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Biomedical subjects

T Nishihara

Publications and source records attributed to T Nishihara.

At least 109 records · Page 6Linked to original sources
PubMed1997

Biodegradation of aniline, anthracene, chlornitrophen, fenitrothion and linear alkylbenzene sulphonate in pond water.

Biodegradation of five chemicals (aniline, anthracene, chlornitrophen (CNP), fenitrothion (FNT) and linear alkylbenzene sulphonate (LAS)) by aquatic bacteria in three different types of ponds was determined according to the cultivation method developed by this group. The degradability toward these chemicals was varied among the ponds, except for LAS which was decomposed well in all samples. Higher degradability towards the two agrochemicals, CNT and FNT, was found in the pond surrounded by paddy fields, whereas aniline and anthracene were decomposed more rapidly in the pond located in the industrial area. Water from the pond in the botanical garden, with the least exposure to any chemicals, exhibited the lowest degradation toward all chemicals tested. There was no significant seasonal variation in the biodegradation of chemicals in these ponds. It was deduced that biodegradability toward certain chemicals could be a result of acclimatization of the microbial community by chemical contamination present and past, suggesting the possible use of biodegradation profiles as an indicator for chemical pollution in the aquatic environment.

Biodegradation, Environmental↗
PubMed1997

The role of N-terminal glycosylation in the human oxytocin receptor.

The human oxytocin receptor includes three N-glycosylation sites in its extracellular N-terminal domain. We have established permanent cell-lines in which the gene for the human oxytocin receptor (OTR) has been introduced into HeLa cells. These cells differ by the disruption of one or more of the N-terminal N-glycosylation sites by site-directed mutagenesis of the transfected OTR constructs. The binding capacity of each transfectant, calculated per mg membrane protein, was 5-17 times higher than that of human term myometrium. The pharmacological characteristics of the transfected wild-type OTR are very similar to those of native myometrial OTR. The mutation of N-glycosylation sites (Asn-X-Ser/Thr), namely OTR-D8N15N26 (Asn8-->Asp8), -N8D15N26(Asn15-->Asp15), -N8D15D26(Asn15-->Asp15, Asn26-->Asp26) and -D8N15D26 (Asn8-->Asp8, Asn26-->Asp26) appear to affect neither their dissociation constant (Kd), nor the affinities for various oxytocin related ligands. As a high level of cell surface binding was retained for each clone, receptor trafficking appears to be normal. This suggests that the full glycosylation of OTR observed in vivo is not essential for its activity. These results indicate also that these cell lines may prove very useful for pharmacological screening of oxytocin related products.

Gene Transfer Techniques↗
PubMed1997

Identification of a novel vitamin D response element from the rat genome.

1 alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active form of vitamin D3, has been thought to be a multifunctional agent. In order to discover novel roles of 1,25-(OH)2D3, we have been looking for new genes that are regulated by 1,25-(OH)2D3. Because the actions of 1,25-(OH)2D3 are mediated through the vitamin D receptor (VDR), that is a DNA binding transcription factor, vitamin D regulated genes should have VDR binding sites in their regulatory regions. In this paper, we describe a novel vitamin D response element (VDRE)-containing sequence, clone 3, which was isolated through binding to VDR. DNA sequence analysis of clone 3 did not reveal any significant similarity with sequences reported previously. Clone 3 had two regions consisting of a direct repeated sequence of AGTTCA motifs, both of which bound to VDR independently. Whereas each direct repeat sequence alone could not mediate transcriptional activation efficiently, with their co-existence there was a strong response to 1,25-(OH)2D3, indicating that these two direct repeated sequences act cooperatively.

Animals↗
PubMed1997

Nuclear factor 1 family proteins bind to the silencer element in the rat glutathione transferase P gene.

