centers. Structural components of dNTP and PPi playing key roles in forming complexes with pro- and eukaryotic DNA polymerases were identified.

[Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs]. / Vzaimodeistvie dNTP-sviazyvaiushchikh uchastkov DNK-polimerazy alpha cheloveka i fragmenta Klenova DNK-polimerazy I iz Escherichia coli s nukleotidami, pirofosfatom i ikh analogami.

AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.

[Modification of human DNA polymerase alpha by N-acetylimidazole]. / Modifikatsiia DNK-polimerazy alpha cheloveka N-atsetilimidazolom.

The modification of tyrosine residues of the human placenta DNA-polymerase alpha by N-acetylimidazole was investigated. The poly(dT)-template and the r(pA)10-primer a each added separately or simultaneously do not influence the rate of enzyme inactivation. In the presence of poly(dT)-r(pA)10 no effect of dCTP and dTTP (noncomplementary to template) and of dAMP and dADP (complementary to template) on the rate and the level of the enzyme inactivation was found. However dATP revealed practically complete protection. Orthophosphate, pyrophosphate each taken separately do not influence the rate of enzyme inactivation with this reagent. The presence of dADP with either ortho- or pyrophosphate, or dAMP with the one of these ligands leads to half protective action in comparison with dATP. Imidazolides of phosphonoacetic acid and 5'-adenylyl++ 1(phosphonoacetic acid) do not inactivate DNA-polymerase alpha from human placenta and the Klenov fragment of DNA-polymerase I from E. coli. All data obtained allow to suggest that the tyrosine residue in the dNTP binding site of DNA-polymerase reveals stacking with the nucleotide only if dNTP is complementary to the template.

[Collagen and fibronectin of the liver in children with congenital fibrosis, an extrahepatic portal circulation block and post-hepatitis cirrhosis (an immunohistochemical study)]. / Kollagen i fibronektinn v pecheni detei pri vrozhdennom fibroze, vnepechenochnoi blokade portal'nogo krovoobrashcheniia i postgepatitnom tsirroze (immunogistokhimicheskoe issledovanie).

Immunofluorescence techniques were used to examine the distribution of Types I, III, IV and V collagen and fibronectin in the liver biopsy specimens from children with congenital liver fibrosis (CLF), extrahepatic portal blood circulation block, and cirrhosis developed following viral hepatitis. Both common patterns and characteristic features of the development of sclerotic processes were established. The common feature is the development of periportal connective tissue (CT) fibrosis mainly at the expense of Types I, III and V collagen and the accumulation of Types I, III, IV, V collagen and fibronectin in the walls of sinusoids during their "collagenization". The distinctive characteristic of posthepatic cirrhosis is the location of fibronectin and Type IV collagen in fibrous CT. Characteristic of CLF is high fibronectin concentrations in the areas of CT neoplasms along the periphery of portal tracts.

[Modification of tyrosine residues of the Klenow fragment of DNA-polymerase I from Escherichia coli by acetylimidazole]. / Modifikatsiia klenovskogo fragmenta DNK-polimerazy I iz Escherichia coli atsetilimidazolom po ostatkam tirozina.

The modification of tyrosine residues of DNA polymerase I Klenow fragment from E. coli by acetylimidazole has been investigated. This reagent was shown to inactivate both polymerization and 3',5'-exonuclease activities but with different velocity. The poly(dT)-template and r(pA)10-primer each added separately to the enzyme have no notable influence on the rate of enzyme inactivation. Simultaneous presence of both template and primer increases the rate of inactivation. In the presence of poly(dT).r(pA) 10 there is not effect of dCTP and dTTP (noncomplementary to the template) on the rate of inactivation of polymerization activity. However, dATP complementary to the template, provides a complete protection. A weak protective action is detected in the presence of dADP. Orthophosphate, pyrophosphate and dAMP each taken separately increase the rate and the level of the enzyme inactivation. dAMP together with either ortho- or pyrophosphate have the same protective action as ATP. All data obtained allow to suggest the functional significance for polymerization activity of tyrosine located in the dNTP binding site of DNA polymerase I.

[Pathomorphology of congenital liver fibrosis in children]. / Patomorfologiia vrozhdennogo fibroza pecheni u detei.

Pathomorphology of congenital liver fibrosis (CLF) was studied basing on histological evaluation of 42 primary and secondary liver biopsies obtained from 26 CLF children aged 1-13. CLF age-specific complications, their variants and incidence rate as well as morphogenesis are outlined, e. g. cholangiolitis and secondary cirrhosis which are not rare in 7-13-year-old children. Presented is CLF staging.