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Biomedical subjects

N Horie

Publications and source records attributed to N Horie.

At least 19 recordsLinked to original sources
PubMed1995

Identification and characterization of two nitrogen-regulated genes of the cyanobacterium Synechococcus sp. strain PCC7942 required for maximum efficiency of nitrogen assimilation.

Two nitrogen-regulated genes were found in the genomic DNA region upstream of the nirA operon involved in uptake and utilization of nitrate in Synechococcus sp. strain PCC7942. The two genes (nirB and ntcB) are transcribed divergently from nirA and encode proteins of 349 and 309 amino acid residues, respectively. The levels of nirB and ntcB transcripts were low in cells growing on ammonium and increased upon transfer of ammonium-grown cells to nitrate-containing medium. The deduced NirB protein sequence has no similarities to other known proteins, whereas the deduced NtcB protein sequence is homologous to bacterial transcriptional activators of the LysR family. Defined mutants constructed by interrupting nirB or ntcB with a drug resistance marker grew as fast as the wild-type strain on ammonium but grew slower than the wild-type strain on nitrate or nitrite. The nirB mutant had higher activities of nitrate reductase, glutamine synthetase, and glutamate synthase than the wild-type strain, but its nitrite reductase activity was 40% of the wild-type levels. The mutant excreted nitrite into the medium during growth on nitrate, showing that nitrite reductase limits nitrate assimilation. These findings suggested that nirB is required for expression of maximum nitrite reductase activity. When grown on ammonium, the nirB mutant grew normally but cultures of the ntcB mutant still showed a yellowish-green color typical of nitrogen-limited cells. NtcB seems to regulate utilization of fixed nitrogen by controlling the expression of a certain gene(s) involved in nitrogen metabolism.

Amino Acid Sequence↗
PubMed1995

Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase.

Triple tandemly repeated sequences and the corresponding complementary sequence are known to exist in the 5'-terminal regulatory region of the human gene for thymidylate synthase (TS). To examine the function of these sequences, a set of deletion mutants was prepared and used in a transient expression assay. The results showed that at least one repeated sequence and its complementary sequence were necessary for the efficient expression of the gene. As another approach to understanding the function of this unique structure, DNA polymorphism in the same region was analyzed. In addition to the TS gene with the triple tandem repeat, the TS gene with a double tandem repeat was found in genomes of normal human subjects at an estimated frequency of 19% when genomes of 21 unrelated Japanese were analyzed. The expression activity of a reporter gene linked to the promoter region of the human TS genes with the two types of repeated sequence was examined and the result showed that the expression activity of the gene with the double repeat was lower than that of the gene with the triple repeat in the transient expression assay. Thus, it appears that the unique repeated sequences in the 5'-terminal region of the human TS gene are polymorphic and contribute to the efficiency of expression of the gene.

Base Sequence↗
PubMed1994

Recognition of UUN codons by two leucine tRNA species from Escherichia coli.

Codon recognition by Escherichia coli tRNA(Leu)4 and tRNA(Leu)5 was investigated by analysis of the competition between two aminoacyl-tRNA species in an in vitro protein synthesis. Both tRNA species strictly obey the wobble rule when they are in competition with other tRNA species. This is probably due to the post-transcriptional modifications at the first position of the anticodon of these tRNA(Leu) species, supporting the proposal that the conformational rigidity of post-transcriptionally modified pyrimidine nucleotides guarantees the correct codon recognition.

Amino Acid Sequence↗
PubMed1993

Characterization of regulatory sequences and nuclear factors that function in cooperation with the promoter of the human thymidylate synthase gene.