We have previously identified a silencer region in the glutathione transferase P (GST-P) gene, of which the expression is completely repressed in liver of the rat. At least three trans-acting factors bind to multiple cis-elements in this region. Since GST-P silencer 4 (GSP4) is a dominant element in this silencer, we purified the GSP4 binding protein, called Silencer Factor A (SF-A). Purified SF-A was separated into several proteins on an SDS-polyacrylamide gel, and the amino acid sequences of four major components of SF-A were determined. The amino acid sequences of three fragments were identical to those of rat NF1-L, and that of the other fragment was the same as that of hamster NF1/Red1. It is known that nuclear factor 1 (NF1) family proteins are encoded by at least four independent genes in vertebrates, and NF1-L and NF1/Red1 are derived from different genes, NFI-A and NFI-B, respectively. The microsequencing of SF-A revealed that at least two types of NF1 existed in rat liver. Functional analysis by using GAL4-fusion protein in HepG2 cells revealed that NFI-A represented the transcription activity from human metallothionein IIA promoter. Our findings indicate that multiple forms of the NF1 family bind to the silencer region and contribute to the negative regulation of the GST-P gene expression.

Amino Acid Sequence↗
PubMed1997

CCAAT/enhancer-binding protein delta gene expression is mediated by APRF/STAT3.

The CCAAT/enhancer-binding protein delta (C/EBP delta) transcription factor is known to be rarely expressed but sharply induced at an early stage of the acute phase response. To investigate the regulation mechanisms for this induction, the 5'-flanking region of the rat C/EBP delta gene was isolated. Functional analyses involving transfection and footprinting indicated that the upstream region up to - 175 bp is sufficient for the full basal activity in rat fibroblast 3Y1 cells. At least three cis-elements including a GC box are involved in this activity. When HepG2 cells were treated with interleukin-6 (IL-6), C/EBP delta mRNA was rapidly induced. Transfection and gel shift analyses identified the binding site for the acute phase response factor/signal transducers and activators of transcription (APRF/STAT3). These findings strongly indicate that C/EBP delta gene expression is mediated by APRF/STAT3, which is phosphorylated for the activation through the IL-6 receptor when cells are treated with IL-6, and trans-activates the other acute phase response genes.

Animals↗
PubMed1997

Genomic organization of the rat nuclear factor I-A gene.

The nuclear factor 1 (NF1) protein family functions as a cellular transcription factor as well as an adenovirus DNA replication factor. This family consists of four subtypes, NFI-A, NFI-B, NFI-C, and NFI-X, each encoded by a different gene. Each subtype possesses different isoforms generated by alternative splicing. To date, only a porcine NFI-C gene has been cloned, and the gene structures of the other NF1 proteins have not yet been identified. We recently isolated four kinds of NFI-A cDNA clones from the rat liver. To gain additional insight into the structure of NFI-A, we isolated the rat NFI-A gene. This gene is composed of 11 exons spanning over 70 kb. All of the exon/intron boundaries are consistent with the GT/AG rule, and consensus sequences surrounding the splice boundaries are also found. The 5'-flanking region lacks a canonical TATA box, but contains several GC-box and AP2 binding sites. A 5'-rapid amplification of cDNA end analysis indicated that the transcription of the NFI-A gene is initiated at multiple sites. We also found conservation in the genomic structure between the rat NFI-A and the porcine NFI-C, suggesting that duplication of an ancestral gene occurred rather recently to produce the NFI-A and NFI-C genes.

Animals↗
PubMed1997

Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites.

Interleukin-1 (IL-1) molecules, IL-1 alpha and IL-1 beta are cytokines involved in the acute-phase response against infection and in the pathogenesis of periodontal destruction. Administration of exogenous IL-1 receptor antagonist (IL-1ra) is effective in reducing the inflammatory reactions mediated by IL-1. However, the relationship between these three naturally occurring IL-1 molecules and periodontal diseases has been poorly characterized. We investigated the correlation of gingival crevicular IL-1 molecules and the clinical status of patients with different severities of periodontitis. IL-1 alpha, IL-1 beta, IL-1ra and the total IL-1/IL-1ra ratio (IL-1 activity index; IL-1AI) were measured in 75 gingival crevicular fluid (GCF) samples from non-inflamed gingiva sites in 2 healthy subjects and diseased sites in 7 patients with several types of periodontitis. IL-1 alpha, IL-1 beta and IL-1ra were measured by specific non-cross-reactive enzyme linked immunosorbent assay. The probing depth, gingival index and alveolar bone loss of each site was recorded at the time of GCF sampling. The total amount of IL-1 alpha, IL-1 beta and the IL-1AI, but not total IL-1ra, were found to be correlated with alveolar bone loss score. Three IL-1 molecules were also measured in the gingival tissue of patients with periodontitis. A similar progressive decrease of the IL-1AI was detected in gingival tissue with periodontitis. These results suggest that the amounts of both crevicular IL-1 and IL-1AI are closely associated with periodontal disease severity.

