Biomedical subjects

T Kubota

Publications and source records attributed to T Kubota.

At least 343 records · Page 19Linked to original sources

PubMed1998

Expression of IL-6 receptor and GP130 in mouse bone marrow cells during osteoclast differentiation.

Interleukin-6 (IL-6) has been postulated as a possible mediator of bone loss after estrogen deficiency, and its signal is transduced via glycoprotein 130 (gp130) after binding IL-6 receptor (IL-6R) in the membrane of target cells. In this study, the expression of IL-6R and gp130 in bone marrow cells during osteoclast differentiation was investigated. Mouse bone marrow cells were isolated and cultured with or without 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. During the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), IL-6R and gp130 expression in the mononuclear cells, stromal cells, and TRAP-positive MNCs were quantitated, using a laser cytometer with a fluorescence confocal microscopy. With 1,25(OH)2D3 stimulus, the level of gp130 significantly increased, but that of IL-6R did not in the stromal cells. In contrast, the levels of both gp130 and IL-6R significantly increased in the mononuclear cells by the treatment with 1,25(OH)2D3. The high expression of both gp130 and IL-6R was observed in the TRAP-positive mononuclear cells. Moreover, both IL-6R and gp130 were expressed in the TRAP-positive MNCs and isolated murine osteoclasts. The treatment of TRAP-positive MNCs with IL-6 caused enhancement of the resorbing activity in a dose-dependent manner, and the effect was prevented by a neutralizing antibody against IL-6R. These data suggest that gp130 and IL-6R, as well as IL-6, are involved in the formation and activation of osteoclasts.

Acid Phosphatase↗

PubMed1998

Sensitization to hyperthermia by intracellular acidification of C6 glioma cells.

Hyperthermia has been introduced as a new modality of treatment for glioma. In these experiments, the cytotoxicity of hyperthermia in C6 glioma cells was enhanced by increasing the intracellular acidity with amiloride and/or 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS). Intracellular pH (pHi) is regulated mainly by Na+/H+ and HCO3-/Cl- antiports through the cell membrane, and amiloride acts on the former, DIDS on the latter to lower pHi. The cellular thermosensitivity to clinically achievable brain hyperthermia at 42 degrees C was enhanced by 0.5 mM amiloride (Na+/H+ antiport inhibitor). T0 values (T0 = the heating period required to reduce experimental survival rate by 1/e) at 42 degrees C without and with amiloride was 192 and 81 min, respectively. The addition of DIDS (HCO3-/Cl- antiport inhibitor) further enhanced. T0 value was 25 min. Fluorophotometric measurement of pHi was employed using the pH sensitive dye, bis(carboxyethyl)carboxyfluorescein, which is trapped in viable cells. The average pHi in control C6 glioma cells in pH 7.2 media was 7.21. In the untreated cells heated at 42 degrees C for 1 hour, the pHi was 7.12. The pHi of the cells heated in the presence of amiloride was decreased to 6.83. The pHi was further lowered to 6.67 by the treatment with amiloride in combination with DIDS for 2 hours. Hyperthermia with amiloride and DIDS may be a more effective treatment for malignant gliomas.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

Characterization of tomato PHYB1 and identification of molecular defects in four mutant alleles.

The structure of the gene encoding the apoprotein of phytochrome B (PHYB1) in tomato has been determined from genomic and cDNA sequences. In contrast to PHYA, PHYB1 lacks an intron upstream of the first ATG. A single transcription start site was found by 5' RACE at -116. Tomato PHYB1 spans 7 kb starting from the first ATG. The coding region is organized into four exons as for other angiosperm PHY. The deduced apoprotein consists of 1131 amino acids, with a molecular mass of 125.4 kDa. Tomato phytochrome B1 shares 78% and 74% identity with Arabidopsis phytochromes B and D, respectively. Along with the normally spliced full-length transcripts, sequences of reverse transcriptase-PCR clones revealed five types of alternative transcripts. Each type of alternative transcript was missing a considerable part of the coding region, including the chromophore-binding site. The four putative PHYB1 mutants in tomato, which are temporarily red-light insensitive (tri), were each confirmed to have a mutation in PHYB1. Each mutation arose from a different, single-base substitution. Allele tri1 is presumably a null because the mutation introduces a stop at codon 92. In tri3, val-238 is replaced by Phe. The importance of this valine residue is evidenced by the fact that the tri3 phenotype is as strong as that of tri1. Alleles tri2 and tri4 encode proteins truncated at their C-termini. The former lacks either 170 or 438 amino acids, depending upon which of two types of splicing occurs during transcript maturation, while the latter lacks 225.

