Biomedical subjects

S Nishimura

Publications and source records attributed to S Nishimura.

At least 19 recordsLinked to original sources

PubMed1992

Modulation of C2 and C3 gene expression of human peripheral blood monocytes by interleukin 1 beta, interferon gamma, tumor necrosis factor alpha and lipopolysaccharide.

The effect of interleukin 1 beta (IL-1 beta), interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF alpha) and lipopolysaccharide (LPS) on the expression of the C2 and C3 genes in human adherent monocytes was studied. Stimulation of monocytes with IFN-gamma increased both C2 and C3 mRNA. IL-1 beta also increased C2 mRNA level, whereas C3 gene expression was not enhanced. TNF alpha failed to increase either C2 or C3 mRNA. LPS increased C2 mRNA, but suppressed C3 gene expression. These results suggest that C2 and C3 production by monocytes is regulated by IL-1 beta and IFN-gamma in the local tissues.

Complement C2↗

PubMed1992

Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II.

The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.

Amino Acid Sequence↗

PubMed1992

DNA strand cleavage at 8-hydroxyguanine residues by hot piperidine treatment.

The two findings indicate that 8-hydroxyguanine(8-OH-Gua) is a hot piperidine-sensitive lesion in DNA. These are cleavages of DNA containing 8-OH-Gua at the site of this residue and decomposition of 8-hydroxydeoxyguanosine when the DNA and nucleoside were treated in 1 M piperidine for 30 min at 90 degrees C. However, no cleavage was observed in DNA containing 8-hydroxyadenine or O6-methylguanine. 8-OH-Gua was found to be different from apurinic sites that are also alkali-labile lesions since the former was more resistant to alkali treatment. This property of 8-OH-Gua can be used as a check for the incorporation of this base into DNA after the synthesis of DNA containing 8-OH-Gua at a specific position or possibly can be one of the markers for the identification of 8-OH-Gua formed in DNA exposed to reactive oxygen species.

Base Sequence↗

PubMed1992

Deficiency of 8-hydroxyguanine DNA endonuclease activity and accumulation of the 8-hydroxyguanine in mutator mutant (mutM) of Escherichia coli.

In this report, we have characterized mutM mutant with respect to its ability to repair 8-hydroxyguanine (oh8Gua) in DNA. The oh8Gua DNA endonuclease activity in mutM strain was minimal as compared with that in the wild-type cells. The presence of oh8Gua in DNA of mutM was 6-fold that of the wild-type strain corresponding to a characteristically higher frequency of G.C-->T.A transversions in this mutant strain. These results suggest that mutator phenotype of mutM is at least partially due to a spontaneous accumulation of oh8Gua resulting from a greatly reduced oh8Gua DNA endonuclease activity.

Base Sequence↗

PubMed1992

Photosensitized formation of 8-hydroxydeoxyguanosine in cellular DNA by riboflavin.

Photosensitized formation of 8-hydroxydeoxyguanosine (oh8dG) in DNA by riboflavin has recently been shown in vitro. The present study describes the formation of oh8dG in cellular DNA by photo-irradiation of cultured mammalian cells in the presence of riboflavin. Formation of oh8dG was dependent on the concentration of riboflavin as well as the duration of photoirradiation. These results suggest that photosensitized formation of oh8dG in DNA by riboflavin may be involved in photocarcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1992

Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca channel.

A polyclonal antibody, CR2, prepared using the C-terminal peptide of the alpha 1 subunit of the rabbit cardiac DHP-sensitive Ca channel, specifically immunoprecipitated the [3H]PN200-110-labeled Ca channel solubilized from cardiac microsomes. The antibody recognized 250 and 200-kDa cardiac microsomal proteins as determined by immunoblotting, and cAMP-dependent protein kinase phosphorylated the 250-kDa, but not the 200-kDa protein in vitro. CHO cells, transfected with the cardiac alpha 1 subunit cDNA carried by an expression vector, synthesized a 250-kDa protein which was recognized by CR2. Adding db-cAMP or forskolin to the transformed CHO cells induced phosphorylation of the 250-kDa protein and stimulated the DHP-sensitive Ba current under patch-clamp conditions. These results suggested that the cardiac DHP-sensitive Ca channel was regulated by cAMP-dependent phosphorylation of the alpha 1 subunit.

