RSH ANTIOXIDANTS INCREASE INTRACELLULAR ROS LEVEL AND PROLIFERATION OF SP2/0 MYELOMA CELLS IN SERUM-FREE MEDIUM.

At present research we have demonstrated the link between introducing of low molecular weight RSH-antioxidants (N-acetylcysteine, glutathione) into serum-free medium composition, reactive oxygen species (ROS) generation and mouse myeloma SP2/0-SF cells proliferative activity. The presence of these compounds in the medium changed the pattern of ROS-activity in cells by concentration-dependent manner, and affected their proliferative characteristics. The optimal value of the proliferative activity was related to 0.2 mM for both thyols and not depended from the thyol-compound nature. Further increasing up from the found concentration optimum lead to growth inhibition with different expression for N-acetylcysteine and glutathione.

Modern principles of classification and development of nutrient media for culturing of human and animal cells.

Analysis of the main principles of classification and development of nutrient media used for culturing of human and animal cells in biology and medicine is presented. The key moments of induction and regulation of mitogenic cascades and their differences in cells of continuous lines, diploid cells, and primary cultures are discussed. Some variants of classification of nutrient media for various cell culture types are described. Peculiarities of composition and the prospects of using serum-free media are discussed. Practical results obtained by the authors in the development of diverse purpose nutrient media are presented.

[New feasibility of using a tissue culture technique in diagnostic oncomorphology in case of neuroblastoma and breast cancer].

The paper gives the results of investigating the cultured tissues from patients with breast cancer (BC) and those with neuroblastoma, by applying both the traditional studies and molecular genetic techniques (FISH). The cultures from the patients with neuroblastoma represented as three cell types--N, S, and I. There are certain similarities between the cell composition in the culture medium and the histological structure of the tumor. Mature neoplasms of the ganglioneuroma type contain S-type cells only while intermediate type I cells are a predominant element of immature neuroblastomas. The presence of a considerable number of I cells in the explants appears to suggest a poor prognosis even when the tumor has a comparatively mature structure as a whole. The results of FISH on explant cells presented a means of detecting a broad range of points that are not recorded on histologic specimens--amplification on the metaphase plate, localization of an amplified product, amplification on one chromosome, and double acentric chromosomes. The results of cultivation of BC cells are difficult to systematize although they offer the advantage of performing the FISH reaction. The authors recommend that the tissue culture method should be more extensively used in the day-to-day diagnostic work of pathology laboratories.

[Influenza virus reproduction in the MDCK cells adapted to growth in serum-free Hybris-2 medium].

Whether the MDCK cell line might adapt to grow in serum-free Hybris-2 medium and influenza viruses might be reproduced in the adapted cells was studied. Seventeen passages using the Hybris-2 medium yielded cells adapted to growth in this medium (MDCK-BS). The reproduction of influenza A (H1N1 and H3N2) and B viruses versus the cells cultured in Eagle's medium was studied. The laboratory strain of influenza A/Aichi/1/68 (H3N2) and the strain B/Ohio/01/05 of influenza B in equal titers were shown to be reproduced in both control cells on Eagle's medium and MDCK-BS cells adapted to growth in the Hybris-2 medium. The reproduction of the strains A/Brisbane/10/07 (H3N2) and A/Solomon Islands/3/06 (H1N1) was less active in the MDCK cells. Each strain of influenza viruses displayed varying infective activities. The developed serum-free Hybris-2 medium may be used for cultivation of monolayer continuous MDCK cells and for their reproduction of influenza A and B viruses.

Studying the proliferation of human peripheral blood T lymphocytes in serum-free medium.

We compared the cultivation of human peripheral blood lymphocytes in serum-free medium Hybris-2 and RPMI 1640 medium with 10% fetal bovine serum in the presence of phytohemagglutinin and interleukin-2. The optimal concentration of phytohemagglutinin significantly differed in serum-free and serum-containing media (0.5 and 5 microg/ml, [corrected] respectively). Both mitogens were more potent in stimulating the proliferation of lymphocytes in serum-free medium than in serum-containing medium. Strong proliferation of CD3(+) and CD4(+) T lymphocytes was observed in both media. The dynamics of other markers was similar in serum-free and serum-containing media. However, significant differences were revealed between individual donors. Our results indicate that the developed serum-free medium may be used in lymphocyte cultivation for scientific, diagnostic, and therapeutic purposes.

