Biomedical subjects
Publications and source records attributed to M Seiki.
Intraduodenally administered 14C-urokinase (14C-UK) was located in Macaca Irus by autoradiography. Sagittal sections, approximately 40 micron thick, of a frozen monkey were applied against X-ray films. Autoradiograms thus obtained indicated that a part of intraduodenally administered 14C-UK was incorporated into the systemic blood circulation by transintestinal mucosal membrane.
Envelope gene product of human T-cell leukemia virus was identified as a glycoprotein with an apparent molecular weight of 62,000 daltons, by using rabbit antiserum against a synthetic decapeptide whose structure had been predicted from the nucleotide sequence. Sera from patients with adult T-cell leukemia also reacted with this glycoprotein.
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The first replicating fragment of the Bacillus subtilis chromosome, B7, inhibited the replication of the plasmid that carried this fragment. In earlier work using sequential cleavage by Alu I, the suppressor function was located within a 489-base-pair segment. The nucleotide sequence of the entire segment now has been determined. The sequence is characterized by two promoter-like structures and several putative recognition sequences, such as termination signals, 2-fold symmetries, inverted repeats, and repeats. By means of sequential cleavage with exonuclease BAL-31, the essential region for suppression was located in a 200-base-pair region that contains the two promoters with the same orientation. Specific transcription was produced in vitro by using B. subtilis or Escherichia coli RNA polymerases. The transcription was mostly from the second promoter. Elimination of the -35 region of the second promoter dramatically affected both inhibitory activity and in vitro transcription, suggesting that the transcriptional activity of the second promoter is involved in the cis-inhibition of DNA replication. The significance of the suppressor sequence in the region of the replication origin of the B. subtilis chromosome is discussed.
Adult T-cell leukemia virus (ATLV) is a human retrovirus closely associated with adult T-cell leukemia. The integrated provirus DNA and cDNA from virion RNA were molecularly cloned and their structures were analyzed. Clone lambda ATM-1 of an integrated provirus DNA in the MT-1 cell line, established from adult T-cell leukemia cells by cocultivation with cord lymphocytes, contained DNA about 13,000 base pairs (bp) long and long terminal repeats (LTR) at both ends of the viral sequence that were about 8,000 bp long. These two LTR sequences were linked to cellular sequences with direct repeats of 7 bp. Each LTR consisted of 754 bp including inverted repeats of 2 bp at the ends and the T-A-T-A-A box, characteristics in common with those of LTRs of other known retroviruses. Adjacent to the 5' LTR there was a sequence identical to the tRNAPro binding site in murine leukemia virus, suggesting that tRNAPro is a primer for reverse transcription of the viral genome. From these structural features, the mechanism of ATLV replication was suggested to be the same as that of other known animal retroviruses. However, the length of the small terminal repeats at the ends of the RNA genome, 228 +/- 1 bases, is much longer than the lengths, up to 80 bases, of those in avian, mouse, or primate retroviruses so far analyzed. These findings suggest that ATLV should be classified in a distinct group of retroviruses with bovine leukemia virus which also makes unusually long strong-stop cDNA.
A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E. coli and B. subtilis. Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment. The cloned plasmid pMS102'-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B. subtilis chromosome. (1) E. coli cells harboring this plasmid stuck to each other and to glass. This property was more apparent when cells were grown in poor media. (2) E. coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid. (3) The frequency of transformation of B. subtilis by pMS102'-B7 was less than 1/1,000 of that by the vector plasmid pMS102'. The number of copies of pMS102'-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B. subtilis. This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts. Both in E. coli and B. subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system. The implication of the function of the B7 fragment in the initiation of the B. subtilis chromosome will be discussed.
A BamHI restriction endonuclease fragment B7, which contains the replication origin of the Bacillus subtilis chromosome, showed inhibitory effects on cell growth and plasmid replication in Escherichia coli and Bacillus subtilis, when B7 was inserted into a composite plasmid pMS102' and introduced into these cells. In order to localize these properties in more limited regions within the B7 fragment, we developed a new and widely applicable method for deletion of DNA segments of various lengths from one or other end of a given region of the plasmid molecule. Using a set of deletions in the B7 fragments of pMS102'-B7, we determined the loci responsible for the inhibitory effects of B7 as described below. (1) Stickiness appearing in E. coli cells was caused by a segment residing in a region of approximately 2.2 kilobase pairs (kbp) overlapping the E19 and E22 fragments. (2) instability of the plasmid in E. coli was due to a segment localized in the 440 bp region of the E19-side terminal portion of the 2.2 kbp region. (3) The same 440 bp were also responsible for inhibition of the replication of the plasmid in B. subtilis. Hybridization of the cloned DNA fragments containing the 2.2 kbp region with the whole B. subtilis chromosome revealed that several regions of the chromosome are homologous to this characteristic sequence.
