Biomedical subjects

T Negishi

Publications and source records attributed to T Negishi.

At least 37 records · Page 2Linked to original sources

PubMed1998

Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout.

The anticarcinogenic activity of chlorophyllin (CHL), a water-soluble derivative of chlorophyll, was first reported in rainbow trout. This review describes certain experiments which set the stage for long-term tumor bioassays, in trout and other species, using CHL and various food-borne carcinogens. Initial work with trout and rat liver enzymes in the Salmonella assay showed that CHL was a potent antimutagen towards heterocyclic amines, polycyclic aromatic hydrocarbons, aflatoxins and other classes of mutagen. Antimutagenic activity was further demonstrated using the corresponding direct-acting mutagens in the absence of an exogenous metabolizing system. Mutagen-inhibitor interaction (molecular complex formation) was identified in spectrophotometry studies, suggesting that CHL acts as an 'interceptor molecule'. In vivo, CHL reduced hepatic AFB1-DNA adducts and hepatocarcinogenesis when the inhibitor and carcinogen were co-administered in the diet. Finally, co-injection of inhibitor and AFB1 into trout embryos established that CHL was more effective than chlorophyll a in reducing AFB1-DNA adducts 2 weeks after injection, and liver tumors after 1 year.

Aflatoxin B1↗

PubMed1998

Ceramide selectively inhibits calcium-mediated potentiation of beta-adrenergic-stimulated cyclic nucleotide accumulation in rat pinealocytes.

Interaction between sphingomyelin metabolism and cyclic nucleotide synthesis in rat pinealocytes was investigated by determining the effect of ceramide on adrenergic-stimulated cAMP and cGMP accumulation. Although C2-, C6-, and C8-ceramide had no effect on basal, isoproterenol-, or norepinephrine-stimulated cAMP and cGMP accumulation, they inhibited the potentiation caused by depolarising concentrations of K+ or BayK 8644. Similar inhibition was observed when ceramide metabolism was inhibited by a glucosylceramide synthase inhibitor. In contrast, the potentiation of cAMP and cGMP accumulation caused by other intracellular Ca(2+)-elevating agents such as ionomycin or thapsigargin or by an activator of protein kinase C was not affected by ceramide. Taken together, our results suggest that ceramide selectively inhibits cyclic nucleotide synthesis when the nucleotide synthesis is potentiated by an increase in intracellular Ca2+ through L-type Ca2+ channels and that the sphingomyelin cycle probably plays an important role in the regulation of these channels.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Specific fluorescent labeling of two functional domains in RNA polymerase alpha subunit.

A monomercury derivative of fluoresceine acetate (FMMA) was previously suggested as a specific reagent reacting with only one of four cysteine (Cys) residues in the alpha. subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated alpha subunit and alpha subunit assembled in the holoenzyme. In both cases, the highest reactivity was identified for Cys-269 positioned in the regulatory helix of C-terminal domain (CTD) which includes the contact sites for both class-I transcription factors and DNA UP elements. Substitution of Ala for both Cys-269 and Cys-176 completely eliminates the reactivity of alpha subunit against the fluorescent dye, supporting the prediction that another reactive amino acid under native conformation is Cys-176, which is positioned within or near the region important for alpha dimerization and its binding of beta' subunit. In the isolated alpha subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant alpha-subunits, bearing only one Cys residue at either 269 or 176, could be reconstituted into locally modified and active enzymes. This FMMA modification system may provide a tool suitable for studies of intra- and intermolecular interactions of this subunit.

Cysteine↗

PubMed1998

Inhibition of N-nitrosation of secondary amines in vitro by tea extracts and catechins.

Inhibition of nitrite-mediated N-nitrosation of dimethylamine, morpholine and N-methylaniline by tea extracts and by 6 individual catechins in the extracts was studied. The inhibitions were detected by quantifying the nitrosamines formed. Eight different kinds of teas (5 green teas, a roasted green tea, an oolong tea, and a black tea) were examined for their inhibitory abilities and for their catechin contents, with an attempt to correlate the inhibitory activities to the catechin contents. The results showed that (1) the green tea extracts inhibit strongly the N-nitrosation of the three secondary amines tested, (2) the 6 catechins, notably epigallocatechin, are capable of blocking the N-nitrosations very efficiently, even more efficiently than ascorbic acid, and (3) the inhibition activities of green tea extracts are mostly ascribable to the catechins present in the extracts. These inhibitions occur by rapid reactions between nitrite and the catechins. It was observed that no mutagenicity results from the reaction between the tea extracts and nitrite.

