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Biomedical subjects

Y Inagaki

Publications and source records attributed to Y Inagaki.

At least 127 records · Page 7Linked to original sources
PubMed1995

Substrate-induced acceleration of N-ethylmaleimide reaction with the Cys-65 mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter depends on the interaction of Asp-66 with the substrate.

We previously reported that the reaction of [14C]N-ethylmaleimide (NEM) with the S65C mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA(B)) is competitively inhibited by tetracycline [Yamaguchi, A. et al., FEBS Lett. 322 (1993) 201-204]. However, this observation has been revealed to be a mistake. The reaction of [14C]NEM with S65C TetA(B) was significantly and reproducibly accelerated by tetracycline, i.e. not inhibited. When Asp-66 was replaced by Ala, the reaction of NEM with the Cys-65 residue was no longer affected by tetracycline. In contrast, when Arg-70 was replaced by Ala, the acceleration of the reaction was unaltered. The tetracycline acceleration of the reaction to the Cys-65 residue was further stimulated with energization of the membrane on the addition of NADH. On the other hand, the tetracycline-induced acceleration was not observed in the absence of a divalent cation. These observations indicated that the Cys-65 locus is exposed to the medium according to the interaction of a divalent cation-tetracycline chelation complex with Asp-66.

Amino Acid Sequence↗
PubMed1995

Overlapping pathways mediate the opposing actions of tumor necrosis factor-alpha and transforming growth factor-beta on alpha 2(I) collagen gene transcription.

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are multifunctional peptides intimately involved in the process of extracellular matrix remodeling. We recently showed that TGF-beta stimulates the human alpha 2(I) collagen gene by increasing the affinity of an Sp1-containing transcriptional complex bound to an upstream sequence termed the TbRE (Inagaki, Y., Truter, S. and Ramirez, F. (1994) J. Biol. Chem. 269, 14828-14834). Here, we report that the TbRE-bound complex also mediates the inhibitory signal of TNF-alpha. Nuclear proteins from cells treated with TNF-alpha bind to the TbRE sequence substantially more strongly than those from untreated cells. Additionally, TNF-alpha increases binding of a second protein complex that recognizes the negatively cis-acting element located immediately next to the TbRE. Thus, we postulate that TNF-alpha counteracts the TGF-beta-elicited stimulation of collagen gene expression through overlapping nuclear signaling pathways. One modifies the TGF-beta-targeted transcriptional complex, probably by reducing its stimulatory effect on collagen transcription. The other acts on the binding of the adjacent factor, presumably by increasing its effectiveness in repressing the activity of the collagen promoter. The convergence of the TGF-beta and TNF-alpha pathways on the same sequence of the alpha 2(I) collagen promoter is yet another example of combinatorial gene regulation achieved through composite response elements.

Base Sequence↗
PubMed1995

Structural and functional analysis of the promoter of the human alpha 1(XI) collagen gene.

In order to eventually elucidate the mechanisms regulating alpha 1(XI) collagen expression in cartilaginous and non-cartilaginous tissues, we performed an initial analysis of the structural-functional features of the promoter of the human gene (COL11A1). After cloning and sequencing the 5' portion of COL11A1, primer extension and nuclease protection assays identified several minor transcriptional start sites clustered around a major one located 318 base pairs from the ATG codon. Consistent with this finding, analysis of the upstream sequence revealed the absence of a TATA motif and the presence of several GC boxes. Transient transfection experiments delineated the smallest promoter sequence directing relatively high expression of a reporter gene in a cell type-specific manner. Nine nuclear protein-bound areas were located within this promoter sequence of the COL11A1 gene. Sequence homologies suggested that the majority of the footprints correspond to potential binding sites for ubiquitous nuclear proteins, such as AP2 and Sp1. Additional experimental evidence indicated that one of the protected areas may bind a transcriptional complex that is identical or closely related to the one that regulates tissue specificity in the coordinately expressed alpha 2(V) collagen gene.

Animals↗
PubMed1995

Amino acid sequences of porcine Sp38 and proacrosin required for binding to the zona pellucida.

We previously purified a boar sperm protein, sp38, and demonstrated that this protein bound to the 90-kDa family of zona pellucida (ZP) glycoprotein in a calcium-dependent manner. Sp38 competed with proacrosin for the binding to the zona pellucida. Herein we have isolated cDNA clones encoding sp38 from a boar testis cDNA library in lambda gt11. The amino acid sequence deduced from the cDNA sequence indicated that sp38 is initially synthesized as a 350-residue precursor protein. The N-terminal 51-residue sequence preceded the N-terminus of the mature sp38. Thus, the sp38 precursor is post-translationally modified to produce the mature protein of 299 residues. Immunostaining of sperm cells using an antibody prepared against a fusion protein of sp38 with T7 gene 10 protein suggested that sp38 is localized at the intraacrosomal region and is released after the acrosome reaction. The 11-residue sequence, KRLSKAKNLIE, in sp38 shared a significant degree of similarity with the 8-residue sequence, KRLQQLIE, in the C-terminal region of porcine proacrosin. Both synthetic oligopeptides corresponding to these two sequences inhibited the binding of 125I-labeled sp38 to zona pellucida glycoprotein.

