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Biomedical subjects

S Inoue

Publications and source records attributed to S Inoue.

At least 433 records · Page 24Linked to original sources
PubMed1998

Development of a highly sensitive chemical method for detecting alpha2-->8-linked oligo/polysialic acid residues in glycoproteins blotted on the membrane.

A highly sensitive chemical method for detecting alpha2-->8-linked oligo/polysialic acid (oligo/polySia) chains was developed, including (i) periodate oxidation, reduction with sodium borohydride, and subsequent acid hydrolysis, giving rise to C7 analogues and intact C9 compounds from nonreducing terminal and internal sialic acid residues, respectively; (ii) fluorescent labeling of these C7 and C9 compounds with 1,2-diamino-4,5-methylenedioxybenzene (DMB); and (iii) quantitation of theseDMB derivatives on fluorometric high-performance liquid chromatography. As little as 1 ng of internal sialic acid residues of oligo/polySia chains, the existence of which indicates the presence of oligo/polySia structure, was detectable by this method. This fluorometric C7/C9 analysis was successfully applied to glycoproteins blotted onto a slit of polyvinylidene fluoride membranes and suggested the presence of some novel oligoSia-containing glycoproteins in pig embryonic brains.

Animals↗
PubMed1998

Oxidative DNA damage and apoptosis induced by metabolites of butylated hydroxytoluene.

DNA damage by metabolites of a food additive, butylated hydroxytoluene (BHT), was investigated as a potential mechanism of carcinogenicity. The mechanism of DNA damage by 2,6-di-tert-butyl-p-benzoquinone (BHT-quinone), 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHT-OOH), and 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO) in the presence of metal ions was investigated by using 32P-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. BHT-OOH caused DNA damage in the presence of Cu(II), whereas BHT-quinone and BHT-CHO did not. However, BHT-quinone did induce DNA damage in the presence of NADH and Cu(II). Bathocuproine inhibited Cu(II)-mediated DNA damage, indicating the participation of Cu(I) in the process. Catalase also inhibited DNA damage induced by BHT-quinone, but not that induced by BHT-OOH. The DNA cleavage pattern observed with BHT-quinone plus NADH was different from that seen with BHT-OOH. With BHT-quinone plus NADH, piperidine-labile sites could be generated at nucleotides other than adenine residue. BHT-OOH caused cleavage specifically at guanine residues. Pulsed field gel electrophoresis showed that BHT-OOH and BHT-quinone induced DNA strand breaks in cultured cells, whereas BHT-CHO did not. Both BHT-quinone and BHT-OOH induced internucleosomal DNA fragmentation, which is the characteristic of apoptosis. Furthermore, flow cytometry analysis revealed an increase of peroxides in cultured cells treated with BHT-OOH or BHT-quinone. These results suggest that BHT-OOH participates in oxidative DNA damage directly, whereas BHT-quinone causes DNA damage through H2O2 generation, which leads to internucleosomal DNA fragmentation.

Apoptosis↗
PubMed1998

Molecular cloning and characterization of human estrogen receptor betacx: a potential inhibitor ofestrogen action in human.

We have identified and characterized a novel human estrogen receptor (ER) beta isoform, ERbetacx, which is truncated at the C-terminal region but has an extra 26 amino acids due to alternative splicing. The ERbetacx transcript is expressed in testis, ovary, thymus and prostate as well as in human cultured cell lines such as HEC-1, HOS-TE85 and Saos-2 cells. ERbetacx protein is also immunodetectable in these human cells. Biochemical analysis reveals that the average dissociation constants ( K d) of ERalpha and ERbeta for 17beta-estradiol (E2) are 0.2 and 0.6 nM respectively, but ERbetacx has no ligand binding ability. ERalpha and ERbeta proteins bind to the estrogen response element, whereas ERbetacx does not form any shifted complex in gel shift assays. In a transient expression assay, ERbetacx shows no ligand-dependent transactivation ability of a basal promoter and also cannot interact with a cofactor, TIF1alpha, in the presence or absence of E2. ERbetacx preferentially forms a heterodimer with ERalpha rather than that with ERbeta, inhibiting DNA binding by ERalpha. Interestingly, however, it shows a significant dominant negative activity only against ERalpha transactivation. Thus, this study indicates that ERbetacx potentially inhibits ERalpha-mediated estrogen action and that alternative splicing of the C-terminal region and its inhibitory properties are characteristic of several members of nuclear receptor isoforms.

