Biomedical subjects

Takeshi Yamazaki

Publications and source records attributed to Takeshi Yamazaki.

At least 19 recordsLinked to original sources

PubMed2007

Dephosphorylation of TORC initiates expression of the StAR gene.

Cyclic AMP responsive element (CRE) binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. However, transducer of regulated CREB activity (TORC), a CREB specific co-activator, upregulates CREB activity in a phospho-Ser133-independent manner. Interestingly, TORC is also regulated by phosphorylation; the phospho-form is inactive, and the dephospho-form active. When PKA phosphorylates CREB, it inhibits TORC kinases simultaneously and accelerates dephosphorylation of TORC. We show in this report that staurosporine, a kinase inhibitor, induces the expression of the StAR gene in Y1 adrenocortical cells, possibly a result of an increase in the population of dephospho-TORC. The expression of the StAR gene is known to be regulated by SF-1 and CREB, and the co-activators CBP/p300 may mediate the actions of both factors. Our experiments using KG501, a disruptor of the interaction between phospho-CREB and CBP/p300, also support the importance of TORC in the regulation of StAR gene expression.

Animals↗

PubMed2006

Comparison between basal and apical dendritic spines in estrogen-induced rapid spinogenesis of CA1 principal neurons in the adult hippocampus.

Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Here, we demonstrated the rapid effect of 17beta-estradiol on the density and morphology of spines in the stratum oriens (s.o., basal side) and in the stratum lacunosum-moleculare (s.l.m., apical side) by imaging Lucifer Yellow-injected CA1 neurons in adult male rat hippocampal slices, because spines in s.o. and s.l.m. have been poorly understood as compared with spines in the stratum radiatum. The application of 1nM estradiol-induced a rapid increase in the density of spines of pyramidal neurons within 2h. This increase by estradiol was blocked by Erk MAP kinase inhibitor and estrogen receptor inhibitor in both regions. Effect of blockade by agonists of AMPA receptors and NMDA receptors was different between s.o. and s.l.m. In both regions, ERalpha agonist PPT induced the same enhancing effect of spinogenesis as that induced by estradiol.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2006

Allosteric inhibition of rat neuronal nitric-oxide synthase caused by interference with the binding of calmodulin to the enzyme.

A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the L-citrulline formation of nNOS from L-arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.

Allosteric Regulation↗

PubMed2006

Mitochondrial processing of bovine adrenal steroidogenic acute regulatory protein.

Steroidogenic acute regulatory (StAR) protein is an important regulatory protein in steroidogenesis and rapidly undergoes proteolysis after import into the mitochondria. In this study, we determined the proteolytic cleavage sites and investigated the effects on the stimulation of steroidogenic activity of the blockage of these sites by mutation. The cleaved StAR proteins, which were purified using an anti-StAR immobilized column, reacted with antiserum against the StAR C-terminal oligopeptide. The molecular weights of the purified proteins were determined by MALDI-TOF mass spectrometry, and were found to be identical to those of the 40-285 and 55-285 amino-acid-regions of the StAR protein. To confirm the identification of the cleavage sites, we constructed site-directed mutants of bovine StAR cDNA, which contained the amino acids R37A/R38A/L40A and/or R53A/R54A/R55A. These mutant StAR proteins expressed in COS-1 cells were not cleaved at positions 39-40 and 54-55, and were processed at sites different from those in the wild-type StAR protein. These mutant proteins stimulated pregnenolone formation at almost the same rate as the wild-type StAR protein in COS-1 cells, which suggests that the cholesterol transfer activity was not affected by the mutation.

Adrenal Glands↗

PubMed2006

The interaction of cytochrome P450 17alpha with NADPH-cytochrome P450 reductase, investigated using chemical modification and MALDI-TOF mass spectrometry.

The lysine residues of guinea pig P450 17alpha were acetylated by acetic anhydride in the absence and presence of NADPH cytochrome P450 reductase (CPR). Eight acetylated peptides were identified in the MALDI-TOF mass spectra of the tryptic fragments from the P450 acetylated without CPR in the limited reaction time of 15 min at ice temperature. The presence of CPR during the acetylation of P450 17alpha prevented double acetylations at K326 and K327 in the J-helix. The activity of P450 17alpha was decreased to 35% by the acetylation, but almost no inactivation was detected in the P450 after acetylation in the presence of CPR. This protection from inactivation shows the importance of K326 and/or K327 in the J-helix of P450 17alpha in the interaction between the two enzymes. Our results provided the first experimental evidence for the importance of the J-helix of P450 in the interaction with CPR. The interaction of P450 17alpha with CPR on the membrane is discussed based on the results of this study, which used molecular modeling.