Animals↗

PubMed1992

High-performance liquid chromatographic separation of human apolactoferrin and monoferric and diferric lactoferrins.

A simple chromatographic separation of the different molecular forms of human milk lactoferrin according to their iron contents is described. The appropriate conditions were developed with a Mono-S cation-exchange column linked to an automated Urtrochrom GTi system. The pure lactoferrin with different iron binding (apolactoferrin, monoferric lactoferrin and diferric lactoferrin) separated into three peaks. The three peaks were eluted from the Mono-S column with 0.88, 0.97 and 1.05 M sodium chloride in 10 mM phosphate buffer (pH 7.0).

Apoproteins↗

PubMed1992

c-Ha-ras containing 8-hydroxyguanine at codon 12 induces point mutations at the modified and adjacent positions.

To determine the type of mutation induced by 8-hydroxyguanine in a mammalian system, we examined the mutations induced by a synthetic c-Ha-ras protooncogene containing 8-hydroxyguanine in the second position of codon 12 (GGC) in NIH3T3 cells. Transfection of this gene significantly increased the number of transformed foci. The c-Ha-ras gene present in these foci was analyzed by the polymerase chain reaction-restriction enzyme method. Interestingly, sequence analysis revealed random mutations at the modified site (G----T, G----A, and G----C) as well as mutations of the adjacent G on the 5'-side of 8-hydroxyguanine (G----A and G----T).

3T3 Cells↗

PubMed1992

Two nucleotides next to the anticodon of cytoplasmic rat tRNA(Asp) are likely generated by RNA editing.

The nucleotide sequences of major cytoplasmic tRNA(Asp) from rat liver and rat ascites hepatoma comprise a U32 and C33 next to the anticodon as was confirmed by different procedures. Additionally we identified a tRNA(Asp) with C32 and U33 in a minor proportion. We have shown earlier that the tRNA(Asp) gene is part of a cluster of tRNA genes which is amplified at least ten times in the rat nuclear genome. Six independent isolated clones display identical sequences in the coding region of the tRNA(Asp) gene which differ from tRNA(Asp) in having C32 and T33. Using a combination of single-strand conformation polymorphism (SSCP) analyses and direct sequencing of polymerase chain reaction (PCR) products we have now demonstrated that no variant allele of the tRNA(Asp) gene with T32 and C33 exists in the rat genome. Together with the RNA sequencing data these findings strongly indicate that major rat tRNA(Asp) is generated by post-transcriptional pyrimidine transitions at positions 32 and 33 and that the minor tRNA(Asp) is its unedited precursor.

Animals↗

PubMed1992

Isolation of genes specifically expressed in flat revertant cells derived from activated ras-transformed NIH 3T3 cells by treatment with azatyrosine.

We previously reported that mouse NIH 3T3 cells transformed by transfection of activated human c-Ha-ras become apparently normal upon treatment with the antibiotic azatyrosine. The revertant cells maintain their normal phenotype during prolonged culture in the absence of azatyrosine, although activated p21ras is still expressed. The normal phenotype induced by azatyrosine could be due to activation of expression of some cellular gene(s) in the cells that results in suppression of ras function. To identify the genes with increased expression in the revertant cells, we adopted differential screening of recombinants from a phage cDNA library made from mRNA of the revertant cells, hybridized with 32P-labeled cDNAs made from mRNAs of the ras-transformed NIH 3T3 cells and the revertant cells. Two clones thus isolated were found to be almost identical to the ras recision gene (rrg), which was identified as a tumor-suppressor gene by Contente et al. [Contente, S., Kenyon, K., Rimoldi, D. & Friedman, R. M. (1990) Science 249, 796-798]. Other genes identified were the collagen type III and rhoB genes. Approximately half the clones were found to contain a sequence corresponding to that of the murine retrovirus-like intracisternal A particle. We speculate that azatyrosine activates several cellular genes in the ras-transformed cells and that some of these genes, including rrg, act cooperatively to counteract ras function, resulting in reversion of the ras-transformed cells to the normal phenotype.

3T3 Cells↗

PubMed1992

Alcohol interaction with high entropy states of macromolecules: critical temperature hypothesis for anesthesia cutoff.

