Biomedical subjects
Publications and source records attributed to T Shibata.
The titled compounds were synthesized and evaluated for in vitro antibacterial activity. The (3R, 4S)-3-aminomethyl-4-trifluoromethyl derivative (S-34109) was confirmed to be optimal because of its superior activity against quinolone and methicillin-resistant Staphylococcus aureus and low side effect potential.
To develop methods for assessing the intrinsic cellular radiosensitivity, it is important to evaluate the relationship between DNA ploidy of cells and frequencies of micronuclei (MN) or chromosome aberrations after irradiation. From the original human fibrosarcoma cell line HT-1080, we isolated two clones which have different chromosome ploidy: clone 5 is pseudodiploid and clone 1 is heteroploid. We examined the radiosensitivity of the two clones using a clonogenic cell survival assay and a cytokinesis-block MN assay, and by scoring chromosome aberrations using the premature chromosome condensation (PCC) method combined with a fluorescence in situ hybridization (FISH) procedure immediately and at 24 h after irradiation. The MN frequency increased according to the irradiation dose in both clones. The MN frequency of clone 1 was significantly higher than that of clone 5 regardless of whether the assay was performed immediately or 24 h after irradiation. However, when the numbers of MN were normalized by the DNA index of each clone, a significant difference in the frequency of MN was not observed. In the PCC and FISH studies, there was a linear relationship between the radiation dose and the initial breaks of chromosome 4, but the breaks of clone 1 were much more frequent than those of clone 5. Twenty-four h after irradiation, the chromosome 4 breaks of clone 1 were observed much more frequently than those of clone 5 at the same radiation dose. When the numbers of chromosome 4 breaks were normalized by the number of chromosome 4 in each clone without radiation, no such difference in the number of breaks was observed. These findings demonstrated that the DNA content or chromosome ploidy influenced the induction of the MN or chromosome aberrations in HT-1080 cells after irradiation.
Escherichia coli RecA is a representative of proteins from the RecA family, which promote homologous pairing and strand exchange between double-stranded DNA and single-stranded DNA. These reactions are essential for homologous genetic recombination in various organisms. From NMR studies, we previously reported a novel deoxyribose-base stacking interaction between adjacent residues on the extended single-stranded DNA bound to RecA protein. In this study, we found that the same DNA structure was induced by the binding to Saccharomyces cerevisiae Rad51 protein, indicating that the unique DNA structure induced by the binding to RecA-homologs was conserved from prokaryotes to eukaryotes. On the basis of this structure, we have formulated the structure of duplex DNA within filaments formed by RecA protein and its homologs. Two types of molecular structures are presented. One is the duplex structure that has the N-type sugar pucker. Its helical pitch is approximately 95 A (18.6 bp/turn), corresponding to that of an active, or ATP-form of the RecA filament. The other is one that has the S-type sugar pucker. Its helical pitch is approximately 64 A (12.5 bp/turn), corresponding to that of an inactive, or ADP-form of the RecA filament. During this modeling, we found that the interconversion of sugar puckers between the N-type and the S-type rotates bases horizontally, while maintaining the deoxyribose-base stacking interaction. We propose that this base rotation enables base pair switching between double-stranded DNA and single-stranded DNA to take place, facilitating homologous pairing and strand exchange. A possible mechanism for strand exchange involving DNA rotation also is discussed.
Oral squamous-cell carcinoma (SCC) is the most common neoplasm in Sri Lanka, accounting for approximately 30% of all cancers in males. Epidemiologic evidence indicates that there is an unequivocal relationship between betel chewing and oral carcinogenesis, suggesting that there may be specific genetic targets of betel-quid ingredients. The p53 gene has been indicated to be a tumor-suppressor gene that is found in mutated form in common human cancers; however, there are few reports about "carcinogen-specific" p53 mutation. Because of this background, primary resected specimens from 23 oral SCCs, 7 leukoplakias and 2 oral submucous fibrosis were collected from oral SCC patients in Sri Lanka and were used for p53 mutation analysis. Exons 5 through 8 of the p53 gene were examined by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. Mutations in the p53 gene were frequent (10/23) in oral SCC specimens from Sri Lanka. Moreover, the mutations clustered significantly in exon 5 (7/10) of the p53 gene, and small deletions and inclusions other than point mutations were observed. These results indicate that 1) betel-quid chewing may cause specific genetic changes, including mutation in the p53 gene; 2) mutations in the p53 gene are not rare events in SCC patients who are betel-quid chewers, which contrasts with other reports; 3) exon 5 of the p53 gene could be one of the specific targets for some betel-quid ingredients; and 4) betel-quid chewing may be a critical environmental factor in the development of oral SCC.