Amino Acid Sequence↗
PubMed1998

A novel phospholipase A2 inhibitor with leucine-rich repeats from the blood plasma of Agkistrodon blomhoffii siniticus. Sequence homologies with human leucine-rich alpha2-glycoprotein.

The phospholipase A2 (PLA2) inhibitor PLIbeta, purified from the blood plasma of Chinese mamushi snake (Agkistrodon blomhoffii siniticus), is a 160-kDa trimer with three 50-kDa subunits; and it inhibits specifically the enzymatic activity of the basic PLA2 from its own venom (Ohkura, N., Okuhara, H., Inoue, S., Ikeda, K., and Hayashi, K. (1997) Biochem. J. 325, 527-531). In the present study, the 50-kDa subunit was found to be glycosylated with N-linked carbohydrate, and enzymatic deglycosylation decreased the molecular mass of the 50-kDa subunit to 39-kDa. One 160-kDa trimer of PLIbeta was found to form a stable complex with three basic PLA2 molecules, indicating that one basic PLA2 molecule would bind stoichiometrically to one subunit of PLIbeta. A cDNA encoding PLIbeta was isolated from a Chinese mamushi liver cDNA library by use of a probe prepared by a polymerase chain reaction on the basis of the partially determined amino acid sequence of the subunit. The cDNA contained an open reading frame encoding a 23-residue signal sequence followed by a 308-residue protein, which contained the sequences of all the peptides derived by lysyl endopeptidase digestion of the subunit. The molecular mass of the mature protein was calculated to be 34,594 Da, and the deduced amino acid sequence contained four potential N-glycosylation sites. The sequence of PLIbeta showed no significant homology with that of the known PLA2 inhibitors. But, interestingly, it exhibited 33% identity with that of human leucine-rich alpha2-glycoprotein, a serum protein of unknown function. The most striking feature of the sequence is that it contained nine leucine-rich repeats (LRRs), each of 24 amino acid residues and thus encompassing over two-thirds of the molecule. LRRs in PLIbeta might be responsible for the specific binding to basic PLA2, since LRRs are considered as the motifs involved in protein-protein interactions.

Agkistrodon↗
PubMed1998

Anionic Polymerization of 1,3-Cyclohexadiene with Alkyllithium/Amine Systems. Characteristics of n-Butyllithium/N,N,N',N'-Tetramethylethylenediamine System for Living Anionic Polymerization.

The n-butyllithium (n-BuLi)/N,N,N',N'-tetramethylethylenediamine (TMEDA) system (with the molar ratio of TMEDA to n-BuLi higher than 4/4) has been found to polymerize 1,3-cyclohexadiene to produce "living" polymer having a narrow molecular weight distribution with well-controlled polymer chain length. The rate of polymerization and polymer yield increased with increasing of the ratio of TMEDA to n-BuLi. The molecular weight distribution of obtained polymers became narrower with the ratio of TMEDA to n-BuLi. The formation of benzene generated by termination reaction was found to decrease with the ratio of TMEDA to n-BuLi. In contrast to 1,3-cyclohexadiene, low yield and broad molecular weight distribution were observed in the polymerization of 1,3-hexadiene and 2,4-hexadiene initiated by the n-BuLi/TMEDA (4/5) system. The microstructure of poly(1,3-cyclohexadiene) was determined by 2D-NMR. A high content of 1,2-units was found in the polymerization initiated by the alkyllithium/TMEDA system, while the high content of 1,4-units was observed in the cases of alkyllithium or alkyllithium/1,4-diazabicyclo[2,2,2]octane system. The 7Li NMR signals of the n-BuLi/TMEDA systems and poly(1,3-cyclohexadienyl)lithium /TMEDA were in higher magnetic field than the signals of n-BuLi and poly(1,3-cyclohexadienyl)lithium, respectively, indicating the disaggregation of the lithium species.

