PubMed Health⌕ Search

Biomedical subjects

T Tobe

Publications and source records attributed to T Tobe.

At least 37 records · Page 2Linked to original sources
PubMed1995

Thermoregulation of virB transcription in Shigella flexneri by sensing of changes in local DNA superhelicity.

Transcription of the virB gene, a transcriptional regulator of invasion genes on the large plasmid of Shigella flexneri, is strictly regulated by growth temperature; when bacteria are grown at 37 degrees C, virB transcription is highly activated, while at 30 degrees C the level of virB transcription decreases to less than 5% of that at 37 degrees C. Transcription from the virB promoter is activated by VirF, which is encoded on the same plasmid, in a DNA superhelicity-dependent manner (T. Tobe, M. Yoshikawa, T. Mizuno, and C. Sasakawa, J. Bacteriol. 175:6142-6149, 1993). Here we provide evidence supporting the involvement of negative superhelicity in the thermoregulation of virB transcription. A local negatively supercoiled domain in the virB promoter region was created by activating a divergent transcription from the T7 RNA polymerase-dependent promoter, phi 10, which was placed upstream of the virB promoter in the opposite orientation. Transcription from the virB promoter was activated even at 30 degrees C by induction of divergent transcription. Levels of virB transcription correlated with levels of expressed T7 RNA polymerase. Transcriptional activation of virB by the system depended completely upon VirF function. The level of virB transcription achieved by introducing a negatively supercoiled domain was enough to give rise to expression of invasion capacity at 30 degrees C. These results indicated that the repression of virB transcription at 30 degrees C was caused by a reduction in negative superhelicity around the virB promoter region at 30 degrees C.

Bacterial Outer Membrane Proteins↗
PubMed1995

Mapping of human inter-alpha-trypsin inhibitor family heavy chain-related protein gene (ITIHL1) to human chromosome 3p21-->p14.

Inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) is a novel glycoprotein isolated from human plasma. The cDNA encoding IHRP has recently been cloned from human liver cDNA libraries. We report the mapping of this gene (ITIHL1) by fluorescence in situ hybridization using a 2.5-kb cDNA fragment as a probe. ITIHL1 was localized to chromosome region 3p21-->p14 where the genes of heavy chain 1 and 3 of inter-alpha-trypsin inhibitor are located. This result, together with significant homology between the nucleotide sequences of ITIHL1 and the heavy chain genes, supports ITIHL1 as being a member of an evolutionary related gene family of ITI heavy chains. Northern blot analysis indicated that IHRP was predominantly synthesized in liver. From Southern blot analysis, it was tentatively concluded that ITIHL1 is a single copy gene.

Blood Proteins↗
PubMed1995

[Effect of human interferon-beta on reconstruction of the choriocapillaris in monkeys following laser photocoagulation].

We studied the effect of human interferon-beta on reconstruction of the choriocapillaris following laser photocoagulation in monkey eyes. Moderate dye laser photocoagulation caused the occlusion of the choriocapillaris by the intraluminal thrombus in the photocoagulated lesions on the retina. After 3 days, immature endothelial cells began to migrate towards the center from the edge of the lesions. After 7 days capillaries were newly formed, and after 14 days the choriocapillaris in the lesions was almost reconstructed. Systemic administration of interferon-beta after photocoagulation suppressed the reconstruction of the choriocapillaris remarkably. These results suggest that interferon-beta inhibits proliferation and migration of the capillary endothelium on the retina. Interferon-beta may be effective as medication for the treatment of age-related macular degeneration.

Animals↗
PubMed1995

[The effect of interferon-beta on experimental choroidal neovascularization].

We studied histologically the effect of systemic administration of human interferon-beta (IFN-beta) on experimental choroidal neovascularization which was caused by intensive laser photocoagulation at the posterior pole of monkey eyes. The regression of choroidal neovascularization was observed in IFN-treated monkeys. Histologically, retinal pigment epithelium (RPE) was remarkably abundant around choroidal neovascularization in the subretinal space, and the activity of neovascularization was weaker in IFN-treated monkeys than in untreated monkeys. These results indicate that IFN-beta promotes the proliferation of RPE and suppresses the activity of endothelial cells of neovascularization to cause regression of choroidal neovascularization. The results suggest that IFN-beta is clinically useful to treat the choroidal neovascularization of age-related macular degeneration.

Animals↗
PubMed1995

[Expression of fibroblast growth factor receptor 1 in experimental choroidal neovascularization with in situ hybridization].