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The initiation of DNA replication of small replicons in vitro involves conformational changes in the whole DNA molecule or in the region near to the replication origin. One striking finding has been the role of DNA gyrase (that is, the necessity for supercoiled structure) in the initial stage of ColE1 replication in vitro. However, little is known about the effect of gyrase on the initiation of replication of bacterial chromosomes in vivo. We have constructed a map of cleavage sites of restriction enzymes at the region of the origin of replication of the Bacillus subtilis chromosome (accompanying paper). This has now enabled us to examine the effect of novobiocin, a selective inhibitor of DNA gyrase, on the replication of the specific chromosomal segments near the origin and to seek a possible role for the gyrase in the initiation of chromosomal replication. We have found that only a limited segment of the chromosome at the origin region was replicated in the presence of novobiocin. This effect allowed us to locate the site of the origin of replication to within a DNA fragment of molecular weight 3.4 x 10(6).
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The clinical and pathological features of T-cell type malignant lymphoma related to human T-cell leukemia virus (HTLV) were investigated in eight patients presenting lymphadenopathy. Biopsy of lymph nodes showed an histology of diffuse non-Hodgkin's lymphoma. All patients were positive for anti-ATLA antibody and HTLV proviral DNA in the lymph node cells. Most patients showed pronounced hypercalcemia and high serum levels of lactic dehydrogenase. All patients died between 3 and 17 months (mean 8 months) after the onset of disease. HTLV-related malignant lymphoma should be added to the spectrum of ATL, being classified as a lymphoma type ATL.
Human T-cell leukaemia virus (HTLV), previously also reported as ATLV, is a recently identified retrovirus which is closely associated with adult T-cell leukaemia (ATL) endemic in southwestern Japan and the Caribbean. Determination of the total nucleotide sequence of the HTLV genome has revealed no typical onc gene acquired from the cellular sequence. Screening of the HTLV provirus genome in tumour cells has shown that in all cases of ATL examined, the primary tumour cells contained the provirus genome and were monoclonal with respect to the integration site of the provirus. These findings suggest that ATL leukaemogenesis may be due to insertional mutagenesis in which the provirus genome is integrated into a specific locus on the chromosomal DNA and then activates an adjacent cellular onc gene, a mechanism already demonstrated in avian lymphoma and erythroblastosis induced by avian leukosis viruses. A common site of HTLV provirus integration in leukaemic cells among some ATL patients was reported by Hahn et al. but subsequently retracted. However, this retraction does not imply the random integration of the proviruses. Independently, we have been testing this insertional mutagenesis model in ATL and report here that the provirus did not have a common locus of integration in 35 ATL patients and did not integrate on the same chromosome in 2 ATL patients.
Matrix metalloproteinase (MMP) expression is associated with advanced-stage cancer and contributes to tumor progression, invasion, and metastasis. Membrane type matrix metalloproteinase (MT-MMP) has a potential transmembrane domain at the C terminus and activates pro-MMP-2, which is mainly produced from interstitial fibroblasts. Its expression on the membrane of invasive tumor cells results in the pericellular space degradation at cell-matrix contact sites and renders cancer cells more invasive at the migration front. To elucidate the relationship between MT-MMP expression and metastasis and prognosis in gastric cancer patients, MT-MMP expression was analyzed immunohistochemically in 127 primary tumors and results were correlated with several prognostic parameters and patient's survival. MT-MMP immunoreactivity was stained on the cell membrane of cancer cells and fibroblasts in the invasion front. MT-MMP was detected in 72 tumors (57%) (MT-MMP-positive). MT-MMP expression was closely associated with macroscopically invasive type, nodal involvement, lymphatic invasion, vessel invasion, and peritoneal dissemination. Patients with MT-MMP-positive tumor had a significantly worse prognosis than those with MT-MMP-negative tumor (p<0.001). Multivariate analysis showed MT-MMP overexpression as an independent prognostic factor in gastric cancer patients. Immunohistochemical analysis for MT-MMP may be an indicator of metastatic potential or the prognosis of gastric cancer patients.