Reconstitution of calcium-triggered membrane fusion using "reserve" granules.

Calcium-gated secretion of proteins involves the transfer of "reserve" granules, exocytotic vesicles that are cytoplasmic and, hence, plasma membrane-naive, from the cell interior to the surface membrane where they dock prior to fusion. Docking and subsequent priming steps are thought to require cytoplasmic factors. These steps are believed to induce fusion competence. We have tested this hypothesis by isolating reserve granules from sea urchin eggs and determining under which conditions these granules will fuse. We find that isolated reserve granules, lacking soluble cofactors, support calcium-dependent membrane fusion in vitro. Preincubation with adenosine 5'-3-O-(thio)triphosphate and guanosine 5'-3-O-(thio)triphosphate did not prevent fusion. Thus, isolated reserve granules have all the necessary components required for calcium-gated fusion prior to docking.

Membrane-bound phenylalanine hydroxylase of human liver.

Phenylalanine hydroxylase (EC 1.14.16.1) antigen and activity have been identified among proteins extracted with a buffer containing 0.4% Triton X-100 from adult human liver bioptate fraction, which was sedimented at 105,000 x g (n = 4). This enzyme fraction was designated as a 'membrane-bound form of phenylalanine hydroxylase'. It amounted to 5-15% of phenylalanine hydroxylase activity and 15-25% of phenylalanine hydroxylase antigen content. After immunoblotting two-dimensional gels, the soluble (cytoplasmic) form of phenylalanine hydroxylase antigen displayed three spots: one spot corresponded to the L-subunit with a molecular weight of 55,000, the two other spots corresponded to the H-subunit with a molecular weight of 57,000. Only the L-subunit was revealed in the membrane-bound enzyme form. Both phenylalanine hydroxylase activity and antigen were also demonstrated in extracts from human embryonic livers (n = 7). However, in this case the membrane-bound phenylalanine hydroxylase amounted to 85% of the antigen content. Subunit compositions of the enzymes were similar in adult and embryonic livers. The differences in the subunit compositions and enzyme activities of membrane-bound and cytoplasmic forms of phenylalanine hydroxylase in adults and embryos may be due to other functions of this enzyme in the hepatocyte membrane.

[Phenylalanine hydroxylase-like antibody in human chorionic villi]. / Fenilalaningidroksilaz-podobnyi antitel v vorsinakh khoriona cheloveka.

An antigen similar by electrophoretic mobility to liver phenylalanine hydroxylase (PH) and cross-reacting with monoclonal antibody PH8 against liver PH was detected in extracts of soluble proteins in 6 from 23 samples of chorionic villi. An antigen with electrophoretic mobility corresponding to 40-41 kDa was detected in extracts of membrane proteins from these 23 samples by immunoblotting with monoclonal antibody PH8. Its molecular weight was similar to that of major chymotryptic peptide of human liver PH. The content of the antigen varied with samples and was less than 20 ng/mg of the extracted protein. Two-dimensional gel electrophoresis revealed only 1 spot of the antigen. The antigen did not react with monoclonal antibodies PH7 and PH9 epitopes of which were located in N-terminal fragment of liver PH. These data suggest that the antigen of membrane fraction could be a PH protein without N-terminal domain.

[The use of highly sensitive immunoblotting for detecting the phenylalanine hydroxylase antigen in human platelets]. / Primenenie immunoblottinga povyshennoi chuvstvitel'nosti dlia vyiavleniia fenilalaningidroksilaznogo antigena v trombotsitakh cheloveka.