Fibroblast growth factor (FGF) is an important factor for neovascularization in vivo. In order to clarify the role of FGF in experimentally produced choroidal neovascularization, we demonstrated mRNA for FGF receptor 1 in situ hybridization. Krypton laser photocoagulation was applied to the posterior retina of colored rats to produce choroidal neovascularization experimentally. These eyes were removed at several different intervals after photocoagulation. Chorioretinal section were used for in situ hybridization. FGF receptor 1 cDNA fragment was used to make antisense and sense probes for in situ hybridization. In normal chorioretinal tissue, staining indicating the existence of FGF receptor 1 mRNA was seen in the ganglion cell layer and inner nuclear layer. After the photocoagulation, the staining was seen in the retinal pigment epithelial cells, melanocytes in the choroid, and choroidal blood vessel wall in the photocoagulated lesions. FGF receptor 1 mRNA was expressed through the development of choroidal neovascularization, and it appears that FGF is necessary for development of choroidal neovascularization. Previous workers showed that the capillary endothelial cells and retinal pigment epithelial cells produce basic FGF in vitro. It seems that FGF effects those cells in an autocrine or paracrine manner in vivo.

Animals↗
PubMed1995

[A long-term course of experimentally produced choroidal neovascularization in the rat].

We studied morphologically a long-term course of experimental choroidal neovascularization (ChNV) induced by krypton laser photocoagulation in the rat retina. Fifty-two weeks after photocoagulation, ChNV was enveloped completely by the retinal pigment epithelium. Vascular endothelial cells of ChNV were thin, with many fenestrations and wide lumen. The ChNV maintained the morphological characteristics of mature leaky capillaries similar to choriocapillaris. The lumen of the neovascularizations tended to be compressed by massive collagen fibers produced by the retinal pigmented epithelium. We found that experimental ChNV in the rat retina retains the characteristics of leaky capillaries for a long time unlike that in the monkey ChNV.

Animals↗
PubMed1995

[Effects of interferon-beta on repair of the retinal pigment epithelium after laser photocoagulation].

We studied the morphological effects of human interferon-beta on repair of the retinal pigment epithelium (RPE) after moderate dye laser photocoagulation in monkey eyes. In the control eyes, RPE cells were proliferating towards the center from the margin of the laser burn 3 days after photocoagulation. At day 14 after photocoagulation, a newly formed monolayer of RPE cells covered Burch's membrane to repair the lesion. In the eyes treated with interferon-beta by systemic administration, RPE cells had proliferated remarkably 3 days after photocoagulation. The RPE cells proliferated to form multiple layers on Burch's membrane even at day 14 after photocoagulation. These results suggest that interferon-beta promotes the proliferation to repair damaged RPE.

Animals↗
PubMed1994

The prognostic role of the DNA ploidy pattern in colorectal cancer analysis using paraffin-embedded tissue by an improved method.

To assess the prognostic value of DNA ploidy in colorectal cancer, compared with the histopathological findings, paraffin-embedded surgical specimens from 330 patients who underwent resection for primary adenocarcinoma were studied using a new modified method of flow cytometry. Of these specimens, 141 were DNA diploid and 189, DNA aneuploid, among which there were 3 DNA hypodiploid lesions. Of the ten variables studied in curative resection, DNA ploidy ranked fourth in prognostic significance according to the linear trend by the chi 2 test, after nodal status, grade of cellular differentiation, and degree of invasive growth, if the DNA ploidy pattern was classified into three categories. Conversely, DNA ploidy was the sixth most significant factor if DNA hypodiploidy was included in the DNA aneuploidy. The Cox multivariate analysis showed that DNA ploidy was one of the five significant factors independently determining prognosis; however, if adjustment for the modified Dukes' stage was made by the Mantel-Haenszel test, the survival difference between the diploid and aneuploid groups did not reach a statistically significant level. Thus, we conclude that from a practical point of view, DNA ploidy is not an essential factor which must be combined with histopathological variables for a better prediction of patient outcome.

Colorectal Neoplasms↗
PubMed1994

Selective expression of monocyte chemotactic and activating factor/monocyte chemoattractant protein 1 in human blood monocytes by Mycobacterium tuberculosis.