Journal Article↗
PubMed1998

A Case of Sclerosing Lymphocytic Lobulitis of the Breast Associated with Diabetes Mellitus.

BACKGROUND: The benign breast disease sclerosing lymphocytic lobulitis is thought to result from autoimmune diseases causing insulin-dependent diabetes mellitus (IDDM) due to insulinitis. In cases of sclerosing lymphocytic lobulitis accompanied by IDDM, the clinical term gdiabetic fibrous breast disease hhas been proposed. METHODS: A case of sclerosing lymphocytic lobulitis of the breast is described RESULTS: The patient was a 43-year-old woman diagnosed with non insulin-dependent diabetes mellitus (NIDDM) 8 years previously. Insulin therapy was thought to be necessary because treatment with glibenclamide was not effective. She visited our facility complaining of a lump in her right breast that was 5 cm in diameter, painless, rock-hard, discrete, and irregularly outlined. Biopsy was performed because breast cancer was strongly suggested by its hardness and its irregular internal echo on ultrasonography. Histopathological findings demonstrated marked stromal sclerosis and lymphocyte infiltration in the perivascular and perilobular areas. Sclerosing lymphocytic lobulitis was diagnosed. CONCLUSION: Referring physicians should avoid performing unnecessary repeated biopsies by recognizing this disease entity, which often occurs bilaterally.

Journal Article↗
PubMed1998

Transforming growth factor-beta1-induced degradation of activated Src tyrosine kinase in rat fibroblasts.

Transforming growth factors-beta (TGF-betas) play pivotal roles in the regulation of cell growth and differentiation, although little is known regarding the regulation of cytoplasmic components by TGF-betas. Src tyrosine kinase is required for signal transduction of various cytokine receptors, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and G-protein coupled receptors. Moreover, Src tyrosine kinase is important in signal cross-talk between these receptors. To determine whether Src kinase is also involved in TGF-beta signaling, we examined the effects of TGF-beta1 on Src in the rat fibroblast cell line 3Y1 and in v-Src-transformed 3Y0 (SR-3Y1). TGF-beta1 inhibited mitogen-activated protein kinase activity and inhibited growth in SR-3Y1 cells, while it did not affect the growth of 3Y1 cells. TGF-beta1 significantly decreased v-Src kinase activity and protein abundance in SR-3Y1 cells, mainly by accelerating the degradation of v-Src. In contrast, in 3Y1 cells, TGF-beta1 did not affect c-Src abundance or kinase activity. However, upon activation of c-Src in 3Y1 cells by PDGF, TGF-beta1 decreased Src abundance. Additionally, in 3Y1 cells transfected with an activated c-Src mutant which lacks the SH3 domain, its level was decreased by TGF-beta1 treatment. These findings suggest that TGF-beta1 specifically induces degradation of activated Src kinase. This may be a novel mechanism for cross-talk between growth factors and TGF-beta1.

Animals↗
PubMed1998

High-performance capillary electrophoretic characterization of different types of oligo- and polysialic acid chains.

We have carried out comparative structural analysis of novel oligo- and polysialic acid chains from diverse sources. Controlled acid hydrolysates of (a) colominic acid, alpha2-->8-linked homopolymer of N-acetylneuraminic acid (Neu5Ac), (b) alpha2-->8-linked oligo/polyNeu5Gc chains present in rainbow trout egg polysialoglycoprotein, and (c) alpha2-->8-linked oligomers of deaminoneuraminic acid (KDN) residues of KDN-rich glycoprotein derived from rainbow trout vitelline envelope were analyzed by high-performance capillary electrophoresis (HPCE). The results showed that three different types of alpha2-->8-linked oligosialic acids having same degree of polymerization can be separated by HPCE. A partial hydrolysate of colominic acid with mild acid was shown by CE to form intramolecular esters during the controlled hydrolysis and the subsequent workup procedure. In contrast, lactonization of (-->5-Oglycolyl-Neu5Gcalpha2-->)n, alpha2-->5-Oglycolyl-linked homopolymer of N-glycolylneuraminic acid (Neu5Gc) present in the egg jelly coat of sea urchin, did not take place as readily as in (-->8Neu5Acalpha2-->)n.