Amines↗

PubMed1998

Differential effects of two protein kinase C inhibitors, calphostin C and Gö6976, on pineal cyclic nucleotide accumulation.

In rat pinealocytes, protein kinase C (PKC) is involved in the alpha1-adrenergic-mediated potentiation of beta-adrenergic-stimulated cyclic nucleotide responses; however, the specific PKC isozyme(s) involved in the potentiation mechanism remain unknown. In the present study, we compared the effects of two PKC inhibitors, calphostin C, a specific inhibitor of PKC, and Gö6976, a selective inhibitor of PKC alpha and PKC beta1, on the adrenergic-stimulated cyclic nucleotide accumulation in rat pinealocytes. Surprisingly, Gö6976 was found to have an enhancing effect on basal cyclic GMP and isoproterenol-stimulated cyclic AMP and cyclic GMP accumulation, an effect not shared by calphostin C. Gö6976 also increased the norepinephrine- and ionomycin-induced potentiation of isoproterenol-stimulated cyclic AMP and cyclic GMP accumulation, whereas the effect of calphostin C was inhibitory. The enhancing effect of Gö6976 was abolished in the presence of isobutylmethylxanthine or zaprinast, but not rolipram, suggesting that this effect of Gö6976 may be mediated through type V or the retinal type of phosphodiesterase. Based on these observations, we propose that some of the PKC isozyme(s) inhibited by calphostin C are involved in the potentiation of beta-adrenergic-stimulated cyclic nucleotide responses and that they act by enhancing synthesis. However, PKC isozymes inhibited by Gö6976 appear to be basally active and tonically inhibit cyclic nucleotide accumulation through their stimulatory action on phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1998

B-90063, a novel endothelin converting enzyme inhibitor isolated from a new marine bacterium, Blastobacter sp. SANK 71894.

A novel endothelin-converting enzyme (ECE) inhibitor, B-90063, was isolated from the culture supernatant of the newly discovered marine bacterium Blastobacter sp. SANK 71894. Based on spectral analyses and chemical reactions, the structure of B-90063 was determined to be bis[6-formyl-4-hydroxy-2-(2'-n-pentyloxazol-4'-yl)-4-pyridon -3-yl]-disulfide (1a). Human and rat ECEs were inhibited more potently by B-90063, with respective IC50 values of 1.0 and 3.2 microM, than were other neutral endopeptidases such as NEP and type-I and -IV collagenases. B-90063 also inhibited the binding of ET-1 to rat ET(A) and bovine ET(B) receptors, though its antagonistic activities were weak. B-90063, thus, may abolish the physiological actions of endothelins through the ECE inhibitory and receptor antagonistic mechanisms.

Animals↗

PubMed1998

[Usefulness of magnetic resonance imaging for evaluation of therapeutic efficacy of bone metastases from bladder cancer: a case report].

A 70-year-old man had a prostate-invading bladder cancer with multiple bone metastases. The bladder cancer seemed to have metastasized via the vertebral vein system because there was no metastasis in other organs. Transurethral resection of bladder tumor was performed followed by one course of M-VAC (methotrexate, vinblastine, doxorubicin) therapy and MEP (methotrexate, etoposide and cisplatin) therapy. After chemotherapy, magnetic resonance imaging (MRI) of the spine revealed further progression of disease, although bone scintigraphy did not, and the patient died of disease.

Aged↗

PubMed1997

Antigenotoxic activity of natural chlorophylls.

Chlorophyllin, a man-made water-soluble form of chlorophyll, is a focus of intensive studies from many laboratories for its antimutagenic and anticarcinogenic properties. Natural chlorophylls, in contrast, have been little studied in this regard. Since yellow-green vegetables are implicated to be protective against human cancers by epidemiological studies, it is important to explore the antigenotoxic properties of natural chlorophylls. Previously, we reported that a chlorophyll sample prepared from Chlorella vulgaris inhibited the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, a direct-acting mutagen, in Salmonella, and that the chlorophyll also showed inhibition of wing spot formation in Drosophila induced by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). We have now prepared several samples of chlorophyll from spinach and chlorella, and studied their effect on the genotoxicity of 4-nitroquinoline 1-oxide (4NQO) in Drosophila. The results showed that the genotoxicity of orally given 4NQO was suppressed by simultaneous administration of the chlorophylls. The mechanisms of this inhibition are discussed.