Acute-Phase Reaction↗
PubMed1997

Prevalence of Actinobacillus actinomycetemcomitans serotypes in Japanese patients with periodontitis.

Oral Actinobacillus actinomycetemcomitans strains are serologically classified into 5 distinct groups, a to e. We examined the distribution of A. actinomycetemcomitans serotypes in Japanese patients with periodontitis. A total of 157 A. actinomycetemcomitans clinical isolates from diseased sites of 39 patients with periodontitis were serotyped by using serotype-specific rabbit antisera against A. actinomycetemcomitans serotypes a, b, c, d and e strains. In the immunodiffusion assay, autoclaved extracts of 42, 6, 39, 9 and 41 A. actinomycetemcomitans clinical isolates reacted with serotypes a, b, c, d and e antisera, respectively. Although 37 patients were infected with a serotype strain, 2 patients harbored 2 different serotype strains, b/e and b/untypeable. To establish a correlation between serotype and genotype of A. actinomycetemcomitans clinical isolates from 2 patients who had different serotype strains, we used arbitrarily primed polymerase chain reaction (AP-PCR) to fingerprint clinical isolates of different serotypes. The AP-PCR genotypes among 4 clinical isolates (b/e and b/untypeable) were identical to that of A. actinomycetemcomitans Y4 (serotype b), indicating the presence of multiple A. actinomycetemcomitans serotypes which are genetically homogeneous in the periodontally diseased sites of patients with periodontitis.

Actinobacillus Infections↗
PubMed1997

Role of CD14 molecules in internalization of Actinobacillus actinomycetemcomitans by macrophages and subsequent induction of apoptosis.

We report the evidence for apoptosis in J774.1 cells by the periodontopathic bacterium Actinobacillus actinomycetemcomitans, suggesting that the ability of A. actinomycetemcomitans to promote apoptosis might be important in the initiation and development of periodontitis. In this study, we examined the role of macrophage CD14, anchored by a glycerophosphatidylinositol tail, in the induction of apoptosis by A. actinomycetemcomitans infection by using the parent J774.1 cells and CD14-defective mutant (LR-9) cells. A small number of A. actinomycetemcomitans Y4 cells inside the LR-9 cells compared with the number in J774.1 cells was detected by confocal scanning microscopy. We found that LR-9 cells showed a weak cytotoxic effect after being infected with A. actinomycetemcomitans Y4. Apoptotic cell death of LR-9 cells infected with A. actinomycetemcomitans Y4, compared with that of the parent J774.1 cells was almost undetectable, as shown by the proportion of fragmented DNA in agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP end-labeling method. Flow cytometric cell cycle analysis of J774.1 cells infected with A. actinomycetemcomitans Y4 revealed the increased percentage of apoptotic cells with hypodiploid DNA. However, LR-9 cells infected with A. actinomycetemcomitans Y4 showed no increase in population of apoptotic nuclei compared with the noninfected cells. These findings suggest that the CD14 molecules may contribute to the phagocytosis of A. actinomycetemcomitans by J774.1 cells and regulate, at least in part, apoptotic cell death of macrophages infected with A. actinomycetemcomitans.

Actinobacillus Infections↗
PubMed1997

Activin A induction of cell-cycle arrest involves modulation of cyclin D2 and p21CIP1/WAF1 in plasmacytic cells.

Activins, members of the transforming growth factor-beta family, have been implicated in the regulation of growth and differentiation of various types of cells. We have recently found that activin A induces apoptotic cell death of plasmacytic cells including B cell hybridoma cells and myeloma cells. In the present study, we demonstrated that activin A caused cell-cycle arrest in the G1 phase before appearance of apoptotic cells in mouse B cell hybridoma cells. Phosphorylation of retinoblastoma protein (Rb) and in vitro Rb kinase activity of cyclin-dependent kinase (CDK)4 was inhibited in activin A-treated cells. Analysis of expression of genes regulating Rb phosphorylation revealed that activin A suppressed cyclin D2, the sole D-type cyclin gene expressed in the hybridoma cells, and activated p21CIP1/WAF1 but had no effect on expression of cyclin-dependent kinases (CDK2, CDK4, CDK6) and other CDK inhibitors (p27KIP1, p16INK4a, p15INK4b). Modulation of cyclin D2 and p21CIP1/WAF1 expression resulted in a decrease in level of cyclin D2-CDK4 complex and an increase in level of CDK4 complexed with p21CIP1/WAF1. Moreover, overexpression of cyclin D2 partially abrogated inhibition of Rb phosphorylation and G1 arrest in the hybridoma cells.