Incubation of nitrocellulose filters containing proteins in solutions of organic alcohols (ethanol, methanol, isopropanol) enabled to increase the immunoblotting sensitivity after denaturating electrophoresis in presence of SDS. Maximal elevation of the label sensitivity (4-6-fold) was observed after incubation of these filters in 30% isopropanol within 2 hrs. The effect of sensitivity elevation appears to be caused by the antigen renaturation due to SDS washing off. The modified procedure of immunoblotting allowed to detect phenylalanine hydroxylase antigen in human thrombocytes. The antigen had electrophoretic mobility similar to that of phenylalanine hydroxylase from liver tissue; its concentration constituted less than 0.1 microgram per 1 mg of protein in thrombocytes.

[Detection of phenylalanine hydroxylase protein in human platelets. Immunochemical detection]. / Obnaruzhenie belka fenilalaningidroksilazy v trombotsitakh cheloveka. Immunokhimicheskaia identifikatsiia.

A protein reactive with anti-phenylalanine hydroxylase monoclonal antibody PH8 has been recovered from human platelet extracts. Two bands corresponding to molecular masses of about 60 kDa and 55 kDa were revealed by immunoblotting after electrophoresis according to Laemmli. Using the same antibody, a single band with a molecular mass of 60 kDa was demonstrated in extracts from human pineal gland; two similar antigens were found in human liver extracts and no antigen was found in adrenal gland extracts. Monoclonal antibodies, PH1 and PH3, did not react with these antigens during immunoblotting. Monoclonal antibodies, PH7 and PH9, reacted with the 55 kDa antigen in platelet extracts. The antigen content in platelet extracts was measured by ELISA with monoclonal antibodies relative to its content in the liver. The antigen content in platelet extracts was about 100 times as low as that in liver extracts and amounted to 100 ng/mg of protein. These findings suggest that the phenylalanine hydroxylase antigen is present in human platelets.

[The dynamics of the properties of liver phenylalanine hydroxylase in human embryogenesis]. / Dinamika svoistv fenilalaningidroksilazy pecheni v émbriogeneze cheloveka.

Content, subunit composition and activity of phenylalanine hydroxylase's (PH) (EC 1.14.16.1) in cytoplasmic and membrane proteins extract of embryonic liver on week 6-11 of pregnancy were studied. PH enzymatic and antigenic activities were detected starting from the week 6 of pregnancy. Liver cytoplasmic PH antigen content increased gradually during development while its enzymatic activity remained practically unchanged. Concomitantly, relative content of L-subunit increased. Content of liver membrane PH antigen was constant during development. Samples of liver with relatively low specific PH activity were characterized by high content of PH in cytoplasm and vice versa. Since PH activity in extracts prepared from mixture of these samples decreased, an unknown PH inhibitor must be present in cytoplasmic protein extracts with relatively low specific PH activity.

[Phenylalanine hydroxylase of human embryos: its homology with the enzyme from the liver of adults]. / Fenilalaningidroksilaza émbrionov cheloveka: gomologiia s fermentom iz pecheni vzroslykh liudei.

Phenylalanine hydroxylase (PH) activity was discovered in the liver of 7-12 week old human embryos. Embryonic and adult PHs were identical, as shown by immunoelectrophoresis. Unlike the adult liver PH, the PH content of the extract of cytoplasmic proteins of embryonic liver was reduced but the specific activity was increased more than by one order of magnitude. H (57,000 D) and L (55,000 D) subunits were detected by immunoblotting. The L subunit predominates in the extract of membrane proteins of embryonic liver. Hence, the major part of phenylalanine oxidizing activity in the embryonic liver is related to the enzyme immunochemically identical with the PH of adult liver but differing from it in some structural and functional properties.

[Immunochemical detection and characteristics of the subunit composition of phenylalanine hydroxylase in the brain of man]. / Immunokhimicheskoe vyiavlenie i kharakteristika sub"edinichnogo sostava fenilalaningidroksilazy v mozge cheloveka.