4-Nitroquinoline-1-oxide↗

PubMed1997

Expression and nocturnal increase of type II iodothyronine deiodinase mRNA in rat pineal gland.

It has been demonstrated that thyroxine deiodinating activity is present in rat pineal gland, and its activity increases significantly during the night time. We have studied whether mRNA for type II iodothyronine deiodinase is expressed in rat pineal gland and whether the nocturnal rise of pineal T4 deiodinating activity is due to the change in type II iodothyronine deiodinase mRNA level. Reverse transcription-polymerase chain reaction amplification and Northern blot analyses have demonstrated that type II iodothyronine deiodinase mRNA is expressed in rat pineal gland and its mRNA level increases markedly at midnight. These results suggest that the nocturnal rise in pineal T4 deiodinating activity is due to the change in type II iodothyronine deiodinase mRNA level.

Animals↗

PubMed1997

[Study of directional differences on static and stress urethral pressure profiles of female urethra].

PURPOSE: We investigated the female continence mechanisms by comparing directional differences of static and stress urethral pressure profiles (UPP) in urinary continent females with those in stress incontinent females. Also, the mechanisms of bladder neck suspension were investigated by comparing directional differences of UPP pre- and post-operatively. METHODS: UPP at rest and under stress were recorded by means of double lumens microtip transducer catheter in 21 females without urinary incontinence (normal group) and 38 females with stress urinary incontinence (SUI group). And UPP were recorded pre- and postoperatively in 19-females of SUI group who had surgical cure of SUI (ope group). These measurements were performed on the urethral directions (anterior-direction of symphysis pubis, lateral and posterior) to which the pressure sensor in the catheter were pointed. Pressure transmission ratios (PTR) were calculated in each quartile dividing functional urethral length (FUL) into four equal lengths. We compared the parameters (the maximum urethral closure pressure = MUCP, FUL and PTR) when the sensor lies at the anterior, lateral and posterior direction in each group. The parameters in normal group were compared with those in SUI group in each direction and those in ope group were, compared pre- and postoperatively. RESULTS: In all groups, MUCP is always highest in the anterior direction but FUL shows no differences in the three directions. In all directions, MUCP and FUI are higher in the normal group than in the SUI group and there is no significant change in MUCP and FUL following successful bladder neck suspension. In the normal group, PTR of anterior, lateral and posterior urethra were approximately equal, but SUI group patients demonstrated significantly decreased PTR in the lateral and posterior urethra in comparison with PTR observed in the anterior urethra. Also, PTR of the anterior urethra in the SUI group approximates that in the normal group but PTR of the lateral and posterior urethra are lower in the SUI group than in the normal group. In the ope group, in the proximal three-quarters of the FUL, PTR in the lateral and posterior urethra approximated to those in the anterior urethra postoperatively. CONCLUSION: These findings suggest that urethral support is destructed in the SUI group as mentioned in DeLancey's hammock hypothesis and lateral and posterior weakness were corrected by bladder neck suspension. Bladder neck suspension restored the continence by constructing posterior support of urethra as the substitution for destructed urethral support.

Aged↗

PubMed1996

Thymic hyperplasia in patients with Graves' disease. Identification of thyrotropin receptors in human thymus.

Thymic size and density were studied in 23 untreated patients with Graves' disease and 38 control subjects using computed tomography. Both thymic size and density were higher in untreated patients with Graves' disease than in control subjects in the age-matched group. After treatment with antithyroid drugs, both thymic size and density were significantly reduced, with a concomitant decrease in thyrotropin receptor antibodies. PCR of human thymic cDNA using primers for human thyrotropin receptor amplified a fragment in a size expected for the receptor, and its nucleotide sequence was identical to human thyrotropin receptor cDNA in the thyroid. Northern blot analysis of human thymic poly(A)+ RNA demonstrated the presence of the full length form of thyrotropin receptor mRNA. Western blot analysis of human thymic membrane using anti-thyrotropin receptor peptide antibodies demonstrated a band of 100 kD that was also observed in the thyroid membrane. Immunohistochemistry of thymic tissue using mouse antihuman thyrotropin receptor monoclonal antibodies demonstrated the immunostaining of epithelial cells. These results indicate that thymic hyperplasia is apparently associated with Graves' disease and suggest that thymic thyrotropin receptor may act as an autoantigen that may be involved in the pathophysiology of development of Graves' disease.

