L-Homoserine: a novel excreted metabolic marker of hepatitis B abnormally produced in liver from methionine.

The hepatitis B infection leads to various profound pathological processes in liver metabolism. Some biochemical alterations detectable by blood analysis are currently used for a preliminary evaluation of the infection. Based on existing data we present here evidence that non-protein amino acid L-homoserine is a pathological, hepatitis B-induced metabolite that is formed and excreted into urine from methionine via splitting S-adenosylmethionine. The urine L-homoserine is proposed as a new marker in the pre-diagnosis examinations that is easier for the clinical analysis than currently used blood test, and is applicable to large-scale epidemiological surveys of the probability of hepatitis B.

Post-panning computer-aided analysis of phagotope collections selected with neurocysticercosis patient polyclonal antibodies: separation of disease-relevant and irrelevant peptide sequences.

The homology of peptide sequences selected from a 7mer phage display library with antibodies elicited by the multicelled parasite Taenia solium in cerebrospinal fluid and serum of neurocysticercosis (NCC) patients and by antibodies of uninfected control patients with similar neurological complications of other ethiology (non-NCC) were analyzed using a PILEUP-Tudos sequence alignments program. The analysis generated dendrograms bearing two types of sequence clusters, those containing (1) only NCC patients-derived peptides and (2) both NCC- and control non-CC -- patient derivatives. By using ELISA, peptides that were selected by the antibodies were identified predominantly in the NCC-derived clusters. In repeated analysis in which sequences were added or removed, the first type of clusters maintained their structure, while the second type of clusters were split into many separate homology units dispersed throughout the guide tree. These results are interpreted as the ability of the analysis to segregate NCC-specific peptide sequences from other sequences. Altogether, this study demonstrates the high potential of the PILEUP-Tudos computer program to analyze phagotope collections recovered through biopanning with polyclonal antibodies elicited in patients by complex and as yet unknown multiple pathogenic antigens and to separate all phagotopes that are disease-relevant on the basis of the sequence homology.

Epitope mapping on N-terminal region of taenia solium paramyosin.

Epitope mapping of the amino-terminal 20aa sequence from Taenia solium paramyosin (TPmy), an immunodominant protein involved in the complex host-parasite relationship in human and porcine cysticercosis is reported. A 12-mer random peptide phage display library was screened with antibodies raised against a synthetic peptide corresponding to the amino-terminal 20aa sequence of TPmy, its highly immunodominant region. In total, 57 clones isolated in two panning conditions were analyzed, of which a single group of 14 sequences found in 25 clones shared a consensus motif showing structural similarity with the antigen Arg10-Thr16 region.

[Targeting the gene for platelet-derived growth factor A-chain (PDGF-A) and preparation of chimeric mice with a "knockout" genome]. / Targeting gena A-tsepi trombotsitarnogo faktora rosta (PDGF-A) i poluchenie khimernoi myshi s "nokautirovannym" genom.

A vector was selected for homologous recombination to generate mouse embryonic stem cell clones with disrupted platelet-derived growth factor A (PDGF-A) chain gene. A chimeric mouse with targeted PDGF-A gene has been created.

[Determination of homoserine kinase activity by chromatographic separation and measurement of reaction products]. / Opredelenie aktivnosti gomoserinkinazy putem khromatograficheskogo razdeleniia i izmereniia produktov reaktsii.

A highly specific procedure for quantitative assay of the homoserine kinase activity in an optimized enzymatic reaction using 14C-labelled homoserine or [gamma 33P]-ATP as substrates, and paper or thin-layer chromatography for separation of the formed o-phosphohomoserine, is described. The procedure is simple, sensitive and allows the assay for the activity of both purified and non-purified homoserine kinases.

[Cloning and structural characteristics of human hair keratin genes rich in sulfur]. / Klonirovanie i strukturnaia kharakteristika genov bogatykh seroi keratinov volos cheloveka.

Two human genomic genes for the hair high-sulphur keratins were for the first time cloned in a 15 kb fragment. The primary structures of the coding regions of the genes and their 5'- and 3'-flanks were determined. In the 5'-flanking region, TATA boxes, initiating codons and a 18 nucleotide sequence, previously described in sheep keratin genes and designated as "the matrix-specific" sequence was revealed. Basing on the nucleotide sequences, the encoded amino acid sequences of the high-sulphur keratins were determined for the first time. The suggested functional role of the structural elements (regions) revealed in the proteins primary structure and problems concerning their evolution tendencies are discussed.

[Influence of the expression of the human growth hormone releasing factor gene on hormone levels in a transgenic rabbit]. / Vliianie ékspressii gena rilizing-faktora gormona rosta cheloveka na uroven' gormonov u transgennogo krolika.