Nerve excitation generates heat and decreases the entropy (review by Ritchie and Keynes (1985) Q. Rev. Biophys. 18, 451-476). The data suggest the existence of at least two thermodynamically identifiable states: resting and excited, with a thermotropic transition between the two. We envision that nerve excitation is a transition between the two states of the excitation machinery consisting of proteins and lipids, rather than the sodium channel protein alone. Presumably, both proteins and lipids change their conformation at excitation. We proposed (Kaminoh et al. (1991) Ann. N.Y. Acad. Sci. 625, 315-317) that anesthesia occurs when compounds have a higher affinity to the resting state than to the excited state of excitable membranes, and that there is a critical temperature above which the affinity to the excited state becomes greater than to the resting state. When the temperature exceeds this critical level, compounds lose their anesthetic potency. We used thermotropic phase-transition of macromolecules as a model for the excitation process. Anesthetic alcohols decreased the main transition temperature of dipalmitoylphosphatidylcholine (DPPC) membranes and also the temperature of the alpha-helix to beta-sheet transition of poly(L-lysine). The affinity of alcohols to the high- and low-temperature states of the DPPC membranes were separately estimated. The difference in the affinity of n-alcohols to the liquid (high-temperature) and solid (low-temperature) states correlated with their anesthetic potency. It is not the total number of bound anesthetic molecules that determines the anesthesia, rather, the difference in the affinity between the higher and lower entropy states determines the effects. The critical temperatures of the long-chain alcohols were found to be lower than those of the short-chain alcohols. Cutoff occurs when the critical temperature of long-chain alcohols is below the physiological temperature, such that the anesthetic potency is not manifested in the experimental temperature range.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1992

Ligand-induced functions of the epidermal growth factor receptor require the positively charged region asymmetrically distributed across plasma membrane.

Many plasma membrane proteins, including the epidermal growth factor (EGF) receptor, possess basic regions on the cytoplasmic surface of the membrane. To examine the function of these positively charged regions, we constructed mutated EGF receptor genes lacking this region by substitution of the basic amino acid residues with 3 approximately 8 neutral Asn residues, or by their complete deletion. There was no significant difference in the affinities for EGF of the wild-type and mutant receptors which are produced in rodent fibroblasts through transfection. However, EGF-induced tyrosine phosphorylation of the receptor was strongly inhibited by removal of the 3 approximately 8 positively charged residues. On addition of EGF, cells expressing the mutant EGF receptors did not show morphological changes, whereas cells expressing the wild-type receptor did. These findings suggest that the positively charged regions of membrane proteins that are asymmetrically distributed on the cytoplasmic surface of the membrane may be required for the functions of membrane proteins in general.

3T3 Cells↗

PubMed1992

Post-inhibitory excitation of adenosine on neurotransmission in guinea pig hippocampal slices.

The postsynaptic field potential (population spike potential; PS) was recorded from the granule cell layer of guinea pig hippocampal slices. Adenosine at low concentrations ranging from 10 nM to 1 microM enhanced the amplitude of PS, whereas at concentrations over 10 microM it inhibited the neurotransmission. There appeared to be a rebound phenomenon after the removal of adenosine at inhibitory concentrations and the amplitude of the PS overshot the initial amplitude (we called this post-inhibitory excitation; PIE). Neither depressants such as gamma-aminobutyric acid (GABA; 1 mM) nor sodium pentobarbital (100 microM) by itself induced PIE. After application of GABA or sodium pentobarbital together with adenosine (0.1 microM), however, removal of all agents could induce the PIE. PIE as well as the excitatory effect of adenosine at low concentrations was counteracted by application of H-7 (100 microM), melittin or polymyxin B, potent protein kinase C (PKC) inhibitors, suggesting that the excitatory effect of adenosine is mediated by a metabolic process involving PKC. These results indicate that PIE induced by adenosine at high concentrations is due to a mechanism similar to the excitatory effect induced by adenosine at low concentrations, and that during application of adenosine at high concentrations the excitation is masked by its potent inhibitory effect.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP hydrolysis.

RAS genes isolated from human tumors often have mutations at positions corresponding to amino acid 12 or 61 of the encoded protein (p21), while retroviral ras-encoded p21 contains substitutions at both positions 12 and 59. These mutant proteins are deficient in their GTP hydrolysis activity, and this loss of activity is linked to their transforming potential. The crystal structures of the mutant proteins are presented here as either GDP-bound or GTP-analogue-bound complexes. Based on these structures, a mechanism for the p21 GTPase reaction is proposed that is consistent with the observed structural and biochemical data. The central feature of this mechanism is a specific stabilization complex formed between the Gln-61 side-chain and the pentavalent gamma-phosphate of the GTP transition state. Amino acids other than glutamine at position 61 cannot stabilize the transition state, and amino acids larger than glycine at position 12 would interfere with the transition-state complex. Thr-59 disrupts the normal position of residue 61, thus preventing its participation in the transition-state complex.