Alleles↗

PubMed1998

A patient-like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer.

Clinically relevant animal models of human cancer are important for studies of cancer biology, invasion and metastasis, and for investigating new forms of prognostic diagnosis and therapy. An ovarian tumor line (RMG-1: human clear cell carcinoma of the ovary) previously grown subcutaneously was implanted orthotopically as intact tissue into the ovarian capsule of 22 nude mice. The tumors showed progressive growth at the orthotopic site in all animals. Tumor-associated serum galactosyltransferase (GAT) tended to be positive in all nude -mice. The tumors invaded or metastasized to the contralateral ovary, retroperitoneum, mesentery and peritoneum, and omentum, and metastasized to the subcutaneous tissue, lymph nodes and distant organs including the liver, kidney, pancreas, and diaphragm. In striking contrast, subcutaneous transplantation of this tumor resulted in growth in only 2 of 5 animals with local lymph node and kidney involvement but no retroperitoneal or peritoneal involvement. These findings suggest that orthotopic implantation provides a suitable micro-environment in which ovarian cancer can express its intrinsic clinically-relevant properties. This approach is relevant to the clinical features of ovarian cancer and is thought to be a useful model for studies of therapy for this cancer.

Adenocarcinoma, Clear Cell↗

PubMed1998

Identification of matrix metalloproteinases (MMPs) in synovial fluid from patients with temporomandibular disorder.

Proteolytic enzymes with gelatinolytic activity in the synovial fluid (SF) of temporomandibular joint (TMJ) arthropathies were assayed by gelatin-impregnated gel enzymography. SF samples were collected from 10 TMJs in patients with closed lock (CL) condition and 5 TMJs from asymptomatic healthy volunteers. Two proteinases with gelatinolytic activities at 92 kDa and 72 kDa were detected in both the normal and the diseased TMJs. Also detected were weak bands at molecular weights of 83 kDa and 66 kDa. All of these proteinase activities were inhibited by EDTA and tissue inhibitor of metalloproteinases (TIMP), required Ca2+ for activation, and were detected with gelatin but not casein as substrate, suggesting that these enzymes were matrix metalloproteinases (MMPs). The 72 kDa and 66 kDa bands further reacted with anti-MMP-2 antibody by Western blot analysis, and the proteinases in the TMJ-SF could cleave type IV collagen in vitro without any activation. These four activities identified by enzymography were, therefore, identified as 92 kDa-gelatinase (proMMP-9), 83 kDa-gelatinase (active MMP-9), 72 kDa-gelatinase (proMMP-2) and 66 kDa-gelatinase (active MMP-2). Densitometric analyses of these bands revealed higher levels of the active form of MMP-9 in the CL patients compared to controls. These findings suggest that MMP-2 and -9 could be dominant proteinases in the TMJ-SF and possibly reflect TMJ pathology.

Adolescent↗

PubMed1998

Lymph node metastasis of early gastric cancer with submucosal invasion.

BACKGROUND: A clinicopathological study of early gastric cancer with submucosal invasion was carried out in relation to lymph node metastasis. METHODS: A retrospective study was conducted of 245 patients with submucosal gastric cancer treated by gastrectomy combined with D2 lymph node resection between 1985 and 1994 in a university hospital. RESULTS: Lymph node metastasis was observed in 34 patients (14 per cent). The mortality rate due to recurrence in patients with lymph node metastasis (three of 34) was significantly higher than in those without lymph node metastasis (five (2 per cent) of 211) (chi2 = 3.95, 1 d.f., P < 0.05). Tumour size, depth of invasion, lymphatic involvement of cancer cells and preoperative diagnosis of advanced cancer correlated significantly with the presence of lymph node metastasis. When the submucosal carcinomas were classified into three categories according to depth of invasion by dividing the submucosal (sm) layer into three equal parts, sm1, sm2 and sm3, the incidence of lymph node metastasis increased from 2 per cent to 12 and 20 per cent respectively. CONCLUSION: When the pathological report reveals sm1 invasion after laparoscopic or endoscopic surgery, reoperation should not be necessary because sm1-carcinomas with diameters of less than 2 cm do not usually metastasize to the lymph nodes.

Adult↗

PubMed1998

Double staining of proteoglycans and the HNK-1 carbohydrate epitope in pseudoexfoliation material.