Immunochemical properties and subunit structure of an antigen were characterized in autopsy specimens of human liver and brain, using antiserum against human phenylalanine hydroxylase. An identical antigen was revealed in extracts of organs by immunoelectrophoresis. Its content was 1.5-2.0 mg/g tissue in the liver and 20-40 micrograms/g tissue in the brain. One L enzyme subunit and two H subunits were identified in the liver extracts after two-dimensional electrophoresis followed by immunoblotting. Subunit structure of phenylalanine hydroxylase in the brain was similar to that in the liver. The molecular weight of L subunit was 55,000 and it was located in the same area as albumin isoforms. The molecular weight of H subunits was 57,000 and they differed from L subunits in pI. The antigen was purified from crude extracts of biopsy liver by affinity chromatography on immunoadsorbent to phenylalanine hydroxylase and showed phenylalanine hydroxylase activity. An antigen with similar molecular weight was also purified from the brain extract by the same method. These data suggest that phenylalanine hydroxylase can be present in the human brain.

[Immunochemical detection of phenylalanine hydroxylase in extracts of various human organs]. / Immunokhimicheskoe vyiavlenie fenilalaningidroksilazy v ékstraktakh nekotorykh organov cheloveka.

Phenylalanine hydroxylase was found in extracts from autoptates of human liver, kidney, myocardium, small intestine, lung and spleen. In all the tissues studied, except of spleen, the antigen was detected by immunoelectrophoresis with monospecific antiserum against human liver phenylalanine hydroxylase. As shown by immunoblotting carried out after electrophoresis under denaturating conditions, the antigen, observed in various tissues, exhibited similar electrophoretic mobility which was very close to that of the enzyme purified from human liver tissue. Molecular mass of revealed antigen was estimated 55-57 kDa. Coincidence of immunochemical and chemical properties of the protein suggested that the antigen, detected in the tissue extracts, was a product of phenylalanine hydroxylase gene expression. The antigen concentration did not correlate with the content of albumin in tissue extracts, thus demonstrating that the revealed antigen did not occur in these preparations with blood contaminations. Content of the antigen in the tissue extracts studied was (ug per g): liver - 1500-1900, kidney - 300-575, brain - 20-40, myocardium - 85-105, lung - 40-125, small intestine - 45-70, spleen - 0-12.

[Identification of the membrane form of phenylalanine hydroxylase from the human liver and characteristics of its subunit composition]. / Vyiavlenie membrannoi formy fenilalaningidroksilazy pecheni cheloveka i kharakteristika ee sub''edinichnogo sostava.

Phenylalanine hydroxylase was detected among human liver bioptats and autoptats extracted with 0.4% Triton X-100 from the 105,000 g homogenate fraction. In contrast to the membrane form of the rat liver enzyme, human liver phenylalanine hydroxylase is detected both by its enzymatic activity and immunochemically under non-denaturating conditions. The enzymatic activity of phenylalanine hydroxylase makes 5-15% of that of the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction as can be evidenced from rocket immunoelectrophoresis data. Immunoblotting of proteins performed after denaturating electrophoresis of the membrane and cytoplasmic fractions revealed an antigen band with a similar electrophoretic mobility. The subunit composition of the enzyme in both fractions was characterized by two-dimensional electrophoresis with subsequent immunoblotting. It was found that the membrane fraction of the enzyme is represented only by the L-subunit with Mr of 55 kD, whereas the cytoplasmic fraction, besides the predominant L-subunit, also contains 2H-subunits of the enzyme with Mr = 57 kD. These 2H-subunits differ between themselves as well as from the L-subunit by the pI value.

[Detection and characteristics of membrane form of phenylalanine hydroxylase (EC 1.14.16.1) from the rat liver]. / Vyiavlenie i kharakteristika membrannoi formy fenilalaningidroksilazy (KF 1.14.16.1) pecheni krys.

The proteins extracted with 0.4% Triton X-100 from the 105000 g homogenate fraction were shown to possess the phenylalanine hydroxylase (EC 1.14.16.1) activity. This phenylalanine hydroxylase fraction was designated as the membrane form of the enzyme. However, immunochemical methods of the antigen analysis performed under non-denaturating conditions and employing monospecific antisera to phenylalanine hydroxylase (double immunodiffusion in agar, racket immunoelectrophoresis, enzyme purification on immunoadsorbents) failed to reveal the antigen among the membrane fraction proteins of the liver. In this fraction the antigen was identified only by immunoblotting performed after electrophoresis of the proteins under denaturating conditions. The molecular mass of the cytoplasmic and membrane forms of the enzyme subunits is identical (52 kD). The Km value of phenylalanine for the cytoplasmic form of phenylalanine hydroxylase is 0.32.10(-3) M, that for the membrane form is 1.66.10(-3) M. Both enzyme forms can bind to phenyl-Sepharose after their activation by the substrate, and they dissociate from the carrier after phenylalanine removal from the incubation mixture, which points to the intactness of the phenylalanine binding allosteric center in the membrane form of the enzyme. This finding allowed for the purification of the membrane form of phenylalanine hydroxylase by affinity chromatography on phenyl-Sepharose.

