Origin of buckling-dimer-row formation of Si(001) surfaces.
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Journal Article↗
Biomedical subjects
S Oikawa
Publications and source records attributed to S Oikawa.
Human inducible nitric oxide synthase gene is transcriptionally regulated by nuclear factor-kappaB dependent mechanism.
We previously showed a sequence of the 5'-flanking region of the human inducible nitric oxide synthase (hiNOS) gene that included putative cis-acting elements. A reported plasmid containing the 5'-flanking region of the hiNOS gene upstream from its reporter gene was constructed, and then transiently or stably transfected into human cells known to express the hiNOS gene following cytokine stimulation. The transfected cells showed the inducibility of the reporter activity following interleukin-1beta stimulation. Reporter inducibility disappeared in cells transfected with a plasmid mutated in the putative nuclear factor (NF)-kappaB binding region. In addition the induction was inhibited by a treatment of anti-oxidant, pyrrolidinedithiocarbamate, known as an NF-kappaB inhibitor. Our results demonstrate that the promotor including the NF-kappaB region is functional and that the hiNOS gene is transcriptionally regulated via NF-kappaB activation in human cells.
Base Sequence↗
Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species.
Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.
8-Hydroxy-2'-Deoxyguanosine↗
Roles of the conserved serines of metallothionein in cadmium binding.
The sequence of six amino acid residues -Ser-Cys-Cys-Ser-Cys-Cys- is present in all mammalian metallothionein sequences and has been highly conserved during evolution, although the metallothioneins have divergent primary sequences. To determine whether two serines in the sequence play a crucial role in metal-binding of metallothioneins, a mutant metallothionein with these two serines replaced by leucines was obtained using an Escherichia coli expression system. The expressed protein was analyzed for its chemical and spectroscopic properties. It was confirmed that the mutant metallothionein (MT) bound cadmium through a metal-thiolate complex and that there was no strong difference between the mutant and the wild-type MTs in retaining the metal-binding cluster. However, the metal-binding cluster of the mutant metallothionein was more unstable than that of the wild-type metallothionein. The two conservative serines could play a role in the stability of metal-binding ligands.
Amino Acid Sequence↗
Copper-mediated DNA damage by metabolites of p-dichlorobenzene.
p-Dichlorobenzene (p-DCB) has been reported to be carcinogenic for rodents, although it does not seem to be mutagenic in bacterial test systems. In this study, the mechanism of DNA damage by metabolites of p-DCB in the presence of metals was investigated by a DNA sequencing technique using 32P-labeled DNA fragments and by an electrochemical detector coupled to an HPLC. 2,5-Dichlorohydroquinone (DCHQ), one of the major metabolites, caused DNA damage in the presence of Cu(II). 2,5-Dichloro-p-benzoquinone (DCBQ) slightly induced DNA damage in the presence of Cu(II), but addition of NADH induced DNA damage very efficiently. DCHQ plus Cu(II) induced piperidine-labile sites at thymine residues at high frequency. A similar DNA cleavage pattern was observed with DCBQ plus Cu(II) in the presence of NADH. Both DCHQ and DCBQ plus NADH increased 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Typical hydroxyl radical scavengers showed no inhibitory effects on this Cu(II)-mediated DNA damage. Bathocuproine and catalase inhibited the DNA damage, indicating the participation of Cu(I) and hydrogen peroxide (H2O2) in the DNA damage. UV-visible and ESR spectroscopy has demonstrated that DCHQ is rapidly autoxidized to DCBQ via a semiquinone radical, even in the absence of metal ions, indicating that the semiquinone radical itself is not the main active species inducing DNA damage. These results suggest that a semiquinone radical produced by autoxidation of DCHQ and/or reduction of DCBQ by NADH reacts with O2 to form superoxide and subsequently H2O2. Consequently, it is considered that the active species derived from the reaction of H2O2 with Cu(I) participates in the DNA damage.