Activins↗
PubMed1997

Specific inhibitors of vacuolar H(+)-ATPase trigger apoptotic cell death of osteoclasts.

Osteoclasts are multinucleated bone-resorbing cells that play a critical role in bone remodeling. Specific inhibitors of vacuolar H(+)-ATPase (V-ATPase), concanamycin A and bafilomycin A1, abolish bone resorption by osteoclasts. In this study, we examined whether these V-ATPase inhibitors trigger apoptotic cell death in osteoclasts, using murine osteoclast-like multinucleated cells (OCLs) formed in vitro. Acridine orange staining revealed that the treatment of OCLs with concanamycin A resulted in chromatin condensation and alterations in nuclear morphology within a few hours. The TdT-mediated dUTP-nick-end labeling (TUNEL) reaction confirmed the apoptotic features of OCLs treated with concanamycin A. The accelerated apoptotic cell death induced by concanamycin A occurred in OCLs treated with interleukin-1 alpha or macrophage colony-stimulating factor as well, which are known to elongate the survival time of osteoclasts. In contrast, these inhibitors did not induce cell death of osteoblastic cells isolated from mouse calvaria. These results suggest that functional impairment of V-ATPase triggers apoptotic cell death in osteoclasts.

Animals↗
PubMed1997

[Acute myeloblastic leukemia showing pronounced skin infiltration during administration of low-dose cytarabine and etoposide with granulocyte colony-stimulating factor].

A 26-year-old woman was admitted to our hospital for lumbago on November 29, 1995. The white blood cell count was 6,500/microliter with 26.5% myeloblasts and the bone marrow was hyperplastic due to myeloblasts. Myeloblasts were negative for myeloperoxidase and positive for alpha-naphthyl butylate esterase, CD11a (89%), CD11b (38%), CD11c (92%), CD33 (91%) and HLA-DR (58%). Chromosomal abnormalities were recognized: 46, XX, t(9;11) (p22;q23), 45, XX, -7, t(9;11) (p22;q23) and 47, XX, +19, t(9;11) (p22;q23). Acute myeloblastic leukemia (M5a) was diagnosed. Disseminated intravascular coagulation was also present. The patient received induction therapy and achieved remission on January 9, 1996, but myeloblasts increased to 3.6% in bone marrow despite consolidation therapy. Low doses of cytarabine (AraC) and etoposide were instituted on March 7, granulocyte colony-stimulating factor (G-CSF) was started on March 15, and pronounced skin infiltration developed on March 18. The patient received reinduction therapy from April 16 and administration of G-CSF was combined for 2 days, and a marked increment of myeloblasts in the peripheral blood was observed. After discontinuation of G-CSF, myeloblasts decreased and skin infiltration disappeared. However, the patient died of cerebral infiltration on June 30. The response of myeloblasts to G-CSF by in vitro liquid culture was noteworthy. The present case stresses the requirement for great caution to be exercised in the use of G-CSF in patients receiving low dose AraC.

Adult↗
PubMed1996

Identification of DNA binding-site preferences for nuclear factor I-A.

Nuclear factor I (NFI) proteins constitute a large family of DNA binding proteins. These proteins promote the initiation of adenovirus replication and regulate the transcription of viral and cellular genes. The binding sites for NFI have been reported in a wide variety of promoters, and they exhibit flexibility in their sequences. To clarify the DNA binding site of NFI-A, one of the NFI proteins, we performed a polymerase chain reaction-mediated random site selection, and determined the optimal sequence as 5'-TTGGCANNNN(G/T)CCA(G/A)-3'.

Animals↗
PubMed1996

Extra Y chromosome in T-cell acute lymphoblastic leukemia.