Adult↗

PubMed1996

Monoclonal antibodies specific to the integral membrane protein P0 of bovine peripheral nerve myelin.

Two monoclonal antibodies (mAbs), 58A and 46E, were generated against the major protein P0 of bovine peripheral nervous system myelin (PNSM). The reactivities of the mAbs were assessed by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry. Both mAbs, 58A and 46E, reacted to PNSM of bovine, human, rat and rabbit, but not to chicken PNSM or the brains of rat and rabbit. In the Western blot, these mAbs showed specific binding to bovine P0 as well as deglycosylated P0, but not to myelin-associated glycoprotein (MAG) of bovine spinal cord. The analyses of the lysylendopeptidase-digested peptides of bovine P0 revealed that the epitopes for the mAbs 58A and 46E were located on the amino acid residues 68-79 and 210-216, respectively. Since the mAbs 58A and 46E recognize the extracellular domain and the cytoplasmic domain of P0, respectively, they could be useful for studies on P0's role in myelin formation, its adhesive properties, and functions of the N-terminal extracellular and C-terminal cytoplasmic domains of the protein.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of the human chromosomal gene for prostacyclin-stimulating factor.

Prostacyclin-stimulating factor (PSF) is a protein which acts on vascular endothelial cells and stimulates the production of prostacyclin. Recently, we were able to purify PSF from the conditioned medium of cultured human diploid fibroblasts and clone PSF cDNA. In this study, we screened a human genomic library and isolated genomic clones to determine the structure of the human chromosomal PSF gene. By determining the nucleotide sequence and transcription initiation site of this gene, we found that it comprises 5 exons and 4 introns. Southern hybridization analysis indicated the presence of a single copy of the PSF gene per haploid set of chromosomes. The 300 bp upstream of the transcription initiation site had a very high GC content, and 7 binding sites for the transcription regulating factor Sp1 were present.

Base Sequence↗

PubMed1996

Inhibition of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline-mediated DNA-adduct formation by chlorophyllin in Drosophila.

The effect of chlorophyllin on 2-amino-3,8-dimethyimidazo[4,5-f]quinoxaline (MeIQx)-mediated DNA-adduct formation in Drosophila was studied. Third-instar larvae of Drosophila were fed MeIQx at 1 mg/6.5 g-feed/bottle, with or without chlorophyllin (100-300 mg). After a 6 h feeding exposure to MeIQx, the larvae were divided into 2 groups. The first group was examined for covalent DNA adducts by 32P-postlabeling assay. The second group was assayed for DNA damage by allowing the larvae to develop to adults and measuring the male/female ratio (males, DNA repair-deficient; females, DNA repair-proficient). The 32 P-postlabeling results indicated a significant decrease in DNA adduct levels in larvae treated with MeIQx and 300 mg chlorophyllin (1.7 +/- 0.7 adducts/10(7) nucleotides) as compared with MeIQx-treated larvae 6.5 +/- 2.1 adducts/10(7) nucleotides). The results on male/female sex ratios also indicated a chlorophyllin-induced decrease in DNA damage by exposure to MeIQx. The suppressive effect of chlorophyllin on the genotoxic actions of a polycyclic mutagen, MeIQx, may be a result of complex formation between chlorophyllin and the mutagen.

Animals↗

PubMed1996

[Determination of free type and complex type prostate-specific antigen (PSA): differences in immunorecognition by Delfia PSA, ACS-PSA and Eiken PA kits].