Several studies have indicated that frequent allelic losses in some specific chromosomal regions occur during colorectal cancer (CRC) progression. To clarify the correlation between such allelic losses and metastatic potential, the allelotype of lymph node-positive early CRCs, which are small but extremely malignant cancers consisting of metastatically competent cells, were investigated. Nineteen paraffin-embedded specimens of early CRC (pT1 tumors according to TNM classification) with positive lymph nodes were collected. The tumor tissues were examined for loss of heterozygosity (LOH), using microsatellite markers on chromosomes 1p34-36, 8p21-22, 14q32, 18q21 and 22q12-13. The relationship between p53 protein expression and the metastatic status was also investigated by immunohistochemical staining. A group of 20 early CRCs with negative lymph nodes having a similar distribution of macroscopic appearance were used as controls. Among the 19 node-positive tumors, LOH at 8p21-22 and 18q21 was detected in 11 cases (57.9%) and 17 cases (89.4%), respectively. Allelic losses within these 2 regions in node-positive tumors were significantly more frequent than that in node-negative ones (p < 0.01). No significant correlation was found between LOH at 1p34-36, 14q32 or 22q12-13 and lymph node metastasis. p53 protein expression was not significantly associated with lymph node metastasis. Our results suggest that putative tumor suppressor genes, which may be involved in the metastatic process of CRC, are located on chromosomes 8p21-22 and 18q21. Allelic losses in these regions are possible risk factors for lymph node metastasis of early CRC.
The glutamate transporter GLT-1 is expressed in astrocytes of the mature brain and spinal cord. In the present study, we examined its expression in the developing mouse spinal cord. By in situ hybridization, 35S-labeled antisense oligonucleotide probes for GLT-1 mRNA consistently labeled the mantle zone/gray matter from embryonic day 11 through the adult stage. However, immunohistochemistry with a specific antibody visualized distinct regional and cellular localizations during the time between the fetal and postnatal stages. At fetal stages, GLT-1 immunoreactivity predominated in the marginal zone/white matter, observed as tiny puncta in cross-sections and as thin fibers in longitudinal sections. The GLT-1-immunopositive structures were also labeled for neuron-specific enolase, a glycolytic enzyme specific to postmitotic neurons and endocrine cells. By electron microscopy, GLT-1 immunoreactivity was detected in axons forming frequent enlargements and was focally localized on a small portion of the axolemma, particularly that facing adjacent axons. At early postnatal stages, GLT-1 disappeared from axons in white matter tracts and, instead, appeared in astrocytic processes surrounding various neuronal elements in the gray matter. Therefore, before switching to astrocytic expression, GLT-1 is transiently expressed in neurons and localized in differentiating axons. Together with our previous finding on the localization of glutamate transporter GLAST in radial glial fibers, GLT-1 and GLAST are thus localized during development on distinct directional cellular elements along which young neurons elongate their axons or move their cell bodies, respectively.
Isoxazolidine-3,5-dione 2 (JTT-501), one of the cyclic malonic acid derivatives, was found to decrease blood glucose at an oral dose of 38 mg/kg/day in KKAy mice and is currently undergoing evaluation in phase II clinical trials. Further studies on a series of malonic acids and related compounds showed that the 1,3-dicarbonyl structure was important for insulin-sensitizing activity. Dimethyl malonate 10, which was selected as a successor for 2, was the optimum compound in a series of 1,3-dicarbonyl compounds and was more potent than the corresponding thiazolidine-2,4-dione 1.
We measured the mental sweating response (MSR) and the skin sympathetic nerve activity (SSNA). Mental arithmetic or recall questions first elicited SSNA and then elicited MSR. MSR was used as the trigger point of time 0 ms to average EEGs. The averaged EEGs contained slow wave fluctuations, which occurred 5 s prior to the MSR onset. The current source locations of the MSR-related potentials were estimated by EEG dipole tracing method in two subjects. Mental stress activated the inferior frontal gyrus 5.5 s prior to the MSR and then 0.5 s later, the lateral part of the hippocampus in a subject, and they activated the medial part of the amygdala 5 s prior to the MSR in another subject. Indirect contact of the brain with the mind associated with mental questions was discussed.