PURPOSE: We combined the techniques of both immunogold and cupromeronic-blue staining with electron microscopy to investigate the possible relationship between proteoglycans and the HNK-1 carbohydrate epitope in pseudoexfoliation material. METHODS: The anterior segment was obtained from an enucleated eye with pseudoexfoliation syndrome. After cupromeronic-blue staining, following the method of Scott, specimens were embedded in L. R. White resin. Ultrathin sections were incubated successively in primary monoclonal antibodies, and 10 nm gold-conjugated secondary antibody. RESULTS: The present study showed simultaneous presence of the HNK-1 epitope and proteoglycans associated with zonular fibrils and pseudoexfoliation material on the posterior surface of the iris. The HNK-1 epitope was immunolocalized to the periphery of the pseudoexfoliation fibers, while cupromeronic blue-positive filaments bridged adjacent pseudoexfoliation fibers. CONCLUSIONS: Proteoglycans and the HNK-1 epitope might play a substantial role in the formation of pseudoexfoliation material via their adhesive function. Proteoglycans probably do not directly bear the HNK-1 epitope in pseudoexfoliation material.

Aged↗

PubMed1998

Early changes in iris blood vessels in exfoliation syndrome.

PURPOSE: To look for possible changes in iris blood vessels that might precede the formation of true exfoliation material, and to correlate these changes, if present, to aging of the iris. METHODS: Iris blood vessels of 10 clinically normal irises from 10 eyes (ages: 1 day-80 years), 4 irises with exfoliation (ages: 64-80 years) and 3 irises from primary open-angle glaucoma (POAG) (ages: 27-65 years) were examined with an electron microscope. RESULTS: Pronounced multilayering of the basal lamina around the vessels, abnormal excessive formation of microfibrils, presence of atrophic invaginations in the outer cell membranes of endothelial cells containing interlacing basal lamina, and an apparent decrease in the perivascular collagen fibers were evident in exfoliative iris vessels devoid of true exfoliation material. Similar, but much milder, changes were evident in normal aged and POAG irises. CONCLUSIONS: Abnormal elastic tissue and/or basal lamina production plays a role early in the formation of exfoliation material around iris blood vessels.

Adult↗

PubMed1998

Prostaglandins induce calcium influx in human spermatozoa.

Progesterone, prostaglandin and follicular fluid are reported to enhance the acrosome reaction through the influx of extracellular calcium into the cytoplasm of human spermatozoa. Prostaglandins are present within the male reproductive tract, and high concentrations of prostaglandins exist in seminal fluid. In order to investigate the mechanisms by which prostaglandins enhance the acrosome reaction through calcium influx, the intracellular calcium response induced by progesterone, prostaglandin E1 (PGE1), prostaglandin E2 (PGE2) and follicular fluid was measured using fura-2. PGE1 and PGE2 promoted calcium influx dose dependently through dihydropyridine insensitive calcium channels. Refractoriness of the elevation of intracellular Ca2+ concentration ([Ca2+]i) to a second stimulus occurred when 60 microg/ml PGE1 was administered 100 s after the prior administration of 60 microg/ml of PGE1, and similarly when 1 microg/ml of progesterone was administered 100 s after the prior administration of 1 microg/ml of progesterone. Refractoriness also occurred when 60 microg/ml PGE1 was administered after the prior addition of 60 microg/ml PGE2, but did not occur between PGE1 and progesterone. Pertussis toxin (PTX) did not modify the changes in [Ca2+]i after the addition of PGE1 or PGE2. In conclusion, PGE1 and PGE2 promoted calcium influx through PTX-insensitive calcium channels which appeared to be recognized by a common receptor different from that of progesterone.

Acrosome↗

PubMed1998

Integrated YAC contig map of the Prader-Willi/Angelman region on chromosome 15q11-q13 with average STS spacing of 35 kb.

Prader-Willi syndrome and Angelman syndrome are associated with parent-of-origin-specific abnormalities of chromosome 15q11-q13, most frequently a deletion of an approximately 4-Mb region. Because of genomic imprinting, paternal deficiency of this region leads to PWS and maternal deficiency to AS. Additionally, this region is frequently involved in other chromosomal rearrangements including duplications, triplications, or supernumerary marker formation. A detailed physical map of this region is important for elucidating the genes and mechanisms involved in genomic imprinting, as well as for understanding the mechanism of recurrent chromosomal rearrangments. An initial YAC contig extended from D15S18 to D15S12 and was comprised of 23 YACs and 21 STSs providing an average resolution of about one STS per 200 kb. To close two gaps in this contig, YAC screening was performed using two STSs that flank the gap between D15S18 and 254B5R and three STSs located distal to the GABRA5-149A9L gap. Additionally, we developed 11 new STSs, including seven polymorphic markers. Although several groups have developed whole-genome genetic and radiation hybrid maps, the depth of coverage for 15q11-q13 has been somewhat limited and discrepancies in marker order exist between the maps. To resolve the inconsistencies and to provide a more detailed map order of STSs in this region, we have constructed an integrated YAC STS-based physical map of chromosome 15q11-q13 containing 118 YACs and 118 STSs, including 38 STRs and 49 genes/ESTs. Using an estimate of 4 Mb for the size of this region, the map provides an average STS spacing of 35 kb. This map provides a valuable resource for identification of disease genes localized to this region as well as a framework for complete DNA sequencing.