Animals↗
PubMed1998

Rapid fragmentation of vimentin in human skin fibroblasts exposed to tamoxifen: a possible involvement of caspase-3.

Tamoxifen (TAM), an anti-estrogen compound, is widely used for chemotherapy of breast cancer, although the molecular mechanisms underlying TAM cytotoxicity are obscure. Here, we show that TAM dramatically caused degradation of vimentin (VIM) in human skin fibroblasts, in a time and dose dependent manner. Addition of caspase-3 inhibitor, Z-DEVD-FMK, inhibited formation of some fragments of VIM, and caspase-3 was proteolytically activated by TAM treatment. Expression of functional estrogen receptors were negative in these cells, and neither transcription nor protein synthesis was required for TAM-induced degradation of VIM. Moreover, quinestrol, an ethinyl estradiol derivative, weakly degraded VIM, whereas neither estradiols nor estriol had any effects. Taken together, TAM may induce fragmentation of VIM associated with an activation of caspase-3, which may be attributed to non-genomic actions of TAM.

Antineoplastic Agents, Hormonal↗
PubMed1998

pH dependence of the reaction rate of p-bromophenacyl bromide and of the binding constants of Ca2+ and an amide-type substrate analog to bovine pancreatic phospholipase A2.

pH dependence of the chemical reaction rates of p-bromophenacyl bromide (BPB) and of the binding constants of Ca2+ to bovine pancreatic active- and pro-phospholipases A2 (PLA2s) was studied at 25 degrees C and ionic strength 0.2. The pH dependence curves of the reaction rates of BPB with both enzymes were biphasic. The amino acid residues participating in the two transitions were ascribed to His 48 and the N-terminal alpha-amino group for the active enzyme and to His 48 and Arg -1 for the proenzyme. The pH dependence curve of Ca2+ binding to the active enzyme was interpreted in terms of participation of Asp 49, His 48, and the alpha-amino group. On the other hand, the curve for the proenzyme was interpreted in terms of participation of Asp 49, His 48, and Arg -1. The Ca2+ and pH dependence of the binding constant of a potent competitive inhibitor, monodispersed (R)-2-dodecanoylamino-1-hexanol-phosphocholine (amide-PC), to bovine pancreatic active-PLA2 was also studied. The binding of amide-PC was markedly facilitated by Ca2+ binding to the enzyme, whereas that of a genuine substrate, monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), was independent of Ca2+ binding. The pH dependence curve of the binding constant of the amide-PC showed one transition, and this was interpreted in terms of participation of His 48, whereas the binding of the diC6PC was independent of the ionization state of His 48. The difference in the Ca2+ dependence for the bindings of the diC6PC and amide-PC was considered to arise from the fact that the amide group of amide-PC can form a hydrogen bond with His 48, whereas the genuine substrate cannot form such a hydrogen bond.

Acetophenones↗
PubMed1998

TIP49, homologous to the bacterial DNA helicase RuvB, acts as an autoantigen in human.

TATA-binding protein (TBP), a central component for transcriptional regulation, forms complexes with various transcription regulators. We have isolated a novel human cDNA for a 49-kD TBP-interacting protein (TIP49). The human TIP49 was highly homologous to bacterial RuvB proteins that function as a DNA helicase to promote branch migration of the Holliday junction. Immunofluorescence analysis using anti-TIP49 antibody showed a typical dot-shaped nuclear staining pattern, suggesting that TIP49 is included in a macromolecular structure in the nucleus and may participate in nuclear events such as transcription and recombination. Moreover, glycerol gradient analysis demonstrated that TIP49 is present in a macromolecular complex in nuclear extracts. Interestingly, we detected a high level of autoantibodies against TIP49 in sera of patients with autoimmune diseases such as polymyositis/dermatomyositis and autoimmune hepatitis. This indicates that the autoantibody against this protein is a new marker for particular connective tissue diseases. These findings provide further evidence that the macromolecular structures described above are targeted by an autoimmune mechanism. The anti-TIP49 antibodies can be useful probes for clinical diagnosis and for investigation of intranuclear structure.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Inhibition of membrane-type 1 matrix metalloproteinase by hydroxamate inhibitors: an examination of the subsite pocket.