Animals↗

PubMed2006

Influence of EPIYA-repeat polymorphism on the phosphorylation-dependent biological activity of Helicobacter pylori CagA.

BACKGROUND & AIMS: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3'-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. METHODS: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. RESULTS: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA-SHP-2 interaction. CONCLUSIONS: EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.

Antigens, Bacterial↗

PubMed2006

Application of liquid chromatography-nuclear magnetic resonance spectroscopy for the identification of ethyldimethylpyrazine, a food flavouring agent.

The application of liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR) for the direct identification of ethyldimethylpyrazine, a food flavouring agent, has been studied. The commercial product is a mixture of two regio-isomers, 2-ethyl-3,5-dimethylpyrazine (1) and 2-ethyl-3,6-dimethylpyrazine (2); however, the exact composition of the mixture is unknown. Structural characterization by LC-MS and GC-MS was not possible because both regio-isomers yield the same molecular related ion and ion fragmentation. To rapidly identify the two regio-isomers, the product was analyzed by LC-NMR with on-flow and fraction loop modes. From the results, the structure elucidations of the two regio-isomers could be carried out without the need to isolate the isomers by the usual procedures.

Chromatography, Liquid↗

PubMed2006

Modified extravesical ureteroneocystostomy for completely duplicated ureters in renal transplantation.

INTRODUCTION: Completely duplicated ureters are the most common congenital malformation of the upper urinary tract. However, there are very few reports on transplantation using kidneys with double ureters, and the technique of ureterocystoneostomy for completely duplicated ureters has not yet been established. PATIENTS AND METHODS: We treated 3 patients with completely duplicated ureters at our institutions from January 1998 to October 2005. We modified the extravesical technique for treating these 3 cases and evaluated the 52-month follow-up period for possible urological complications. RESULTS: Neither urological complications nor urinary tract infections occurred in the follow-up period after using our new technique. CONCLUSION: We conclude that this technique is an appropriate procedure for the transplantation of kidneys with completely duplicated ureters.

Cystostomy↗

PubMed2006

[Analysis of constituents in urushi wax, a natural food additive].

Urushi wax is a natural gum base used as a food additive. In order to evaluate the quality of urushi wax as a food additive and to obtain information useful for setting official standards, we investigated the constituents and their concentrations in urushi wax, using the same sample as scheduled for toxicity testing. After methanolysis of urushi wax, the composition of fatty acids was analyzed by GC/MS. The results indicated that the main fatty acids were palmitic acid, oleic acid and stearic acid. LC/MS analysis of urushi wax provided molecular-related ions of the main constituents. The main constituents were identified as triglycerides, namely glyceryl tripalmitate (30.7%), glyceryl dipalmitate monooleate (21.2%), glyceryl dioleate monopalmitate (2.1%), glyceryl monooleate monopalmitate monostearate (2.6%), glyceryl dipalmitate monostearate (5.6%), glyceryl distearate monopalmitate (1.4%). Glyceryl dipalmitate monooleate isomers differing in the binding sites of each constituent fatty acid could be separately determined by LC/MS/MS.

Chromatography, Liquid↗

PubMed2006

Identification of the main constituents in sandarac resin, a natural gum base.

Sandarac resin, a natural gum base, is described as "a substance composed mainly of sandaracopimaric acid obtained from the secretion of sandarac trees" in the List of Existing Food Additives in Japan. To evaluate its quality as a food additive, the main constituents in a sandarac resin product were investigated. Three constituents were isolated and identified as sandaracopimaric acid, sandaracopimarinol and 4-epidehydroabietic acid by MS and 2D-NMR. Quantification of the main constituent, sandaracopimaric acid, was performed by HPLC and its content in the product was determined to be 11.6%.

Chromatography, High Pressure Liquid↗

PubMed2005

Ca2+ signal stimulates the expression of steroidogenic acute regulatory protein and steroidogenesis in bovine adrenal fasciculata-reticularis cells.