A transgene rabbit with the human growth hormone releasing factor gene was produced by a method of microinjections into fertilized oocytes; a mouse metallothionein I gene was used as a promoter. Gene expression was accompanied by a phenotypical effect, expressed in increasing the rates of development. The maximum difference among the transformant, transplants and control was revealed on the 30-45th day of postnatal development. Analysis of the hormonal status of the transgene animal has shown change in the levels of the majority of hormones: a 6-10-fold increase in insulin; a 2-3-fold increase in the level of triiodothyronine, thyroxin; a reduced somatostatin concentration, a two-fold decrease in the level of progesterone, and a four-fold decrease in the level of testosterone. Activation of the promoter zone with Zn++ salts for 5 weeks resulted in a further increase in the transformant body mass by 10%. However blood hormone levels in the transgene rabbit returned to normal. Proceeding from the above it can be assumed that exogenous gene expression probably increased somatotropin secretion which determined dysfunction of most of the endocrine glands; the effect of somatotropin was probably insulin-mediated.

[Biological effect of human erythropoietin in transgenic mice]. / Biologicheskii éffekt éritropoétina cheloveka u transgennykh myshei.

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).

[Localization of fragments binding nuclear proteins in the promotor region of the human alpha-1-antitrypsin gene]. / Lokalizatsiia v promotornoi oblasti gena alpha-1-antitripsina cheloveka uchastkov sviazyvaniia s iadernymi belkami.

Human and mice nuclear extracts from livers and mice spleen extract were analysed in an attempt to find any proteins capable of binding to the human alpha 1-antitrypsin gene promoter. The nuclei of all studied tissues contain such proteins. The proteins were partially purified on DEAE-trisacryl, heparin sepharose and phosphocellulose columns. The multiple sites for liver nuclear proteins binding to the human alpha 1-antitrypsin gene promoter were found by the DNAse I footprinting technique.

[Induction of unstable mutations in Drosophila melanogaster by microinjection of oncogenic virus DNA into polar embryonic plasm. Malignant effect of oncoviral DNA]. / Induktsiia nestabil'nykh mutatsii u Drosophila melanogaster mikroin''ektsei DNK onkogennykh virusov v poliarnuiu plazmu émbrionov. Maligniziruiushchii éffekt onkovirusnykh DNK.

We have demonstrated that the ability to induce benign neoplasms We have dominant mode of inheritance in Drosophila melanogaster is the specific feature of oncoviral DNAs. It is supposed that development of this type of neoplasms in Drosophila is connected with the changes in expression of protooncogenes in mutant genome: firstly, the genetic factors directing the development of neoplasms and Drosophila protooncogenes which shared the homology with v-src are localised in the same regions; secondly, there are structural rearrangements in c-src/fps (29A) protooncogene in mutant stocks which display the ability for neoplastic growth.

[Induction of unstable mutations in Drosophila melanogaster by microinjection of oncogenic virus DNA into the embryo polar plasma. Insertional nature of mutations]. / Induktsiia nestabil'nyukh mutatsii u Drosophila melanogaster mikroin"ektsiei DNK onkogennykh virusov v poliarnuiu plazmu émbrionov. Insertsionnaia priroda mutatsii.

We have demonstrated that mutations induced in Drosophila melanogaster by the microinjections of adenovirus Sa7 DNA in early embryos are of insertional nature. The role of insertional elements is played by the Drosophila transposons, but not by the virus DNA. The ability of oncoviral DNA to induce transpositions of mobile elements in recipient genome is the molecular basis of this system of genetic instability.

[Induction of unstable mutations in Drosophila melanogaster by microinjections of oncogenic virus DNA into the embryo polar plasma. Prolonged genetic instability of mutations at the lobe locus]. / Induktsiia nestabil'nykh mutatsii u Drosophila melanogaster mikroin"ektsiei DNK onkogennykh virusov v poliarnuiu plazmu émbrionov. Prodlennaia genet'icheskaia nestabil'nost' mutatsitii v lokuse lobe.

Mutations Lobe induced by the microinjections of RSV cDNA into Drosophila melanogaster early embryos are characterized by permanent genetic instability; the level of this instability is being changed in time. Based on the results of genetic analyses of Lobe mutations and molecular analysis of white and ADH mutations induced at high frequency in this system of gene instability, we supposed that unstable mutations which arose under the influence of retroviral cDNA are of the insertional nature.

[Design of a gene coding encoding a polypeptide, mixing the enzyme activity of Escherichia coli homoserine kinase and threonine synthetase]. / Konstruirovanie gena, kodiruiushchego polipeptid, sovmeshchaiushchii fermentativnye aktivnosti gomoserinkinazy i treoninsintetaty Escherichia coli.