[Genetic heterogeneity and approaches to the prenatal diagnosis of phenylketonuria (review)]. / Geneticheskaia geterogennost' i podkhody k prenatal'noi diagnostike fenilketonurii (obzor).

Analysis of the data on structure polymorphism of phenylalanine hydroxylase in mammals including man is of importance in elucidation of the enzyme structural alterations in the patients with phenylketonuria. Molecular-genetic approaches are developed for prenatal diagnosis of hereditary diseases; approaches for studies on the phenylketonuria genetic heterogeneity have to be developed. The most promising methods for the prenatal diagnosis appear to be based on the oligonucleotide probes or on use of specific restrictases, recognizing mutant sites in the gene. This suggests that analysis of the intralocus genetic heterogeneity of phenylketonuria is one of important problems in studies of the genetic heterogeneity.

[Isolation of inactive phenylalanine hydroxylase from autopsy specimens of human liver]. / Poluchenie neaktivnogo belka fenilalaningidroksilazy iz autoptatov pecheni cheloveka.

An electrophoretically homogeneous protein has been isolated from human liver autoptats, using a procedure employed for the isolation of phenylalanine hydroxylase from rat liver. The procedure includes chromatography of liver extracts on phenyl-Sepharose and subsequent purification on DEAE-Toyopearl. The activity of phenylalanine hydroxylase in the autoptats was markedly decreased in comparison with that in bioptats. The isolated protein possessed no enzymatic activity. However, the subunit composition of the protein, the molecular masses of protein subunits (55 and 57 kD) and the amino acid composition were close to those of the human enzyme. Antibodies to the protein inhibited the phenylalanine hydroxylase activity in human liver bioptats and weakly inhibited the rat enzyme. The experimental results suggest that the structural organization of phenylalanine hydroxylase does not alter as a result of the loss of enzymatic activity in cadaverous human liver.

[Isolation and characteristics of highly-purified phenylalanine hydroxylase from the rat liver]. / Poluchenie i kharakteristika vysokoochishchennoi fenilalaningidroksilazy iz pecheni krys.

Electrophoretically homogenous phenylalanine hydroxylase was isolated from rat liver tissue using a number of methods involving affinity chromatography on phenyl-Sepharose, ion exchange chromatography on DEAE-Toyoperle and hydrophobic rechromatography on phenyl-Sepharose. Molecular mass of the enzyme was equal to 50,000 Da and Km = 4.4 X 10(-3) M with phenylalanine as a substrate. The enzymatic preparations were used for immunization of rabbits and the monospecific antiserum towards phenylalanine hydroxylase was produced.

[Oligomerization of phenylalanine hydroxylase during its activation with phenylalanine]. / Oligomerizatsiia fenilalaningidroksilazy pri ee aktivatsii fenilalaninom.

Activated and nonactivated forms of phenylalanine hydroxylase varied in the activity within the first minutes of the reaction initiated by means of a synthetic coenzyme 6,7-dimethyl-5,6,7,8-tetrahydropteridine. In activation of the enzyme by phenylalanine the degree of its oligomerization was altered. As shown by chromatography and ultracentrifugation nonactivated enzyme was a dimer with molecular mass 130,000 daltons, whereas the activated form was found to be tetramer of 260,000 daltons. H and L subunits of phenylalanine hydroxylase were equally important in organization of the tetramer. Activation of phenylalanine hydroxylase altered the enzyme affinity to hydroxyapatite. Thus, allosteric activation of phenylalanine hydroxylase occurred due to the enzyme oligomerization.