8-Hydroxy-2'-Deoxyguanosine↗
Independent self-assembly of cadmium-binding alpha-fragment of metallothionein in Escherichia coli without participation of beta-fragment.
We examined the independent self-assembly of the alpha- and beta-fragments of human metallothionein (MT) into cadmium-binding conformation in an Escherichia coli expression system, in addition to wild-type MT expression. The expressed alpha-fragment formed independently the structure of a metal-binding cluster without the aid of the beta-fragment. The alpha-fragment and wild-type MT expressed in E.coli were purified and analyzed for their biochemical and spectroscopic properties. The apparent cadmium binding of the alpha-fragment was approximately 12-fold greater than that for the wild-type MT, whereas in other respects the studied biochemical properties were similar. In contrast, we were unable to obtain any independently expressed beta-fragment as the cadmium-binding form in this study. Possible explanations for this phenomenon are discussed.
Amino Acid Sequence↗
Human arterial smooth muscle cell proliferation in diabetes.
In the present study, we focus on the proliferation of human arterial smooth muscle cells (SMCs) from NIDDM patients (DM-SMCs) to clarify the reactivity to the growth factor(s) in fetal calf serum (FCS) and the factor(s) secreted by T-cells. The proliferation of DM-SMCs was significantly greater than SMCs from nondiabetic patients (nonDM-SMC). DM-SMC conditioned medium (DM-condMed) increased the growth of nonDM-SMCs. These results suggest that the growth factor is secreted from DM-SMCs as an autocrine system, which increases the proliferation of nonDM-SMCs. T-cells increased DNA synthesis of SMCs, and DM-SMCs strikingly reacted to T-cells. The present results support a function of T-cells in stimulating SMC growth. In conclusion, human arterial SMC proliferation is increased in diabetes in the same fashion as in experimentally induced diabetes in animals through responses to growth factors and an increased autocrine system. These results provide a mechanism for the increase in atherosclerotic disease in diabetes.
Adult↗
Retrograde dissection of the temporalis muscle preventing muscle atrophy for pterional craniotomy. Technical note.
A procedure for preventing muscle atrophy in pterional craniotomy by temporalis muscle dissection is described, along with anatomical considerations. The inferior to superior dissection of the temporalis muscle is a very simple technique and is less invasive than other approaches.
Craniotomy↗
[Obesity and vascular diseases].
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Arteriosclerosis↗
Oxidative and nonoxidative mechanisms of site-specific DNA cleavage induced by copper-containing metallothioneins.
DNA cleavage induced by metallothionein (MT) containing copper was investigated by a DNA sequencing technique. Reconstituted Cd7-MT showed no ability to cause DNA cleavage. Commercially available rabbit MT I caused DNA cleavage, suggesting that DNA cleavage is due to the metal contained in commercial Mt. Cu2Cd5-MT and Cu12-MT were prepared by the treatment of commercial rabbit MT I with [Cu(CH3CN)4]CIO4. Cu12-MT frequently induced an alteration of thymine residues, especially in the 5'-GTC-3' sequence, and piperidine treatment led to chain cleavage at the thymine residues. The site specificity was similar to that obtained with Cu(I) plus H2O2. H2O2 enhanced DNA cleavage induced by Cu12-MT. Catalase and a Cu(I)-specific chelating agent, bathocuproine, inhibited DNA cleavage. These results suggest that Cu(I) and H2O2 have important roles in the production of active species causing DNA cleavage. Commercial MT and Cu2Cd5-MT induced DNA cleavage much less than Cu12-MT, but gave particularly specific DNA cleavage. Cu2Cd5-MT induced cleavage specifically at the central guanine residue of the 5'-GGT-3' sequence. A similar cleavage pattern was obtained with commercial MT. No effect of piperidine treatment suggests that the DNA cleavage might not be due to base damage and/or liberation. The DNA cleavage was inhibited efficiently by EDTA, but not by bathocuproine and catalase. Experiments with DNA ligands, albumin, and denatured DNA suggest that commercial MT and Cu2Cd5-MT induce nonoxidative cleavage of the deoxyribose phosphate backbone through its DNA recognition. These two types of cleavage mechanisms are discussed in relation to the possible role of Cu-MT in carcinogenesis.