The functioning of Escherichia coli threonine operon isolated genes thrB and thrC was studied by using the genetic complementation and enzymatic activity determination techniques. A new gene thrBC was obtained by the genes merging. The genes thrB and thrC were shown to function in Escherichia coli cells independent of the operon and the polipeptide encoded by the thrBC gene combined the functions to express the products of both genes in bacterial cell. At the same time the enzyme coded for by the merged genes demonstrates the level of activity compared with the ones of the isolated genes.

[The structure of PstI-repeats adjacent to the growth hormone gene and transcription of the family of PstI-repeats in various bovine organs]. / Struktura PstI-povtorov, prilegaiushchikh k genu gormona rosta, i transkriptsiia semeistva PstI-povtorov v razlichnykh organakh byka.

Previously cloned PstI-repeats located in 5'- and 3'-flanking sequences of the bovine growth hormone gene were studied in details. The size of the repeats (1,200-2,000 b.p.), the degree of homology, the orientation and direction of their transcription were determined. They were shown to be transcribed in thymus, liver, kidneys, pancreas, adenohypophysis, spleen, although with different efficiency. The transcripts are polyadenylated and heterogeneous in size. Partial asymmetry of PstI-repeat family transcription was shown.

[Several copies of angiogenin gene are present in the mammalian genome]. / Gen angiogenina predstavlen neskol'kimi kopiiami v genomakh mlekopitaiushchikh.

Human cDNAs coding for angiogenin were isolated from Li7 hepatoma. Analysis of the nucleotide sequences of isolated cDNA clones and its comparison with recently published sequences of cDNA and human angiogenin gene permitted to suggest that an intron is present in the 5' region of the gene, dividing the coding and 5' untranslating regions. The size of the intron exceeds 1700 b.p. Up to now it has been known that the angiogenin gene is a single copy gene. However our results of blot-hybridization with genomic DNA of some mammals showed that there are 2-3 copies of the gene in their genomes. According to our results the angiogenin gene is conserved in mammalian genomes.

[Cloning of tobacco DNA fragment with promoter properties in a transgenic plant]. / Klonirovanie fragmenta DNK tabaka, obladaiushchego svoistvami promotora v transgennom rastenii.

A selection system for isolating DNA sequences with transcription-promoting activity by their functioning in bacterial cells has been proposed. Tobacco nuclear DNA fragments were inserted in front of the promoterless neomycin 3'-phosphotransferase II (NPT-II) gene and promoter-like sequences were identified by their ability to restore NTP-II activity in E. coli cells. One of these recombinant plasmids was introduced in tobacco protoplasts by direct gene transfer and transformed calli were isolated by kanamycin selection. The NTP-II expression in regenerated transgenic plants were highest in root, slightly lower in stem and were practically absent in leaf. Sequence analysis of cloned segment showed the presence of conserved sequences essential for promoter activity in eukaryotic cells. A transcription start site was observed by S1 mapping. The size of protected fragments corresponds to the initiation of transcription 176 and 179 base pairs upstream the initiation codon in tobacco and 75 base pairs in E. coli.

[Induction of unstable mutations in Drosophila melanogaster by microinjections of DNA from oncogene viruses into the embryonic polar plasm. Part IV. Malignantization of tissues of mutant larvae]. / Induktsiia nestabil'nykh mutatsii u Drosophila melanogaster mikroin''ektsiei DNK onkogennykh virusov v poliarnuiu plazmu émbrionov. Soobshcheniie IV. Malignizatsiia tkanei mutantnykh lichinok.

We have demonstrated that larval tissues of mutant stocks induced by injections of oncogene virus DNA (Sa7 and RSV) show the neoplastic mode of growth after transplantation into the body cavity of wild-type flies. Neoplasms tested were shown to be ordinary benign insect neoplasms, and at the same time they show the dominant mode of heredity typical of the neoplasms of vertebrates. Genetic factors responsible for the neoplastic growth are localised in the 3rd chromosome in the stocks obtained after injections of the adenoviral Sa7 DNA, and in the 2nd and 3rd chromosomes in the mutant stocks induced by the retroviral DNA.

[Characteristic of segments of transgenic animal genomes, adjacent to integrated sequences of foreign DNA]. / Kharakteristika uchastkov genoma transgennykh zhivotnykh, prelezhashchikh k integrirovannym posledovatel'nostiam chuzherodnoi DNK.

DNAs of seven transgenic mice and one transgenic rabbit was divided into fractions according to reassociation kinetics and GC-content. Moderate and/or frequent (reverse) repeated sequences of the genome were revealed in all cases next to different transgenes. DNA fractions containing foreign sequences differed by the GC-content in different transgenic animals.