To identify the essential sequence of the promoter of the human thymidylate synthase (hTS) gene, deletion mutants were constructed and assayed for promoter activity. The essential sequence was located within 65 bp upstream from the major cap site and a sequence that reduces the promoter activity was found in a region upstream from the essential promoter sequence. We previously identified two DNA-binding nuclear factors, NF-TS2 and NF-TS3, that bind to a region around the site of initiation of translation of the hTS gene. In this study, we confirmed the binding site of these factors by gel mobility shift analysis and found that NF-TS2 is the major factor that binds to the hTS gene in HeLa cells, whereas NF-TS3 is the major factor in the TIG-1 line of human fibroblast cells. To clarify the function of these factors, we examined the effects of the binding of these factors on the promoter activity. Our findings suggest that the binding of NF-TS2 enhances the promoter activity of the hTS gene in HeLa cells, whereas the binding of NF-TS3 represses the activity of the same promoter in TIG-1 cells.

Animals↗
PubMed1993

Cloning and structural analysis of a human thymidylate synthase pseudogene splitted by several Alu sequences.

An unidentified genomic DNA fragment of 2.4kb that is weakly hybridizable with thymidylate synthase (TS) cDNA was cloned from a human genomic DNA library. Sequencing of the cloned DNA fragment and comparison of the sequence with that of the known human TS cDNA revealed that the DNA fragment contained a human TS processed pseudogene with unusual features. Based on the rate of nucleotide substitutions for neutral mutations in the 3'-untranslated regions between the gene and the pseudogene, it was estimated that the human TS pseudogene was formed about 16 million years ago.

Biological Evolution↗
PubMed1992

Identification of cellular differentiation-dependent nuclear factors that bind to a human gene for thymidylate synthase.

Three nuclear factors were identified that interact with sequences in the 5'-upstream region of the human thymidylate synthase gene. Two of these factors interact with a sequence around the initiation codon of the thymidylate synthase gene. The amounts of these two factors changed dramatically as human promyelocytic leukemia HL-60 cells differentiated into macrophage-like cells by the treatment with 1,25-dihydroxyvitamin D3. The change was closely correlated with the decrease in the amount of thymidylate synthase mRNA during the differentiation. These findings suggest that the specific nuclear factors are involved in the regulation of the expression of human thymidylate synthase gene during the differentiation of HL-60 cells.

Base Sequence↗
PubMed1992

Regulatory sequences clustered at the 5' end of the first intron of the human thymidylate synthase gene function in cooperation with the promoter region.

A human thymidylate synthase (TS) minigene containing 5'- and 3'-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5' end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CAT gene construct. These results indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5'-flanking sequences.

Gene Expression Regulation, Enzymologic↗
PubMed1989

Highly selective synthetic inhibitors with regard to plasma-kallikrein activities.

The synthetic inhibitors of plasma kallikrein (PK) were found, which are called PKSI-1007, PKSI-0180 and PKSI-0527 in our laboratories. (1) The inhibitors inhibited PK competitively with D-Pro-Phe-Arg-pNA and the Ki values obtained were considerably small, 10(-6) M-10(-7) M. However, the Ki values for glandular kallikrein (GK), plasmin (PL), thrombin (TH) and factor Xa (FXa) were larger. In particular, a selectivity of PKSI-0527 towards PK was very high and the toxicity was weak (i.v. LD50 for mice is over 100 mg/kg). (2) The inhibitors were effective (a) to prevent the bradykinin formation in the kaolin-activated human plasma and the acid-treated ascites taken from the mice bearing Sarcoma 180, (b) to prolong the coagulation time by contact activation, and (c) to inhibit the enhancement of ADP-platelet aggregation by PK. The results indicated that the some PKSI-inhibitors will be much useful for the basic studies, furthermore they deem to be even promising towards the clinical application.

Amino Acid Sequence↗
PubMed1989

Identification and characterization of an L1 family sequence with a very long open reading frame in the third intron of the human thymidylate synthase gene.

A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.

Amino Acid Sequence↗
PubMed1989

Effect of physical training on thrombotic tendency in rats: decrease in thrombotic tendency measured by the He-Ne laser-induced thrombus formation method.

The effect of physical training on thrombotic tendency was assessed in rats. Exercise was done on a flat treadmill for 30 min at a rate of 1,400 m/h (submaximal speed), 5 times a week for either 1.5 or 3 months. The thrombotic tendency was measured by the He-Ne laser-induced thrombus formation method in microvessels of mesentery, i.e. measurement of the number of laser irradiations necessary to induce stasis of blood flow by occlusive thrombus formation. An increase in the number of irradiations necessary to induce occlusive thrombus formation was observed in arterioles, but not in venules after physical training for 1.5 and 3 months.