Albumins↗
The effect of transfection of the CEA gene on the metastatic behavior of the human colorectal cancer cell line MIP-101.
Carcinoembryonic antigen (CEA) has been shown to increase the metastatic potential of some human colorectal cancer cell lines. To investigate further the mechanisms involved we have produced three clones (6, 8 and 17) from the poorly differentiated human colorectal cancer cell line MIP-101 that have been transfected with the full length cDNA encoding for human carcinoembryonic antigen (CEA). They produce CEA with a mol. wt. of 180000 by Western blotting and secrete it into the culture medium. Clone 6 is a high CEA producer, clones 8 and 17 are intermediate producers. The doubling time for clone 8 was similar to that of the parent cell line while clones 6 and 17 had doubling times nearly twice that of the parent cells. These clones are tumorigenic when injected subcutaneously in nude mice are positive for CEA by immunoperoxidase staining and the mice have elevated blood levels of CEA. Clone 6 formed large aggregates in culture while clone 17 formed smaller aggregates. Clone 8 behaved like the parent cell line with rare cell/cell contact. Clones 6 and 17 also adhered to CEA coated plastic while clone 8, a neo-transfected control and the parent cell line did not. A significant increase in the incidence of hepatic tumors was observed with clone 6 (P < 0.01) and clone 17 (P < 0.02) following intrasplenic injection into nude mice. Immunohistopathology of the hepatic tumors showed strong CEA staining from clones 6 and 17 with weak staining from clone 8. The parent cell line was negative for CEA as were the neo-transfected controls. Of the neo controls none of 10 had liver colonies. Mice injected with clone 6 which developed liver metastasis had the highest plasma levels of CEA (37.3 +/- 8.8 ng/ml). We observed strong CEA staining in Kupffer cells in the normal liver adjacent to the CEA producing tumors. This study provides further evidence for the involvement of CEA in the metastatic process.
Adenocarcinoma↗
Structural fluctuation of methyl N,N'-diacetyl-beta-D-chitobioside in vacuo and in aqueous solution: molecular dynamics simulations and proton NMR spectroscopy.
Molecular dynamics (MD) simulations of methyl N,N'-diacetyl-beta-D-chitobioside (GlcNAc beta-(1-->4)GlcNAc beta-OMe) have been performed both in vacuo and in aqueous solution with the explicit inclusion of the solvent water molecules. The beta-(1-->4) glycosidic linkage fluctuates considerably, over a range of +/- 10 degrees, in each of the MD simulations in vacuo and in aqueous solution. The intra- and inter-residue hydrogen bonds in vacuo are replaced by intermolecular hydrogen bonds with the solvent water molecules in aqueous solution. Multiple conformations (gg and gt) exist for the exocyclic hydroxymethyl groups. The results of the MD simulations are compared with those of 1H-1H nuclear Overhauser effect measurements.
Carbohydrate Conformation↗
A reference of the GOLD classification of monoclonal antibodies against carcinoembryonic antigen to the domain structure of the carcinoembryonic antigen molecule.