The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been reported to mediate the activation of pro-gelatinase A (proMMP-2), which is associated with tumor proliferation and metastasis. MT1-MMP can also digest extracellular matrix (ECM) such as interstitial collagens, gelatin, and proteoglycan and thus may play an important role in pathophysiological digestion of ECM. We studied the inhibitory effect of various hydroxamate MMP inhibitors, including known inhibitors such as BB-94, BB-2516, GM6001, and Ro31-9790, on a deletion mutant of MT1-MMP lacking the transmembrane domain (DeltaMT1) to further characterize the enzyme and develop a selective inhibitor for MT1-MMP. The evaluation of the inhibitory activities of various hydroxamates reveals general structural profiles affecting selectivities toward MMPs. In particular, a longer side chain at the P1' position is preferable for the binding to MMP-2, -3, and -9 and MT1-MMP. For the P2' position, an alpha-branched alkyl group is critical for the binding toward DeltaMT1, while the introduction of a bulky group at the alpha-position of hydroxamic acid seems to diminish the activity against DeltaMT1. Summation of the data on the sensitivity of DeltaMT1 to various hydroxamate inhibitors indicates that (1) the volume of the S1' subsite of DeltaMT1 is similar to that of MMP-2, -3, and -9, which is bigger than that of MMP-1, and (2) the S1 and S2' subsites are narrower than those in other MMPs. On the basis of these results, the hydroxamates with a P1' phenylpropyl and P2' alpha-branched alkyl group were synthesized and evaluated for inhibitory activity. These inhibitors (1h,i) showed strong activity against DeltaMT1 over MMP-1, but no selectivity between DeltaMT1 and MMP-9. These results are explained using molecular modeling studies conducted on MT1-MMP.

Amino Acid Sequence↗
PubMed1998

Flow cytometric measurement of benzo[a]pyrene-diol-epoxide-DNA adducts in normal human peripheral lymphocytes and cultured human lung cancer cells.

DNA adducts are mainly detected by 32P-postlabeling and enzyme-linked immunosorbent assays. We have established a method for detection of benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts by flow cytometry, and have clarified the effects of the DNA adducts on cell-cycle progression and the relationship between cell-cycle phases and DNA adduct formation, using human peripheral lymphocytes and three human lung cancer cell lines. We measured the BPDE-DNA adduct levels in both lymphocytes and cancer cells by isolating nuclei, using a nuclear isolation buffer containing Triton X-100 and staining with a BPDE-DNA-specific monoclonal antibody and biotin-streptavidin fluorescein conjugates. BPDE did not affect cell-cycle progression in human peripheral lymphocytes. However, in human lung cancer cells exposed to > 1 microg/ml BPDE, accumulation of cells in the S phase was seen. Cells with DNA content greater than G2M (aneuploid cells) or cells with less than G1 DNA content (apoptotic cells) increased gradually with exposure to increasing BPDE concentrations, suggesting that BPDE may affect cell-cycle progression through binding to DNA. Thus, the measurement of DNA adducts by flow cytometry may provide new insights into carcinogenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

Inhibition of inducible nitric oxide synthase prevents LPS-induced acute lung injury in dogs.