The anti-apoptotic activity of p35s from two baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV) and Bombyx mori NPV (BmNPV), was compared in mammalian cells. AcNPV p35 efficiently blocked apoptosis induced by caspase overexpression, but BmNPV p35 did so very poorly. Analysis of chimeric p35s and in vitro cleavage of wild type p35s suggest that the cleavage efficiency of p35 correlates with the blocking activity. Single amino acid substitutions of BmNPV p35 with those observed in AcNPV p35, however, resulted in significant loss of its anti-apoptotic activity. We speculate that sequences flanking the cleavage site have uniquely evolved during baculovirus evolution.
We used a novel cDNA cloning method based on the cadherin-beta-catenin protein interaction and identified a new human classic-type cadherin, which we named cadherin-15, from adult brain and skeletal muscle cDNA libraries. Sequence analysis revealed that this cadherin was closely related to mouse muscle cadherin and seemed to be its human counterpart. However, its deduced amino acid sequence differed from that of mouse muscle cadherin in that it had an extra 31-amino acid sequence at its C terminus that has been found neither in mouse muscle cadherin nor in any other known classic cadherin. Analysis of cadherin-15 protein expressed in L fibroblasts showed that it was cleaved proteolytically, expressed on the cell surfaces as a mature form of about 124-kDa, and functioned as a cell-cell adhesion molecule in a homophilic and specific manner, but Ca2+ did not protect it against degradation by trypsin. Our findings also suggest that cadherin-15 mediates cell-cell adhesion with a binding strength comparable to that of E-cadherin.
The structure of isohelianol isolated from the seeds of Camellia sasanqua Thunb. was established to be 3,4-seco-19(10-->9)-abeo-8alpha,9beta,10alpha-eupha-4,24-dien-3-ol (1) on the basis of spectroscopic methods.
Percutaneous bone marrow injections were performed on 7 nonunions of the femur. There were 6 hypervascular nonunions and one avascular nonunion. Two nonunions presented with active infections. One other patient had a history of infection which had subsided. One nonunion received the injection twice. After the site of nonunion was curetted and the bone surface was scored, 150 ml of bone marrow aspirated from the iliac bone was injected. Complete union occurred in 4 patients within 9 months; all of them were uninfected hypervascular nonunions following intramedullary nail fixation. One nonunion with a bone defect united partially leaving a 1 x 1 cm defect. The two infected femoral nonunions failed to unite. The results show that percutaneous autologous bone marrow injection for femoral nonunions can be considered for uninfected hypervascular nonunions following intramedullary nail fixation. In these cases stimulation of healing processes of fracture leading to consolidation can be expected from bone marrow injection. However, femoral nonunion with an active infection and loss of fixation is considered to be a contraindication for this technique.
We report on a 2-year-old girl with probable limb-girdle muscular dystrophy associated with an extra-abdominal desmoid tumor of the right mandible. This association is previously undescribed. The tumor was totally removed. Cytogenetic analysis of the tumor showed a clonal karyotypic abnormality: 46,XX,add(1)(p36) in 3 of 20 cells analyzed. Since an association of a neoplasm with limb-girdle muscular dystrophy has previously been reported in 3 cases, the two abnormalities are likely related causally. The chromosome abnormality in our patient may play a role in the occurrence of her desmoid tumor.
Total DNA was extracted from the leaves of a F1 hybrid and its parents, Papaver bracteatum Lindle and P. pseudo-orientale Medw. Analysis of random-amplified polymorphic DNA (RAPD) using ten arbitrary oligonucleotide 10-mers, showed that F1 hybrid was confirmed to be genetically intermediate of both parental plants compared with the genetic distance between F1 hybrid and individual parents. Furthermore, the comparison of the band patterns between a F1 hybrid, P. bracteatum and P. pseudo-orientale clearly showed that part of the bands of both parents were induced into a F1 hybrid. The content of thebaine was determined by HPLC and ELISA used anti-thebaine monoclonal antibody.