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.

Adrenocorticotropic Hormone↗

PubMed2005

Tributyltin disturbs bovine adrenal steroidogenesis by two modes of action.

Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.

Adrenal Cortex Hormones↗

PubMed2005

Helical rosette nanotubes with tunable stability and hierarchy.

The design of nanostructured materials with tunable dimensions and properties that maintain their structural integrity under physiological conditions is a major challenge in biomedical engineering and nanomedicine. Helical rosette nanotubes (HRN) are a new class of materials produced through a hierarchical self-assembly process of low molecular weight synthetic organic modules in water. Here, we describe a synthetic strategy to tune their stability and hierarchy by preorganization of the self-assembling units, control of net charge per unit of nanotube surface area, amphiphilicity, and number of H-bonds per self-assembling module, and through peripheral steric (de)compression. Using these criteria, HRNs with tunable stability and hierarchical architecture were produced from self-assembling modules that (a) persist as individual molecules in solution, (b) self-assemble into HRN but denature at high temperature (<85 degrees C), (c) self-assemble into HRN whose structural integrity persists even in boiling water (>95 degrees C), and (d) self-assemble into well-dispersed short nanotubes, long nanotubes, ribbons, or superhelices. Given the biocompatibility, synthetic accessibility, and chemical and physical tunability of these materials, numerous applications in biomedical engineering, materials science, and nanoscience and technology are envisioned.

Journal Article↗

PubMed2005

Neptune is involved in posterior axis and tail formation in Xenopus embryogenesis.

In order to elucidate the molecular mechanisms underlying the posterior axis and tail formation in embryogenesis, the function of Neptune, a zinc-finger transcription factor, in Xenopus laevis embryos was investigated. Injection of neptune mRNA into the animal pole area of embryos resulted in the formation of an additional tail structure that included a neural tube and muscle tissue. This activity required FGF signaling since coinjection of a dominant-negative FGF receptor RNA (XFD) completely blocked the formation of a tail structure. A loss-of-function experiment using a fusion construct of neptune and Drosophila engrailed (en-neptune) RNA showed that endogenous Neptune is necessary for formation of the posterior trunk and tail. Furthermore, activity of Neptune was necessary for the endogenous expression of brachyury and fgf-8 at the late gastrula stage. These findings demonstrate a novel function of Neptune in the process of anterior-posterior axis formation through the FGF and brachyury signaling cascades. An experiment using a combination explant with ventral and dorsal marginal tissues showed that cooperation of these two distinct tissues is important for the tail formation and that expression of Neptune in prospective ventral cells may be involved in the activation of the process of tail formation.

Animals↗

PubMed2005

Analysis of the constituents in jojoba wax used as a food additive by LC/MS/MS.

Jojoba wax is a natural gum base used as a food additive in Japan, and is obtained from jojoba oil with a characteristically high melting point. Although the constituents of jojoba oil have been reported, the quality of jojoba wax used as a food additive has not yet been clarified. In order to evaluate its quality as a food additive and to obtain basic information useful for setting official standards, we investigated the constituents and their concentrations in jojoba wax. LC/MS analysis of the jojoba wax showed six peaks with [M+H]+ ions in the range from m/z 533.6 to 673.7 at intervals of m/z 28. After isolation of the components of the four main peaks by preparative LC/MS, the fatty acid and long chain alcohol moieties of the wax esters were analyzed by methanolysis and hydrolysis, followed by GC/MS. The results indicated that the main constituents in jojoba wax were various kinds of wax esters, namely eicosenyl octadecenoate (C20:1-C18:1) (1), eicosenyl eicosenoate (C20:1-C20:1) (II), docosenyl eicosenoate (C22:1-C20:1) (III), eicosenyl docosenoate (C20:1-C22:1) (IV) and tetracosenyl eiosenoate (C24:1-C20:1) (V). To confirm and quantify the wax esters in jojoba wax directly, LC/MS/MS analysis was performed. The product ions corresponding to the fatty acid moieties of the wax esters were observed, and by using the product ions derived from the protonated molecular ions of wax esters the fatty acid moieties were identified by MRM analysis. The concentrations of the wax esters I, II and III, in jojoba wax were 5.5, 21.4 and 37.8%, respectively. In summary, we clarified the main constituents of jojoba wax and quantified the molecular species of the wax esters without hydrolysis by monitoring their product ions, using a LC/MS/MS system.