Animals↗
PubMed1988

A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

Anticodon↗
PubMed1986

Proteolytic enzymes in human eccrine sweat: a screening study.

Thermally induced human eccrine sweat was collected both by simple scraping (SS) and by a polyethylene sweat collector that covered the Vaseline-coated skin of the back to minimize epidermal contamination (CS, clean sweat). Using synthetic chromogenic Kabi S-series substrates, we demonstrated a variety of proteases in both SS and CS, the former being higher than the latter to varying extents. The higher enzyme activity in SS is mainly due to epidermal contamination because abundant protease activity was eluted from the nonperspiring stripped and unstripped skin in vivo and from the stripped stratum corneum itself. The electrophoretic separation of sweat protein has shown that CS contains at least 7 and SS 15 gelatinolytic proteinases. Although some CS proteinases could be derived from the sweat duct, the sweat secretory coil itself is responsible for at least two proteinases at 78 and 25 kilodaltons. The identity and function of these enzymes remain to be studied.

Caseins↗
PubMed1985

Two tRNAIle1 species from an extreme thermophile, Thermus thermophilus HB8: effect of 2-thiolation of ribothymidine on the thermostability of tRNA.

From Thermus thermophilus HB8 grown at 65 degrees C, two major tRNAIle species have been purified by column chromatography and polyacrylamide gel electrophoresis. The nucleotide sequence of one of these two tRNAIle1 species (tRNAIle1a) has been determined to be pGGGCGAUUAGCUCAGCUGmGUDAGAGCGCACGCCUGAUt6AAGCGUGAGm7GUCGGUGGs2T psi CAm1AGUCCACCAUCGCCCACCAOH. The nucleotide sequence of the other species (tRNAIle1b) is found to be the same as that of tRNAIle1a except for the modification in position 54; tRNAIle1a has s2T(54) while tRNAIle1b has T(54). The melting temperature of tRNAIle1a is as high as 86.2 degrees C while that of tRNAIle1b is 83.3 degrees C. The single replacement of an oxygen atom (2-carbonyl oxygen) of T(54) by a sulfur atom significantly contributes to the thermostability of the tRNAIle1a species. In addition, the methylation of G(18) and A(58) possibly contributes to the thermostability of T. thermophilus tRNAIle1a and tRNAIle1b species.

Base Sequence↗
PubMed1985

Variation in activities of non-plasmin fibrinolytic proteinase and plasminogen-activator in the lung and spleen induced by bacterial endotoxin in rats with special reference to the effects of MD-805.

The behavior of direct fibrinolytic (non-plasmin) proteinase activity and plasminogen-activator activity in the lung and spleen was investigated in rats after a single intravenous injection of bacterial endotoxin, and the influence of thrombin inhibitors on the effects of the endotoxin was assessed. The non-plasmin fibrinolytic activity was markedly increased following a decrease of plasminogen-activator in the lung. In addition, variations in hematological parameters, i.e. a decrease of platelet count, fibrinogen level and antithrombin III, and an increase of blood urea nitrogen and euglobulin fibrinolytic activity, were induced by the injection, indicating the occurrence of disseminated intravascular coagulation. In comparative studies on the effects of the endotoxin injection and thrombin infusion, in the lung and spleen an increase of fibrinolytic proteinase activity was induced in a similar manner; the plasminogen-activator activity in the lung was decreased by the endotoxin injection but not decreased by the thrombin infusion. In prevention studies with heparin and MD-805, the latter was found to prevent the decrease of either fibrinogen or platelet count. However, the former failed to prevent the decrease of platelet count although that of the fibrinogen level was prevented. Heparin and MD-805 exerted no preventive effect on the endotoxin-induced variations of proteinase activity and plasminogen-activator activity in the lung.

Animals↗