Biomedical subjects
Publications and source records attributed to N Horie.
An unusual case of polypoid carcinoma in a 72-year-old woman is reported. The tumor showed a pedunculated growth on the lateral border of the tongue and was composed of a malignant basaloid component in intimate association with definable foci of epithelial dysplasia of the surface mucosa. It was considered that histologic features of the present case corresponded with those of basaloid squamous carcinoma.
We present an unusual case of glandular odontogenic cyst (GOC) enclosing the crown of an impacted canine that developed in the anterior mandible in a 54-year-old woman. Microscopically, it contained numerous glandular structures and hyaline bodies in the epithelial lining. The present rare case is sufficiently distinctive to be considered a dentigerous variant of GOC.
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.
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.
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The occurrence of sialolithiasis in children is uncommon, while parotid sialolithiasis in children is rare. A case of parotid sialolithiasis arising in a 4-year-old girl is presented.
To identify the essential motifs of the promoter of the human thymidylate synthase (hTS) gene, we constructed a set of deletion mutants that covers the region from -441 to +28 of the hTS gene. (The nucleotide positions are numbered from the first position of the initiation codon of the hTS gene.) From the results of chloramphenicol acetyltransferase (CAT) assay of these mutants, two positive elements for the promoter activity were identified: one contains CACCC box (CCACACCC) that is found in the SV40 enhancer motif and the other contains the sequence that is homologous to the Sp1 binding site in the mouse TS gene. Furthermore, two negative regulatory sequences were identified between the two positive elements and upstream from the CACCC box. Cassette mutations were introduced into these motifs and the function of the motifs was confirmed. From the results of gel mobility shift analysis, we found that three nucleoprotein complexes were formed in the promoter region of the hTS gene. The formation of one of the complexes was competed by the DNA fragment bearing the GC box. The gel mobility shift analyses using the DNA fragments with cassette mutations revealed that the complex was formed on the Sp1 binding site of the hTS gene and the formation of the complex correlated with the promoter activity of the fragments with cassette mutations measured by the CAT assay.
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.
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.
Myeloid calcium binding proteins MRP-8 and MRP-14 were induced, and their genes were coordinately expressed, during differentiation of human leukemia HL-60 cells into macrophage-like cells after treatment with 1,25-dihydroxyvitamin D3 (VD3). Both MRP-8 and MRP-14 mRNAs appeared on the day after VD3 treatment. Their level reached a peak on day 2, and then quickly declined. Nuclear factors that interact with the 5'-upstream regions of MRP-8 and MRP-14 genes were studied with gel mobility-shift assays. Two factors (MP8FI and MP8FII) that interacted with 379 bp (426-48 bp upstream from the transcription-initiation site of MRP-8 gene) and 67 bp (-47 - +20) DNA fragments, respectively, were found in the cells treated with VD3 for 1 day. MP8FI and MP8FII were present neither in the nuclei of untreated HL-60 cells, nor in the nuclei of the cells treated with VD3 for 6 days. Human monocytic leukemia THP-1 cells, which constitutively expressed MRP genes, had MP8FII but not MF8FI. MP8FII was found to interact with the 19-mer sequence located just upstream of the TATA box. Also, two factors that bound to the different upstream regions (-400 - -150 and -149 - +50) of MRP-14 gene were detected in the differentiated HL-60 cells. One of these, MP14FI, appeared on day 1, but on day 6 its concentration greatly decreased. The other, MP14FII, was found in greater quantity on day 6 than on day 1. MP14FI, but not MP14FII, was found in THP-1 cells. These factors may be involved in the expression of MRP-8 and MRP-14 genes in VD3-differentiated HL-60 cells.
A 19-year-old man with myospherulosis of the lower lip is described. The lesion was probably caused by petrolatum-based antibiotic ointment.
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.
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.
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.
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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.
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.
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.