Nitric oxide (NO) is produced by inducible NO synthase (iNOS) after LPS stimulation, and reacts with superoxide to form peroxynitrite. We hypothesize that in LPS-induced lung injury, NO generated by iNOS plays a key role through the formation of peroxynitrite. We developed an acute lung injury dog model by injecting LPS, and examined the effects of selective iNOS inhibitors, aminoguanidine (AG) and S-methylisothiourea sulfate (SMT), on the LPS-induced lung injury. At 24 h after LPS injection, arterial oxygen tension and mean arterial pressure decreased, and shunt ratio and lung wet-to-dry weight ratio increased. On histology, the LPS group had marked neutrophil infiltration and widening of the alveolar septa. On immunohistochemistry, iNOS and nitrotyrosine, a major product of nitration of protein by peroxynitrite, were observed in the interstitium, capillary wall, and neutrophils in the airspaces of the LPS group. Treatments with AG and SMT prevented worsening of gas exchange, hemodynamics, and wet-to-dry weight ratio. On histology, AG and SMT treatments markedly suppressed lung injury, iNOS protein, and nitrotyrosine production. We conclude that NO released by iNOS may play a critical role in the pathogenesis of LPS-induced acute lung injury. This study suggests that iNOS inhibitors may have potential in the treatment of LPS-induced acute respiratory distress syndrome.

Animals↗
PubMed1998

Cross-inhibition of both estrogen receptor alpha and beta pathways by each dominant negative mutant.

Both estrogen receptor alpha (ERalpha) and the recently identified ERbeta are nuclear receptors that are activated by estrogen. It was reported that ERalpha and ERbeta form heterodimers. Here, we show that they activate transcription independently rather than synergistically via estrogen response elements (ERE). To show the cross-talk between ERalpha and ERbeta, we utilized dominant negative mutants of ERs constructed by C-terminal truncation. Interestingly, ERalpha1-530 inhibited transactivation not only by ERalpha but also by ERbeta, whereas ERbeta1-481 inhibited transactivation by ERalpha as well as by ERbeta. The GST pull-down assay also demonstrated the cross-interaction of these mutants with wild-type ERalpha and ERbeta. Thus, we found dominant negative mutants that block both ERalpha and ERbeta signaling pathways.

Amino Acid Sequence↗
PubMed1998

Role of the histone deacetylase complex in acute promyelocytic leukaemia.

Non-liganded retinoic acid receptors (RARs) repress transcription of target genes by recruiting the histone deacetylase complex through a class of silencing mediators termed SMRT or N-CoR. Mutant forms of RARalpha, created by chromosomal translocations with either the PML (for promyelocytic leukaemia) or the PLZF (for promyelocytic leukaemia zinc finger) locus, are oncogenic and result in human acute promyelocytic leukaemia (APL). PML-RARalpha APL patients achieve complete remission following treatments with pharmacological doses of retinoic acids (RA); in contrast, PLZF-RARalpha patients respond very poorly, if at all. Here we report that the association of these two chimaeric receptors with the histone deacetylase (HDAC) complex helps to determine both the development of APL and the ability of patients to respond to retinoids. Consistent with these observations, inhibitors of histone deacetylase dramatically potentiate retinoid-induced differentiation of RA-sensitive, and restore retinoid responses of RA-resistant, APL cell lines. Our findings suggest that oncogenic RARs mediate leukaemogenesis through aberrant chromatin acetylation, and that pharmacological manipulation of nuclear receptor co-factors may be a useful approach in the treatment of human disease.

Antineoplastic Agents↗
PubMed1998

The complete primary structure of human estrogen receptor beta (hER beta) and its heterodimerization with ER alpha in vivo and in vitro.

Human estrogen receptor beta (hER beta) cDNA that encodes the full-length amino acid sequence has been isolated from testis poly(A)+ RNA with the combination of cDNA screening and reverse transcription-PCR. It is composed of a 1590-bp open reading frame and a segment of the 5'- and 3'-untranslated region (UTR) and encodes an additional 53 amino acids in the N-terminal region compared with the previously reported one. Protein interaction between ER alpha and ER beta was demonstrated in vitro by GST pull-down assay and in vivo by immunoprecipitation. Thus, this study indicates that ER alpha and ER beta can interact in vivo, cross-signaling each other.

Amino Acid Sequence↗