Angelman Syndrome↗

PubMed1998

Utilization of proteinase K-treated cells as lipopolysaccharide antigens for the serodiagnosis of Helicobacter pylori infections.

We have evaluated the use of proteinase K (PK)-treated cells isolated from Helicobacter pylori as lipopolysaccharide (LPS) antigens in an immunoblot assay and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of H. pylori infection. The sera from patients with chronic gastritis, gastric ulcer, duodenal ulcer or gastric cancer, and from healthy adults with or without H. pylori infection were assayed with three commercial serodiagnostic kits (HM-CAP, Helico-G, and G.A.P. II) and novel methods relying on the use of PK-treated cells. The PK-treated cells used in these assays were selected on the basis of their possibility to possess a common epitope in the O-polysaccharides of H. pylori, which is known to be highly immunogenic in humans. Of the sera from these patients, 71-94% were positive with the commercial kits, 97% with immunoblot assay, and 90% with ELISA. On the other hand, of the healthy adults infected with H. pylori, 72-97% were positive with the commercial kits, 86% with immunoblot assay, and 72% with ELISA. PK-treated cells that did not contain the common epitope were unsuitable as an antigen for immunoblot assay or ELISA. Furthermore, the reactivity of these sera reacted specifically with H. pylori PK-treated cells but not with LPSs from other gram-negative bacteria, such as Campylobacter, Proteus, Bordetella, and Salmonella. These results demonstrate that the serological assays relying on the use of H. pylori PK-treated cells possessing a highly antigenic epitope are potentially useful as a serodiagnostic test for H. pylori infection.

Adult↗

PubMed1998

Identification of two novel mutations in the OCRL1 gene in Japanese families with Lowe syndrome.

The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked disorder with features of congenital cataracts, Fanconi syndrome of the renal tubule, and mental retardation. The OCRL1 gene has been positionally cloned and shown to encode a phosphatidylinositol 4,5-biphosphate-5-phosphatase. OCRL is thus thought to be an inborn error of inositol polyphosphate metabolism. We analyzed the gene in two Japanese OCRL patients and their families by DNA sequencing and mismatch polymerase chain reaction (PCR) followed by restriction digestion. A novel nonsense mutation (C1399T) replacing the glutamine of codon 391 (Gln 391 Stop) was identified in exon 12 in 1 patient and also in his mother. A novel missense mutation (C1743G) was identified in exon 15 in the second patient. his mother and maternal grandmother. The missense mutation predicts a substitution of serine for arginine (Ser 505 Arg) in a domain highly conserved among the inositol-5-phosphatase family. Our observations expand the range of OCRL1 mutations that cause Lowe syndrome, and will be useful for genetic counseling in these two families.

Amino Acid Sequence↗

PubMed1998

Apoptosis of lymphocytes in mice administered lipopolysaccharide from Leptospira interrogans.

We report that administration of leptospiral lipopolysaccharide (LPS) in mice results in massive surface marker changes in the lymphocytes of the spleen. It appears that many of these changes relate to the large number of cells undergoing apoptosis. It is also shown that tumor necrosis factor-alpha (TNF-alpha) induces similar effects and is produced in large quantities after injection of leptospiral LPS. It is likely that cytokines such as TNF-alpha are involved in apoptosis.

Animals↗

PubMed1998

A protease inhibitor attenuates gastric erosions and microcirculatory disturbance in the early period after thermal injury in rats.