In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-stranded breaks (DSBs) occurring in micrococcal nuclease (MNase)-hypersensitive regions of the chromatin. MNase-sensitive sites also undergo meiosis-specific alterations in chromatin structure prior to the appearance of DSBs. DSB formation requires the products of numerous genes. Herein we have examined the effects of mutations in four such genes, MRE11, RAD50, XRS2, and MRE2, on MNase sensitivity at DSB sites in premeiotic and meiotic cells. Disruption mutations in each of four genes confer greater than wild-type levels of MNase sensitivity in premeiotic cells. In meiotic prophase, all of these mutations affect MNase sensitivity at DSB sites and fall into two distinct phenotypic classes. The type 1 mutations (mre2 and mre11) confer a reduction in MNase sensitivity relative to the wild-type level. The type 2 mutations (rad50 and xrs2) permit a meiotic increase in the MNase sensitivity to reach a final level higher than that observed in wild-type cells. An mre11 disruption mutation (type 1) is epistatic to a rad50 null mutation (type 2) with respect to its meiotic effects on MNase sensitivity, suggesting that the events observed in the type 2 mutants during meiosis are dependent upon type 1 functions. One interpretation of these results is that Mre11, Rad50, Xrs2, and possibly Mer2 (whose splicing is Mre2-dependent) form a complex at recombination hot spots and establish a chromatin/DNA configuration favorable for the induction of DSBs.
It is known that mercuric chloride (HgCl2) is a nephrotoxicant. When HgCl2 (1 mg/kg body weight) was intraperitoneally injected into rats, acute renal failure was induced. Histological changes in the kidneys were exclusively observed in the proximal tubules and the severe necrosis was found as early as 24 h after HgCl2 injection. The heme oxygenase-1 (HO-1) mRNA was strongly and promptly induced at about 2.5 h, the earliest time examined and abruptly decreased after the injection. Whereas the time course of HO-1 protein level was delayed as compared with that of HO-1 mRNA level. The levels of HO-1 mRNA and protein similarly increased with dose-dependent manner. The localization of HO-1 protein was restricted to the tubule cells. These findings suggest the potential involvement of HO-1 induction in the response to HgCl2-induced acute renal injury.
A 60-year-old man was transferred to our hospital for blunt trauma of the chest suffered in a traffic accident. He had a weak pulse, and cardiopulmonary resuscitation was continued in the ambulance. Chest roentgenography revealed left hemothorax, and echocardiography revealed pericardial bleeding. He was immediately transported to the operating room, because of severe hypotension due to massive bleeding from the chest drainage tube. Median sternotomy was performed under stand-by cardiopulmonary bypass. There was projectile bleeding from the anterior wall of the right ventricle. The site of rupture was sutured with felt strip. A tear in the pericardium was also present at the apex. Postoperative recovery was uneventful. Fulda et al. reported that the incidence of combined pericadial and cardiac chamber rupture was 8% for those patients suffering blunt chest trauma, and that the rate of survival was less than 15% for blunt heart rupture. We have reported here successful repair of combined rupture of the right ventricle and pericardium.
We report a case of cardiac synovial sarcoma, a very rare primary tumor of the heart. The patient was a 29-year-old man with chief complaint of dyspnea. On echocardiography, a tumor 42 mm in diameter occupying the most space of the left atrium was found. The tumor was obstructed the opening of the mitral valve. He was diagnosed with heart failure due to disturbance of the pulmonary venous return and hemodynamic mitral valve stenosis, and underwent surgery. The tumor was a 5-cm sphere originating in the left atrial posterior wall, and was covered with a sheath and solid. The atrial posterior wall was covered with a peel that appeared to be tumor tissue, and this tissue expanded toward the openings of the pulmonary veins bilaterally and the posterior cusp of the mitral valve. The tumor was resected together with the posterior wall. On pathologic examination, the tumor was diagnosed as a synovial sarcoma. Since no other primary focus was found, this tumor was judged to be a primary sarcoma of the heart. The patient was discharged with elimination of symptoms. The sarcoma recurred after 4 months, and the patient underwent a second operation. The recurrent tumor began from the site of resection of the previous operation and expanded up to the inferior pulmonary veins bilaterally. A wide area of the left atrial wall including the tumor was resected, and he was discharged again. The sarcoma recurred again, and he died of progression of heart failure and aggravation of general conditions after 8 months from the first operation.