To study immunorecognition of free type and complex type prostate-specific antigen (PSA) by current commercial PSA assays, sera from 3 patients with stage D2 prostate cancer were separated by Sephacryl S-200 chromatography and determined by Delfia PSA, ACS-PSA and Eiken PA kits. Two antibodies used in the 3 kits are 2 monoclonal, 1 monoclonal and 1 polyclonal and 2 polyclonal antibodies, respectively. Following chromatography, two PSA peaks were obtained in all patients. One was about 100 kDa and the other about 30 kDa. The former was considered to be the complex type PSA (complex with alpha-1 antichymotrypsin) and the latter to be free type PSA. As to free type PSA, the ACS-PSA kit and Eiken PA kit quantitated PSA values approximately 5.1 and 2.5 times higher than the Delfia PSA kit. For complex type PSA, the quantity determined by ACS-PSA kit was approximately 1.3 times higher than that determined by the Delfia PSA kit, while the quantity determined by the Eiken PA kit was about one third of that determined by Delfia PSA kit. The ratio of complex type PSA to total PSA (free type PSA + complex type PSA) was 74.8 +/- 14.9% (mean +/- SD) when determined by Delfia PSA kit, 59.3 +/- 18.4% by ACS-PSA kit and 52.9 +/- 13.8% by Eiken PA kit. The range of this ratio determined by ACS-PSA kit was from 47.3% to 80.5% in the 3 patients. These findings suggest that there are qualitative differences in immunorecognition of free type PSA and complex type PSA among current commercial PSA assays and that there are quantitative differences in the ratio of the 2 forms of PSA in serum among prostatic cancer patients. The measurement and follow-up of both free type and complex type PSA might be important for diagnosis and monitoring of prostate cancer.

Aged↗

PubMed1995

Solution structure of the activator contact domain of the RNA polymerase alpha subunit.

The structure of the carboxyl-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alpha CTD), which is regarded as the contact site for transcription activator proteins and for the promoter UP element, was determined by nuclear magnetic resonance spectroscopy. Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins. The UP element binding site was found on the surface comprising helix 1, the amino-terminal end of helix 4, and the preceding loop. Mutation experiments indicated that the contact sites for transcription activator proteins are also on the same surface.

Base Sequence↗

PubMed1995

Structural map of the alpha subunit of Escherichia coli RNA polymerase: structural domains identified by proteolytic cleavage.

The alpha subunit of Escherichia coli RNA polymerase plays essential roles in protein-protein contacts, not only for RNA polymerase assembly, but also for transcription activation by class I factors. To reveal the structure-function relationship of the alpha subunit, we attempted to elucidate the organization of the structural domains by analysis of the pattern of limited proteolysis with two endoproteases, V8 protease and trypsin. The results indicate that one region, Arg235 to Glu244, is highly accessible to endoproteases. We propose that the alpha subunit consists of two major structural domains, the amino-terminal domain upstream from Arg235 and the carboxy-terminal domain downstream from Glu245, each being connected by an inter-domain linker formed by the spacer between these two amino acid residues. The structural organization is in good agreement with its functional map, i.e., the amino-terminal subunit assembly determinants and the carboxy-terminal transcription activation determinants, including the contact sites with class I transcription factors and DNA UP (enhancer) elements. The secondary proteolytic cleavage sites were also determined, in order to analyse intra-domain structures.

Amino Acid Sequence↗

PubMed1995

Effects of bradykinin on Ca2+ mobilization and prostaglandin E2 release in human periodontal ligament cells.

In fura-2-loaded human periodontal ligament (HPDL) cells, bradykinin induced a rapidly transient increase and subsequently sustained increase in cytosolic Ca2+ ([Ca2+]i). When external Ca2+ was chelated by EGTA, the transient peak of [Ca2+]i was reduced and the sustained level was abolished, implying the Ca2+ mobilization consists of intracellular Ca2+ release and Ca2+ influx. Thapsigargin, a specific Ca(2+)-ATPase inhibitor for inositol 1,4,5-trisphosphate (1,4,5-IP3)-sensitive Ca2+ pool, induced an increased in [Ca2+]i in the absence of external Ca2+. After depletion of the intracellular Ca2+ pool by thapsigargin, the increase in [Ca2+]i induced by bradykinin was obviously reduced. Bradykinin also stimulated formation of inositol polyphosphates including 1,4,5-IP3. These results suggest that bradykinin stimulates intracellular Ca2+ release from the 1,4,5-IP3-sensitive Ca2+ pool. Bradykinin stimulated prostaglandin E2 (PGE2) release in the presence of external Ca2+, but not in the absence of external Ca2+. Ca2+ ionophore A23187 and thapsigargin evoked the release of PGE2 in the presence of external Ca2+ despite no activation of bradykinin receptors. These results indicate that bradykinin induces Ca2+ mobilization via activation of phospholipase C and PGE2 release caused by the Ca2+ influx in HPDL cells.

4-Nitrophenylphosphatase↗