We describe a 66-year-old man who developed T-cell acute lymphoblastic leukemia (ALL) with a sole clonal chromosomal abnormality of 47,XY,+Y. Leukemic cells were positive for CD2, CD7, terminal deoxynucleotidyl transferase and cytoplasmic CD3. T-cell receptor beta, gamma, and delta genes remained germline configurations. The bone marrow aspirate was 47,XY,+Y in all metaphase cells observed. The patient achieved complete remission by chemotherapy, and the bone marrow cells and the phytohemagglutinin stimulated peripheral blood lymphocytes showed a normal karyotype of 46,XY at that time. This fact suggests that an extra Y chromosome may be a kind of new chromosomal abnormality of T-cell ALL.

Aged↗
PubMed1996

Title aggregation patterns of argyrophilic nucleolar organizer regions induced by 5-fluorouracil in the nuclei of MCF-7 human breast cancer cells.

The effects of tamoxifen and 5-fluorouracil (5-FU) on the patterns of argyrophilic nucleolar organizer regions (AgNORs) in MCF7 human breast cancer cells were studied. Tamoxifen and 5-FU both inhibited the growth of MCF-7 cells by 18% by day 3 of culture, but each had different effects on the AgNORs. Whereas no significant changes were induced by tamoxifen, effects on the AgNORs of MCF-7 cells by 5-FU were dramatic: 5-FU treatment changed the pattern of AgNORs, reducing the number of satellites by aggregation, typically to a single aggregation around nucleoli in a sphenoidal fashion. We named these morphological changes: fluorouracil induced AgNOR aggregations (FAA). Following treatment with 500 ng/ml 5-FU, FAA developed rapidly. AgNORs forming two or three aggregates in 24% (6 h), 24% (12 h), 40% (24 h) and 34% (48 h) of cells, compared to a control rate of 14%. Single large aggregate was rarely found in untreated cultures but after 6, 12, 24 and 48 h treatment with 500 ng/ml 5-FU, AgNORs had formed a single aggregate in 6, 8, 16 and 22% of cells, respectively. FAA were observed at a concentration of 100 ng/ml 5-FU; 48 h treatment resulted in cells in which two or three aggregates were increased by 24% and single aggregate by 16%. These large single aggregates were larger than nucleoli stained by Papanicolau staining.

Antimetabolites, Antineoplastic↗
PubMed1996

DNA binding specificity of the CCAAT/enhancer-binding protein transcription factor family.

CCAAT/enhancer-binding protein (C/EBP) transcription factor family members are related by a high degree of amino acid sequence identity to the basic leucine zipper DNA-binding domain and show distinct but overlapping patterns of tissue- and stage-restricted expression. Although C/EBPalpha and C/EBPbeta have been shown to recognize a consensus sequence derived from regulatory elements in virus and acute-phase response genes, the potential for more subtle differences in the binding preference of the C/EBP family has not been previously addressed. The consensus sequence of C/EBPdelta has not been reported. By using the method of polymerase chain reaction-mediated random site selection to assess the DNA binding specificity of the C/EBP family in an unbiased manner, we demonstrated the sequence preferences for C/EBP family members. With small variations, these C/EBP family members showed similar sequence preferences, and the consensus sequence was identified as RTTGCGYAAY (R = A or G, and Y = C or T). The phosphorylation of C/EBPdelta by casein kinase II increased the binding activity, but did not affect the binding specificity, whereas it was reported that the phosphorylation of C/EBPalpha and C/EBPbeta decreased the binding affinity. The specificity of action of C/EBP family members may be derived from the characteristics of each factor, including the expression profiles, the DNA binding affinities, the cofactors, and so on, in addition to the DNA binding specificities.

Animals↗
PubMed1996

Isolation of a genomic DNA fragment having negative vitamin D response element.

The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) in the promoter region of target genes and acts as a ligand-dependent transcriptional regulator. In order to identify novel VDREs and new genes that are regulated by the active form of vitamin D [1,25-(OH)2D3], rat genomic DNA fragments bound by VDR were isolated. One of these fragments, designated as VBF5 was transcribed and the transcript was down-regulated by 1,25-(OH)2D3. These data strongly indicate that VBF5 may contain a VDRE regulating negatively an unidentified gene expression.

Animals↗