The epitopes of 42 well-characterized monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) from 10 different research groups were mapped in terms of domain structure (domains N, A1-B1, A2-B2, and A3-B3) of the CEA molecule on the basis of the reactivities with recombinant CEA proteins expressed in Chinese hamster ovary cells. Thirty-six of the 42 MAbs tested have previously been classified into 5 essentially nonoverlapping epitope groups (GOLD 1-5) by cross-competition assays among MAbs for CEA binding (Hammarström S, et al.: Cancer Res. 1989; 49:4852-4858). The epitopes recognized by GOLD 2 MAbs were all present on domain A2-B2, those for GOLD 5 MAbs were all on domain N, and those for GOLD 4 were mapped around domains A1-B1 and A2-B2. On the other hand, the epitopes for GOLD 1 MAbs were distributed into domains N, A2-B2, and A3-B3, and those for GOLD 3 MAbs were separated into domains N and A3-B3. Although the exact reasons for the dispersed patterns of GOLD 1 and 3 MAbs on the domain structure of the CEA molecule are unclear at present, several factors, such as a spatial relation or a close proximity of epitopes, conformation dependency, and repetitivity of epitopes, may be considered as possible explanations. The epitope mapping reported here helps form the basis for understanding the relation between the chemical structure and antigenic activities of the CEA molecule and may be useful to study the functions of the CEA molecule, especially those of the respective domains.
Animals↗
Metal-mediated oxidative damage to cellular and isolated DNA by certain tryptophan metabolites.
The tryptophan metabolites 3-hydroxyanthranilic acid (3-HAA) and 3-hydroxykynurenine (3-HKyn) are carcinogens. DNA damage by 3-HAA and 3-HKyn in the presence of metal ions was investigated as a potential mechanism of their carcinogenicity. Pulsed field gel electrophoresis showed that in the presence of Mn(II), 3-HAA and 3-HKyn induced DNA double-strand breaks in cultured human cells. DNA single-strand breaks were observed with alkali treatment. The enhancing effect of catalase inhibitor and the inhibitory effect of o-phenanthroline on the strand breakage indicated the involvement of H2O2 and endogenous transition metal ion. Damage to DNA fragments obtained from c-Ha-rds-1 protooncogene was investigated by a DNA sequencing technique. 3-HAA and 3-HKyn induced piperidine-labile sites frequently at thymine and guanine residues in the presence of Cu(II). The inhibitory effects of bathocuproine and catalase on Cu(II)-mediated DNA damage suggest that Cu(I) and H2O2 have important roles in the production of active species causing DNA damage. The Cu(II)-mediated DNA damage was enhanced by preincubation of 3-HAA with Mn(II). UV-visible spectroscopy showed that Mn(II) and Cu(II) enhanced the rate of autoxidation of 3-HAA in different ways. These results suggest that in the presence of Mn(II) or Cu(II), these tryptophan metabolites produce H2O2, which is activated by transition metal ion to cause damage to DNA both in the case of isolated DNA and cultured cells.
3-Hydroxyanthranilic Acid↗
Expression of human metallothionein-2 in Escherichia coli: cadmium tolerance of transformed cells.
A genetic approach was undertaken to investigate the physiological roles of human metallothionein-2. A constructed expression plasmid, pEXPMTII, in which human metallothionein-IIA cDNA was inserted downstream of a tryptophan-lactose promoter, was used to transform Escherichia coli JM105 strain. Cadmium-binding metallothionein was successfully expressed in E. coli in the medium containing cadmium, while copper and zinc-metallothioneins were scarcely observed in copper- or zinc-containing medium. The amino acid composition and sequence of the biosynthesized cadmium-metallothionein were analyzed. The selectivity of metals bound to metallothionein and the stability of metal-binding forms of metallothionein in E. coli were discussed. In addition, cadmium, zinc, or copper resistance of the cells expressing metallothionein was examined. Cells transformed with the plasmid pEXPMTII and cultured in a medium containing cadmium exhibited tolerance only to cadmium. It was demonstrated that human metallothionein-2 functioned for cadmium detoxification in E. coli.
Base Sequence↗
Immunochemical analysis of carcinoembryonic antigen (CEA)-related antigens differentially localized in intracellular granules of human neutrophils.