Acrosin↗
PubMed1995

Inhibitory effect of the immunosuppressant FK506 on apoptotic cell death induced by HIV-1 gp120.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 may play a central role in inducing immunoregulatory disorders after HIV infection. The apoptotic death of normal human peripheral blood mononuclear cells was induced by priming with gp120 followed by stimulation with an anti-T cell receptor (TCR) antibody. Tumor necrosis factor-alpha produced by gp120-binding macrophages may be important to induce this cell death. Treatment of gp120-primed cells with an immunosuppressant (FK506) before TCR signaling inhibited apoptotic cell death, and this blocking effect of FK506 was concentration dependent. FK506 did not have any influence on cell growth and viability over the range of concentrations tested. These findings suggest that FK506 is a potentially useful drug in delaying the onset of AIDS after HIV infection.

Antiviral Agents↗
PubMed1995

Antibody-dependent cell-mediated cytotoxicity against cell lines generated by liver-specific idiotype-bearing antibody.

We produced a murine monoclonal antibody (mAb), designated H2-mAb, against a fractionated soluble phase of human liver homogenate which antibody reacted with human liver cells. A human antibody possessing the same idiotype as the H2-mAb, designated LSIA (liver-specific idiotype-bearing antibody), can be measured by a sandwich enzyme-linked immunosorbent assay, using the anti-H2 idiotype antibody. The serum level of LSIA in patients with histologically proven chronic hepatitis (CH) was significantly higher than that in healthy subjects and it was also higher than that in subjects with other diseases, including systemic lupus erythematosus. In a comparison between patients with CH type B and those with CH type C, there was no significant difference in serum levels of LSIA. It was possible to purify LSIA from the sera of patients with CH. The purified LSIA bound to the human cell lines Chang and HCC-M, derived from liver cells and a hepatoma respectively, but not to HeLa cells, a uterine carcinoma derivative. The reactivity of this mAb to HCC-M was weaker than that to Change. Moreover, the presence of LSIA caused an antibody-dependent cell-mediated cytotoxic challenge against Change cells in vitro.

Adult↗
PubMed1995

A new genetic assay for rifampicin susceptibility of Mycobacterium tuberculosis.

A new method for testing rifampicin (RFP) susceptibility of Mycobacterium tuberculosis was developed. This method is based on detection of the internal sequence derived from 71-kDa heat shock protein mRNA in tubercle bacilli heat-treated in the presence of RFP. The target sequence was amplified by reverse transcription and PCR, followed by agarose gel electrophoretic analysis. No amplification occurred in one RFP-susceptible strain by exposure to 45 degrees C for 45 min in Middlebrook 7H9 broth containing RFP (10 micrograms/ml) after overnight incubation at 37 degrees C. On the other hand, an amplified 275-bp product was obtained from the RFP-resistant strain MY-129. In a subsequent trial using 65 clinical isolates, this method defined their RFP susceptibility levels as well as the verification of the MICs obtained by the conventional agar dilution method, with the exception of one RFP-susceptible strain. Thus, this method provides a rapid and practical system to determine RFP susceptibility in M. tuberculosis.

Antibiotics, Antitubercular↗
PubMed1995

Clinical evaluation of bevantolol hydrochloride in patients with severe hypertension. The Cooperative Study Group on Bevantolol in Japan.

The clinical efficacy and safety of bevantolol hydrochloride, a newly developed beta-blocker, used in combination with other types of antihypertensive agents, were evaluated in patients with severe hypertension by multicenter open-label trials. A total of 28 patients were studied at 20 medical centers. Four patients (14.3%) were excluded for some reasons, and the remaining 24 patients (22 outpatients and 2 inpatients) were analyzed. Following the initiation of therapy, blood pressure decreased from 181 +/- 15 (SD)/114 +/- 3 mmHg to 170 +/- 17/102 +/- 8 mmHg (p < 0.01) on the 14th day of the therapy and gradually lowered further thereafter. At the end of the trial (8th week), blood pressure was stabilized at the level of 160 +/- 14/96 +/- 10 mmHg. The antihypertensive efficacy rated by the changes in mean blood pressure was 79.2% (19/24). Pulse rate decreased slightly but significantly from 75 +/- 10 beats/min to 70 +/- 7 (p < 0.05) on the 14th day of the therapy and stabilized at the similar level thereafter. As abnormal laboratory data were detected in 3 patients and 1 patient complained of a mild headache, the safety ratio was 83.3% (20/24 patients). When the usefulness was assessed in terms of antihypertensive efficacy and safety profiles, bevantolol hydrochloride was considered useful in 75.0% of the patients studied. In conclusion, bevantolol hydrochloride, used in combination with other classes of antihypertensive agents, appears to be an excellent drug for the management of patients with severe hypertension.