Amino Acid Sequence↗

PubMed1992

Induction of mutation of a synthetic c-Ha-ras gene containing hypoxanthine.

The second base of codon 61 of a synthetic c-Ha-ras gene was replaced with a hypoxanthine residue in a site-specific manner. Transfection of this gene into NIH3T3 cells by the calcium phosphate procedure resulted in increased focus formation. Total DNA was extracted from transformed cells, and the sequences of the inserted c-Ha-ras DNA were analyzed by the polymerase chain reaction-single-strand conformation polymorphism method. Mutations with A (or hypoxanthine) to G transition were detected exclusively. These results suggest that the synthetic c-Ha-ras gene can be used for investigations of mutagenesis caused by DNA lesions.

3T3 Cells↗

PubMed1992

Inhibition of chemical carcinogenesis in vivo by azatyrosine.

A single painting of 7,12-dimethylbenz[a]anthracene on the skin of transgenic mice harboring the human protooncogene c-Ha-ras induced papillomas at 100% incidence after 20 weeks (M. Izawa et al., unpublished data). Application of L-beta-(5-hydroxy-2-pyridyl)alanine (azatyrosine) to the skin at a dose of 2 mg/mouse once every 3 days after initiation with 7,12-dimethylbenz[a]anthracene greatly reduced the percentage incidence, number per mouse, and size of papillomas. Injection of methylnitrosourea i.p. into transgenic mice induced papillomas in the forestomach after 12 weeks (2 to 12 papillomas/mouse) at 100% incidence (K. Ando et al., Cancer Res., 52: 978-982, 1992). Administration of azatyrosine i.p. at a dose of 2 mg/mouse once every 2 days for 12 weeks after initiation with methylnitrosourea completely prevented the formation of forestomach papillomas. These results clearly indicated that azatyrosine inhibits chemical carcinogenesis in vivo.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1992

Possible role of surface potential in the gating mechanism of Ca2+ channels in cat adrenal chromaffin cells: studies with fura-2 microfluorometry.

The cytosolic free Ca2+ concentration ([Ca]in) in isolated cat chromaffin cells was measured by fura-2 microfluorometry. During 30 mM KCl depolarization or sucrose substitution for NaCl, a reduction in external Ca2+ concentration under optimal conditions paradoxically caused a rise in [Ca]in and, in separate experiments, in catecholamine secretion. The results support a previously suggested role of surface potentials in the gating mechanism of Ca2+ channels.

Adrenal Medulla↗

PubMed1992

Evidence that the ras oncogene-encoded p21 protein induces oocyte maturation via activation of protein kinase C.

The ras oncogene-encoded p21 protein is known to induce cell maturation of Xenopus laevis oocytes and malignant transformation of NIH 3T3 mouse fibroblasts. The pathways involved in oocytes and NIH 3T3 cells appear to be similar to one another. For example, in both cases, the ras p21-induced cellular events involve increased intracellular levels of the second messengers diacylglycerol and inositol phosphates, the former of which activates protein kinase C (PKC). To investigate the pathway of ras-induced oocyte maturation, we have explored the relationship between p21 protein and PKC. We show that the maturation signal from oncogenic p21 microinjected into Xenopus oocytes is completely blocked by the relatively specific PKC inhibitor CGP 41251, a staurosporine analogue that selectively inhibits PKC, but not by an inactive analogue of staurosporine, CGP 42700. Microinjection of purified PKC or of phorbol ester induces maturation of oocytes. PKC-induced maturation is inhibited by CGP 41251 but not by CGP 42700. Maturation induced by microinjected PKC is also not inhibited by two specific anti-p21 agents, the inactivating anti-p21 monoclonal antibody Y13-259 and the amino acid derivative azatyrosine. Both of these agents block p21-induced cell maturation. These results suggest that ras effects depend upon the action of PKC, whose activation is an event that occurs downstream of p21 in the maturation signal pathway.

Alanine↗