The effects of camostat mesilate, a synthetic serine protease inhibitor on gastric microcirculation and active oxygen species generated by leucocytes from the gastric and jugular veins in the early period after thermal injury were assessed. Male Wistar rats were anaesthetized and a 30% full skin-thickness dorsal burn was inflicted. Camostat mesilate (100 mg/kg) was dissolved in distilled water and administered orally to rats 40 min before thermal injury (the camostat group). The control animals (the vehicle group) were administered distilled water orally. Rolling leucocytes as well as Monastral blue B deposits in venules were observed using in vivo microscopy. Active oxygen species were measured by chemiluminescence. Camostat mesilate decreased the total length of gastric erosion, venular deposits of Monastral blue B, and rolling of leucocytes in venules, and relatively increased luminol-dependent chemiluminescence activity generated by zymosan-stimulated leucocytes 15 min after thermal injury. These results suggest that serine proteases are involved in the formation of gastric erosions and gastric microcirculatory disturbance in the early period after thermal injury.

Animals↗

PubMed1998

p53 expression in gastric mucosa with Helicobacter pylori infection.

Expression of p53 was examined immunohistochemically in the Japanese monkey model with Helicobacter pylori infection of the gastric mucosa to investigate the association between H. pylori infection and gastric carcinogenesis for a period of 4 years. In the course of these observations, from 3 years after H. pylori inoculation, nuclear staining for p53 was seen in the glandular cells of the mucosa infected with H. pylori, especially in the neck region of the glands. There was a gradual increase in the number of immunopositive cases among the infected animals. Three years after inoculation, three out of six cases, and 4 years after inoculation, four out of six cases exhibited positive staining for p53. Before inoculation, and up to 2 years after inoculation, the infected group showed no immunoreaction for p53. The non-infected group likewise displayed no immunostaining for p53 through 4 years of observation. These results suggest that p53 alterations occur in the H. pylori-infected gastric mucosa and that H. pylori infection may play an important role in gastric carcinogenesis.

Animals↗

PubMed1998

Effects of evening light on body temperature.

Six healthy male subjects aged 21-35 years participated in the present study. The subjects were exposed to dim light (150 lux) or bright light (3000 lux) at eye level, from 19.00 to 21.30 h for 5 days. Rectal temperature and wrist activity were monitored throughout the study period. Rectal temperature nadir was delayed significantly after the bright light exposure. Ease in sleep initiation and overall sleep quality, measured by questionnaire, were aggravated significantly by the evening bright light exposure. These results suggest that strong illumination at night may disturb nocturnal sleep.

Adult↗

PubMed1998

Upregulation of nucleobindin expression in human-activated lymphocytes and non-Hodgkin's lymphoma.

Nucleobindin (Nuc) was originally found to be an enhancement factor of anti-DNA antibody production secreted by a lymphoid cell line derived from a lymphoproliferative MRL/lpr mouse. It has been shown that Nuc has a unique structure containing a DNA- and two calcium-binding domains, and a leucine zipper motif, but its biological roles have not yet been fully elucidated. Expression of Nuc was first studied in human lymphocytes. Expression of Nuc mRNA in normal peripheral blood mononuclear cells was significantly increased upon mitogen stimulation. Anti-human Nuc monoclonal antibody H-1D8 immunoprecipitated Nuc protein in the nuclear extract of Molt-4 cells. Furthermore, in the immunohistochemical staining of tumor specimens from 108 patients with non-Hodgkin's lymphoma (NHL) with H-1D8, H-1D8-positive cells were observed in nearly all cases in varying frequency. According to the Working Formulation, the percentage of cases in which more than 90% of the tumor cells were stained with H-1D8 was 65% in the high grade of the histological malignancy, 54% in the intermediate grade, and 22% in the low grade; however, normal cells surrounding the tumor cells were virtually negative for H-1D8. These results showed that the level of Nuc expression in human lymphocytes reflects the status of activation or proliferation of the cells, thus providing a clue for the further investigation into biological roles of Nuc. In addition, it might be applicable to the clinicopathological estimation of NHL as a novel indicator.

Adolescent↗

PubMed1998

Successful polymerase chain reaction-based diagnosis of fungal meningitis in a patient with chronic granulomatous disease.

Meningitis is not a common complication of chronic granulomatous disease (CGD). Here, we present details of a 3-year-old boy with X-linked CGD, who suffered from fungal meningitis. While 19 samplings using conventional cerebrospinal fluid (CSF) cultures failed to detect any organisms, fungal DNA was identified in the CSF by a new polymerase chain reaction (PCR)-based method. The patient recovered without any sequelae after treatment with a combination of antifungal agents, interferon-gamma and granulocyte infusions. This case report demonstrates that fungal meningitis must be included in the differential diagnosis of infections in CGD patients and that the PCR-based detection of fungal DNA is a powerful tool for diagnosis.

Child, Preschool↗