Adrenergic beta-Antagonists↗
PubMed1995

Immediate hypersensitive reactions to buckwheat ingestion and cross allergenicity between buckwheat and rice antigens in subjects with high levels of IgE antibodies to buckwheat.

BACKGROUND: Immediate hypersensitive reactions induced by buckwheat ingestion are considered to be IgE-mediated. Some subjects, however, develop no immediate adverse reactions after buckwheat ingestion despite high levels of buckwheat-specific antigens IgE. The mechanism is unknown. OBJECTIVE: To investigate the mechanisms. METHODS: RAST for buckwheat and rice and RAST inhibition between these antigens were performed using sera from 23 buckwheat-sensitive subjects and 30 buckwheat-tolerant subjects who had IgE antibodies for both buckwheat and rice. RESULTS: RAST values for buckwheat and rice were significantly correlated with each other (P < .01) in the buckwheat-tolerant group, but not in the buckwheat-sensitive group. This suggests the IgE antibodies from the subjects without any overt symptoms after buckwheat ingestion recognize the cross-reactive epitope between buckwheat and rice, whereas the IgE antibodies from those with immediate reactions to buckwheat ingestion do not. RAST inhibition assays were performed to evaluate this. RAST inhibition of heterogeneous combination of inhibitor and disc antigen such as rice and buckwheat was significantly smaller than that of homologous combination of rice and rice or buckwheat and buckwheat in the group with immediate symptoms after buckwheat ingestion. There was no significant difference in RAST inhibition between homologous and heterogeneous combinations in the group without the symptoms. CONCLUSIONS: There was cross-reactivity with IgE antibodies between buckwheat and rice and IgE antibodies from the buckwheat-tolerant subjects with high levels of IgE antibodies from the buckwheat might recognize the epitopes on buckwheat antigens which cross-react with rice antigens, whereas IgE antibodies from the buckwheat-sensitive subjects might bind to buckwheat-specific epitopes.

Adolescent↗
PubMed1995

Regulation of the alpha 2(I) collagen gene transcription in fat-storing cells derived from a cirrhotic liver.

Fat-storing cells (FSC) are the main producers of type I collagen in both normal and fibrotic livers. In order to elucidate the molecular mechanisms controlling collagen expression in FSC, we examined the transcription of the alpha 2(I) collagen gene (COL1A2) in two distinct FSC clones, CFSC-2G and CFSC-5H, derived from a single CCl4-induced cirrhotic liver. The phenotype of CFSC-2G resembles freshly isolated FSC, whereas that of CFSC-5H mimics activated myofibroblasts. Cell transfection experiments showed that the upstream sequence between nucleotides -378 and -183 is essential for COL1A2 transcription in both FSC clones. This is the same promoter region that is transcriptionally active and contains the binding site of a multimeric protein complex that mediates TGF-beta stimulation of COL1A2 expression in dermal fibroblasts. We therefore examined the relative levels of endogenous and transfected COL1A2 transcription and their response to TGF-beta treatment in the two FSC clones. The results showed that CFSC-5H expresses a significantly higher level of the COL1A2 mRNA than CFSC-2G. They also showed that TGF-beta treatment increases both endogenous and transfected COL1A2 transcription in CFSC-2G but not in CFSC-5H. Interestingly, nuclear proteins from both FSC clones bind to the TGF-beta-responsive element more strongly than those from dermal fibroblasts. Altogether, the data suggest that collagen production in CFSC-5H cells has been already activated by the autocrine stimulation of TGF-beta. In contrast, CFSC-2G cells are only partially activated but can be easily recruited to produce collagen when stimulated by exogenous TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipocytes↗
PubMed1995

The effect of DU-6859a, a new potent fluoroquinolone, on fecal microflora in human volunteers.