Subcellular localization and antigenic properties of eight carcinoembryonic antigen (CEA)-related proteins (nonspecific cross-reacting antigens, NCAs) in neutrophils, including CD66 antigens, were examined with a panel of CD66 monoclonal antibodies (MAbs), whose specificities were characterized with CHO cells expressing CEA gene family recombinant proteins. Intracellular granules of neutrophils were isolated by cell lysis followed by centrifugation on a sucrose gradient. SDS-PAGE analysis of immunoprecipitates from the granules revealed that NCA-95 (CD66c, NCA-50/90) exists predominantly in the azurophil granule-enriched fraction. NCA-90 was identified in the same fraction and was suggested to be a molecule closely-related to NCA-95. NCA-26 was identified in the specific granule fraction together with NCA-160 (CD66a, BGP), and was likely a splicing variant of NCA-160. NCA-100 (CD66b, CGM6) was also identified in this fraction. NCA-80, -58 and -30, which are detectable in culture medium of neutrophils with polyclonal anti-NCA-50/90, were not recognized by any of the CD66 MAbs tested. These findings indicate that NCA-160, -100, -95, -90 and -26 are the products of the CEA gene family and stored separately in azurophil and specific granules. It remains to be clarified whether or not the other three secretory antigens, NCA-80, -58 and -30, are still unknown members of the CEA family.
Animals↗
Enzyme-linked immunosorbent assay for apolipoprotein A-I in the serum of cattle.
An ELISA was developed to evaluate the concentration of apolipoprotein A-I, a major apoprotein in high-density lipoprotein, in the serum of cattle. Serum apolipoprotein A-I was purified electrophoretically, and antibodies to this protein were raised in rabbits. The specificity of the antiserum was assessed by use of several immunologic techniques including western blotting. The ELISA was sensitive (detection limit was 70 ng of apolipoprotein A-I/ml) and reliable (coefficients of variance were in the range of 3.5 to 8.2%). By use of this method, the serum apolipoprotein A-I concentration was higher in 2- to 6-year-old Holstein cows (mean +/- SD, 0.580 +/- 0.304 mg/ml) than in 7- to 15-month-old heifers (0.339 +/- 0.237 mg/ml), 6-month-old heifers (0.238 +/- 0.188 mg/ml), and 6-month-old steers (0.173 +/- 0.146 mg/ml). The concentration, however, is not largely different in cows in early, middle, and late lactation and in non-lactating stages. Results also indicated that apolipoprotein A-I concentration was decreased in cows with hepatic lipidosis (fatty liver) induced by administration of ethionine, suggesting that this method is a useful tool for the pathophysiologic study of lipid metabolism and its impairment in cattle.
Aging↗
Enzyme-linked immunosorbent assay for serum apolipoprotein B-100, a major triglyceride-transport protein in dairy cows.
An ELISA was developed to determine serum concentration of apolipoprotein B-100, a major triglyceride-binding protein in very low-density lipoproteins and a putative maker for hepatic lipidosis of dairy cows. Serum apolipoprotein B-100 was prepared electrophoretically, and antibodies to this protein were raised in rabbits. The antiserum prepared was further purified by affinity chromatography, using bovine serum albumin-Sepharose 4B, to remove antibodies to albumin. For the ELISA, addition of 2-mercaptoethanol to the coating buffer (50 mM sodium carbonate, pH 9.6) was required to evaluate apolipoprotein B-100 concentration in serum. The ELISA developed was sensitive (detection limit was 300 to 400 ng/ml of serum) and reliable (coefficients of variance were in the range of 3.3 to 7.6%). By use of the established ELISA, the serum apolipoprotein B-100 concentration was found to be significantly (P < 0.01) lower during the early lactating stage than during other stages of lactation. Reduced hepatic synthesis or secretion of apolipoprotein B-100 during the early lactating stage, together with the excess uptake by the liver of serum nonesterified fatty acids, is suggested to be relevant in the accelerated accumulation of triglycerides in the liver of dairy cows during the periparturient period.
Animals↗