Neutrophils are the predominant leukocyte population in acute inflammation. Granulomatous inflammation such as tuberculosis is a specific type of chronic inflammation characterized by the predominant accumulation of macrophages. To clarify the mechanism of cellular recruitment in inflammation, the expression of chemokines, interleukin-8 and monocyte chemotactic and activating factor (MCAF)/monocyte chemoattractant protein 1 (MCP-1), was examined in human blood monocytes in response to lipopolysaccharide of Escherichia coli, which could induce acute inflammation, or purified protein derivative (PPD) or Mycobacterium tuberculosis, which could provoke chronic inflammation. Monocytes stimulated with PPD or M. tuberculosis expressed low levels of antigenic interleukin-8 but high levels of MCAF/MCP-1 compared with monocytes stimulated with lipopolysaccharide. Northern blot analysis showed the early induction of interleukin-8 mRNA and the delayed expression of MCAF/MCP-1 mRNA in response to PPD or M. tuberculosis. Thus, the disparate expression of chemokines may contribute to the cellular recruitment in acute and chronic inflammations.

Adult↗
PubMed1994

Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri.

Intercellular spreading of shigellae is a prerequisite for shigellosis, although the molecular mechanisms underlying the phenomenon are still largely obscure. To elucidate some of these mechanisms, we performed random Tn10 insertion mutagenesis in Shigella flexneri YSH6000T and found a chromosomal locus in the NotI-J segment responsible for bacterial spreading. The locus affected in the mutant, designated vacJ, was neither involved in the invasion of epithelial cells nor in intracellular movement, but was required for intercellular spread. The vacJ mutant was capable of forming bacterium-containing membranous protrusions within the infected cell, but had diminished ability to move from the protrusions into the cytoplasm of the adjacent epithelial cells. Cloning and sequencing of the vacJ region indicated that the vacJ gene encoded a 28.0 kDa protein possessing a signal peptide at the N-terminus, which contained the motif characteristic of lipoproteins. The analysis of the vacJ product indicated that VacJ was exposed on the bacterial surface. The vacJ gene was distributed among shigellae and enteroinvasive Escherichia coli, and the constructed vacJ mutants failed to spread intercellularly, indicating that vacJ is a chromosomal gene essential for the pathogenicity of shigellae.

Amino Acid Sequence↗
PubMed1994

Deregulation of temperature-dependent transcription of the invasion regulatory gene, virB, in Shigella by rho mutation.

Expression of the virB gene, the transcriptional regulator for the invasion genes encoded by the large plasmid of Shigella flexneri, is temperature-regulated. virB transcription is under the control of VirF and H-NS, which act as positive and negative regulators, respectively, and is highly responsive to changes in DNA superhelicity. To further investigate the molecular mechanisms underlying the thermoregulation of virB transcription, a mutant which expressed an invasion phenotype at both 30 degrees C and 37 degrees C was isolated using miniTn10-kan (miniKAN) random insertion mutagenesis. The insertion site was mapped to the rho gene, and resulted in the addition of 11 amino acids to the C-terminus of the Rho protein. Consequently, decreased transcription termination activity at a rho-dependent terminator, lambda tL1, was observed. In the rho mutant, both the transcription of virB and expression of invasion genes were activated at 30 degrees C and were less responsive to changes in temperature. The deregulation of virB expression by the mutation was dependent upon the virB promoter, since the effects of the mutation on virB transcription were abolished when its promoter region was replaced by the tac promoter. Temperature-responsive changes in DNA topology, as determined by linking numbers of a reporter plasmid, showed that changes in DNA superhelicity in the rho mutant were smaller than that in the wild type. Furthermore, when the mutant was grown in medium containing novobiocin, an inhibitor of DNA gyrase, virB transcription at 30 degrees C as well as at 37 degrees C was greatly diminished. These results indicated that Rho protein could have a profound effect on topological temperature-dependent changes in DNA structure, thus contributing to thermoregulation of virB transcription.

Bacterial Outer Membrane Proteins↗
PubMed1994

vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12.