Following the oral administration of DU-6859a to six healthy male volunteers at 100 mg per dose three times a day for seven consecutive days, the degree of disturbances of fecal microflora and fecal drug concentrations were examined. The total viable count decreased transiently during the administration due to changes in the number of members of the family Bacteroidaceae, the most predominant organisms. The other obligate anaerobes and most aerobes including facultative anaerobes were suppressed markedly by DU-6859a, with an exception of yeasts. Members of the family Enterobacteriaceae and most of anaerobic bacteria were reduced to below the detection limit in all subjects from the 3rd day of administration period to one day after the discontinuation of the drug. Clostridium difficile was detected in one of the volunteers at the end of administration. Side effects, such as mild transient soft stool and mild transient diarrhoea, were observed in five and one volunteers, respectively. During the administration, DU-6859a was detected in feces at high concentrations which correlated well with the decrease of susceptible members of fecal flora as well as their detection rate.

Adult↗
PubMed1995

Clinical pathoepidemiology of hepatocellular carcinoma in Japan.

There has been a rapid increase in the number of patients with hepatocellular carcinoma (HCC) in Japan. This may be the result of an increased number of patients with hepatitis C virus (HCV)-related liver disease, and of prolonged survival of these patients due to improvement in the management of cirrhosis. Indeed, 96% of recent HCC patients admitted to our hospital were related to viral hepatitis B or C, and HCV infection accounted for three-fourths of these cases. Since most HCCs develop in the course of chronic liver disease, it is important to identify high risk groups for HCC development and to detect small HCCs early. We have established a screening method by combining various imaging studies and the measurement of serum alpha-fetoprotein; this combination has markedly increased the detection of small HCCs less than 2 cm in diameter. Multiple HCCs can occur in the same patient, either simultaneously or successively. Partly for this reason, the cumulative recurrence rate of HCC is very high even after surgical resection. In order to determine whether such HCCs arise from the common or different clonal origins, we have utilized the restriction landmark genomic scanning method. It remains to be clarified whether HCV plays a direct role in hepatocarcinogenesis, and whether antiviral treatments such as interferon eventually will decrease the incidence of HCC among patients with chronic HCV infection.

Adult↗
PubMed1994

Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site.

Transforming growth factor-beta 1 (TGF-beta) is a strong and rapid inducer of several genes coding for extracellular matrix components, such as type I collagen. We report here that TGF-beta stimulates transcription of the human alpha 2(I) collagen gene (COL1A2) promoter by increasing the affinity of an Sp1-containing protein complex for its cognate DNA-binding site. Cell transfection experiments mapped the TGF-beta-responsive element (TbRE) of the COL1A2 promoter to a 131 bp region that contains at least two cis-acting elements. Insertion of the TbRE upstream of the thymidine kinase promoter conferred TGF-beta inducibility to the otherwise unresponsive thymidine kinase promoter. Footprinting assays revealed that the TbRE contains two neighboring protein-bound sequences, termed Box 3A and Box B. Within Box 3A is an Sp1 recognition sequence whose structural integrity is required for nuclear protein binding in vitro, and for promoter inducibility in vivo. Gel mobility shift assays documented increased binding to the TbRE of nuclear proteins from TGF-beta-treated cells compared with those from TGF-beta-untreated fibroblasts. There was, however, no binding increase with Box 3A alone or with an Sp1 oligonucleotide. Thus, the results strongly suggest a functional interaction between Sp1 and other components of the TbRE complex in mediating TGF-beta stimulation of COL1A2 gene expression.

Base Sequence↗
PubMed1994

Changes of antigen expression on human hepatoma cell lines caused by sodium butyrate, a differentiation inducer.

We have previously reported the effect of a differentiation inducer, sodium butyrate (SB), on human hepatocellular carcinoma (HCC) cell lines, demonstrating that it was a potent inducer of differentiation. In the present study, we investigated the alteration in expression of an antigen defined by a murine monoclonal antibody, H2, as well as alterations in the expression of other antigens, on the HCC cell lines HCC-T, HCC-M, and PLC/PRF/5, since it is known that specific antigenic changes occur during the differentiation of leukemic cells. The expression of the antigen defined by H2 increased immunocytochemically on HCC-T, HCC-M, and PLC/PRF/5 during treatment with SB. A flowcytometric study showed that almost all the HCC-T and HCC-M cells treated with SB highly expressed this antigen after 5 days' treatment. The antigen expression detected by H2 decreased after the removal of SB from the medium. On the other hand, antigen expression detected by another monoclonal antibody, 5C11, was not changed by this treatment. The expression of intracellular adhesion molecule (ICAM)-1 in HCC-T increased slightly, but that of beta 2-microglobulin and HLA-DR did not change. These results demonstrated that some antigen expression was changed by SB treatment and that the antigen defined by H2 seemed to be highly expressed on human HCC cells in the differentiated state.

Antibodies, Monoclonal↗