Chromatography, Liquid↗

PubMed2004

Inhibitory effect of organotin compounds on rat neuronal nitric oxide synthase through interaction with calmodulin.

Organotin compounds, triphenyltin (TPT), tributyltin, dibutyltin, and monobutyltin (MBT), showed potent inhibitory effects on both L-arginine oxidation to nitric oxide and L-citrulline, and cytochrome c reduction catalyzed by recombinant rat neuronal nitric oxide synthase (nNOS). The two inhibitory effects were almost parallel. MBT and TPT showed the highest inhibitory effects, followed by tributyltin and dibutyltin; TPT and MBT showed inhibition constant (IC(50)) values of around 10microM. Cytochrome c reduction activity was markedly decreased by removal of calmodulin (CaM) from the complete mixture, and the decrease was similar to the extent of inhibition by TPT and MBT. The inhibitory effect of MBT on the cytochrome c reducing activity was rapidly attenuated upon dilution of the inhibitor, and addition of a high concentration of CaM reactivated the cytochrome c reduction activity inhibited by MBT. However, other cofactors such as FAD, FMN or tetrahydrobiopterin had no such ability. The inhibitory effect of organotin compounds (100microM) on L-arginine oxidation of nNOS almost vanished when the amount of CaM was sufficiently increased (150-300microM). It was confirmed by CaM-agarose column chromatography that the dissociation of nNOS-CaM complex was induced by organotin compounds. These results indicate that organotin compounds disturb the interaction between CaM and nNOS, thereby inhibiting electron transfer from the reductase domain to cytochrome c and the oxygenase domain.

Amino Acid Oxidoreductases↗

PubMed2004

Propagation of a protease-resistant form of prion protein in long-term cultured human glioblastoma cell line T98G.

Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), a lethal, neurodegenerative condition, occur in sporadic, genetic and transmitted forms. CJD is associated with the conversion of normal cellular prion protein (PrP(C)) into a protease-resistant isoform (PrP(res)). The mechanism of the conversion has not been studied in human cell cultures, due to the lack of a model system. In this study, such a system has been developed by culturing cell lines. Human glioblastoma cell line T98G had no coding-region mutations of the prion protein gene, which was of the 129 M/V genotype, and expressed endogenous PrP(C) constitutively. T98G cells produced a form of proteinase K (PK)-resistant prion protein fragment following long-term culture and high passage number; its deglycosylated form was approximately 18 kDa. The PK-treated PrP(res) was detected by immunoblotting with the mAb 6H4, which recognizes residues 144-152, and a polyclonal anti-C-terminal antibody, but not by the mAb 3F4, which recognizes residues 109-112, or the anti-N-terminal mAb HUC2-13. These results suggest that PrP(C) was converted into a proteinase-resistant form of PrP(res) in T98G cells.

Cell Line, Tumor↗

PubMed2004

[Analysis of residual solvents in natural flavorings by headspace GC using the standard addition method].

Headspace GC using the standard addition method has been developed for the simultaneous determination of organic solvents in natural flavorings. The procedure can be outlined as follows: an aliquot of the sample is transferred to a 10 mL vial. To each vial, a DMSO solution containing solvents at different concentrations is added as the standard solution. The vials are kept at 50 degrees C (for automatic injection) or 40 degrees C (for hand-operated injection) for 40 minutes. One mL of the vapor phase in each vial is injected into a gas chromatograph equipped with an Aquatic-2 column (0.25 mm i.d. x 60 m). To evaluate this method, we conducted a performance study in collaboration with 10 laboratories, using ginger oleoresin. We analyzed 6 solvents (methanol, 2-propanol, acetone, dichloromethane, hexane, and 1,1,2-trichloroethene) for which the maximum residue limits are established in Japan's Specifications and Standards for Food Additives. Methanol and acetone existed in the ginger oleoresin, so only the other that four kinds of solvents were added to it. Eight of the laboratories used automatic injection, while the remaining two used hand-operated injection. Statistical analyses were conducted on the data obtained from the 8 laboratories. Repeatability standard deviations (RSDr) ranged from 4.3 to 11.4%, and reproducibility standard deviations (RSDR) ranged from 8.4 to 19.0%.

2-Propanol↗