The genetic determinants required for invasion of epithelial cells by Shigella flexneri and for the subsequent bacterial spreading are encoded by the large virulence plasmid. Expression of the virulence genes is under the control of various genes on the large plasmid as well as on the chromosome. We previously identified one of the virulence-associated loci near phoBR in the NotI-C fragment of the chromosome of S. flexneri 2a YSH6000 and designated the locus vacC. The vacC mutant showed decreased levels of IpaC, and IpaD proteins as well as transcription of ipa, an operon essential for bacterial invasion (N. Okada, C. Sasakawa, T. Tobe, M. Yamada, S. Nagai, K. A. Talukder, K. Komatsu, S. Kanegasaki, and M. Yoshikawa, Mol. Microbiol. 5:187-195, 1991). To elucidate the molecular nature of the vacC locus, we cloned the vacC region from YSH6000 on a 1.8-kb SalI-BamHI DNA fragment. The nucleotide sequence of the 1,822-bp vacC clone was highly (> 98%) homologous to the tgt region of Escherichia coli K-12, which is located at 9.3 min on the linkage map. Complementation tests indicated that the vacC function was encoded by an open reading frame expressing a 42.5-kDa protein, which corresponded to the tgt gene of E. coli K-12, coding for tRNA-guanine transglycosylase (Tgt) (K. Reuter, R. Slany, F. Ullrich, and H. Kersten, J. Bacteriol. 173:2256-2264, 1991). The cloned tgt gene from E. coli K-12 restored the virulence phenotype to the vacC mutant of YSH6000. Characterization of the vacC mutant indicated that levels of VirG, a protein essential for bacterial spreading, and VirF, the positive regulator for the expression of the virG and ipaBCD operons, decreased significantly compared with those of the wild type. Similar phenotypic changes occurred in vacC mutants constructed by insertion of a neomycin resistance gene in shigellae and enteroinvasive E. coli strains, consistent with the hypothesis that the vacC (tgt) gene contributes to the pathogenicity of Shigella flexneri.

Amino Acid Sequence↗
PubMed1994

Specific PCR amplification for N-ras mutations in neoplastic thyroid diseases.

We analyzed point mutations of N-ras protooncogene codon 61 in thyroid neoplasms by means of a mutation-specific PCR method. In this method, one of the paired primers has a base at the 3' terminal that is complementary to a mutated base of the DNA sequence to be analyzed. With this primer, only alleles which have the same mutation can be amplified. Among 24 thyroid tissues, we detected 2 point mutations out of 7 follicular carcinomas (29%). One tumor had a cytosine to adenine substitution mutation at the first base of codon 61, and the other had an adenine to guanine substitution mutation in the second base of the same codon. The same mutations were not detected in 7 follicular adenomas or 1 papillary carcinoma. These results were confirmed by both dot blot hybridization and direct sequencing method. Mutation of N-ras codon 61 may be significant in malignant transformation of follicular thyroid tumors. Because of its easy availability, the mutation-specific PCR method is a useful screening test for N-ras mutations.

Adenocarcinoma, Follicular↗
PubMed1994

[The role of repeat transurethral resection in stage A1 carcinoma of the prostate].

In order to evaluate the significance of repeat transurethral resection (TUR) in differentiating stage A1 prostatic adenocarcinoma from those with stage A2, we performed repeat TUR in 34 patients with an initial diagnosis of stage A1 prostatic adenocarcinoma. It was found that residual adenocarcinoma was present in five cases (14.7%), but the diagnosis was changed from stage A1 to stage A2 in only one case (2.9%). In one patient with final diagnosis of stage A1 carcinoma, bone metastases were detected seven months after the repeat TUR. It was concluded that repeat TUR for stage A1 prostatic adenocarcinoma did not yield clinically significant information.

Adenocarcinoma↗
PubMed1994

[Laparoscopic pelvic lymphadenectomy for localized prostate cancer].

Between April 1992 and May 1993, 13 patients (age, 66-79) with localized prostate cancer underwent laparoscopic pelvic lymphadenectomy at Asahi General Hospital. Clinical stage comprised A2 for 3 patients, B1 for 1 and C for 9. Lymphadenectomy covered inner half of external iliac nodes and obturator nodes, from pubic bone to proximal end of umbilical ligament. Operating time ranged from 70 minutes to 133 minutes with median of 102 minutes. The number of total lymph nodes dissected ranged from 3 to 17 nodes with median of 7. Lymph nodal involvement was detected in one patient. Two patients needed laparotomy due to bleeding; from abdominal wall caused at insertion of trocar in one, and oozing of blood for 10 hours after procedure in the other. Other serious complications were not observed. In conclusion, laparoscopic pelvic lymphadenectomy was a good staging procedure for localized prostate cancer.

Aged↗
PubMed1994

[Experimental choroidal neovascularization in the rat].

We successfully produced, highly (78%) reproducible experimental choroidal neovascularization (ChNV) in the subretinal space of pigmented rats with intense diode laser photocoagulation. ChNV in the pigmented rat was characterized by rapid development of neovascular membrane and proliferation of retinal pigment epithelium in the subretinal space. This procedure may be useful as an experimental model for ChNV.

Animals↗