Biomedical subjects

M Nishiyama

Publications and source records attributed to M Nishiyama.

At least 91 records · Page 5Linked to original sources

PubMed1999

Decreased type 2 corticotropin-releasing hormone receptor mRNA expression in the ventromedial hypothalamus during repeated immobilization stress.

Chronic or repeated stress results in reduction of food intake and body weight in rats. Stress-induced anorexia has been attributed to increased corticotropin-releasing hormone (CRH) function in the central nervous system. To explore possible roles of other neuropeptides and peripheral hormones involved in food intake and energy utilization during continuing stress, we examined the impact of repeated immobilization stress on expression of mRNAs coding for CRH, neuropeptide Y (NPY), galanin and pro-opiomelanocortin (POMC) mRNAs in such hypothalamic nuclei as the paraventricular nucleus (PVN), arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH), as well as plasma insulin and leptin concentrations. Changes in type 2 CRH receptor (CRHR-2) mRNA in the ventromedial hypothalamus (VMH), a possible target of anorectic CRH effect, were also examined. Rats were immobilized for 2 h daily for 6 days and sacrificed 24 h after the last immobilization. Immobilized rats had lower food intake and body weight and higher levels of PVN CRH mRNA than controls. Repeated immobiliza tion also lowered plasma insulin and leptin concentrations and VMH CRHR-2 mRNA levels. These results provide additional evidence linking VMH CRHR-2 mRNA levels to plasma leptin concentration. ARC NPY and DMH galanin mRNAs increased following repeated immobilization, while ARC POMC mRNA decreased. DMH NPY mRNA and ARC galanin mRNA were unaltered by immobilization. Since NPY and galanin are considered orexigenic, while the POMC-melanocortin-4 receptor system is apparently anorexigenic, the changes in neuropeptide mRNAs and VMH CRHR-2 mRNA may play counterregulatory roles against anorectic CRH effects.

Adrenocorticotropic Hormone↗

PubMed1999

Detection of alternative splicing of fibronectin mRNA in a single cell.

Pre-fibronectin mRNA is subject to alternative splicing at three sites, EDA, EDB and IIICS. We analyzed the alternative splicing of fibronectin mRNA in a single cell. Reverse transcription-polymerase chain reaction analyses showed cells that produced a single form of mRNA at each one of these sites as well as cells that produced multiple forms at a given site: for example, some cells produced either the EDA(+) or EDA(-) form of the mRNA and other cells produced both forms. About 80% of the cells produced both (+) and (-) forms of the mRNA at the EDA and EDB sites, and the remaining cells contained either the (+) or (-) form. Five forms of fibronectin mRNA can result from alternative splicing at the IIICS site. Complex combinations of alternative splicing products were observed among the individual cells: there were ten different combinations of mRNA isoforms with respect to the IIICS site. Statistically significant changes in alternative splicing at the IIICS site were observed during cellular senescence.

Alternative Splicing↗

PubMed1999

Dihydropyrimidine dehydrogenase, multidrug resistance-associated protein, and thymidylate synthase gene expression levels can predict 5-fluorouracil resistance in human gastrointestinal cancer cells.

Gene expression of dihydropyrimidine dehydrogenase (DPD) and newly multidrug resistance-associated protein (MRP) was found to correlate well with primary 5-FU resistance in 7 human gastrointestinal cancer cell lines. Although mRNA and protein levels of thymidylate synthase (TS) did not relate to the resistance, 5-FU treatment revealed a remarkable increase of TS expression. Such enhanced TS expression was more significant than DPD and MRP, and observed less in 5-FU sensitive cells. DPD and MRP expression levels can predict primary 5-FU resistance, and TS may be a potent predictor of cellular 5-FU resistance after 5-FU treatment.

ATP-Binding Cassette Transporters↗

PubMed1999

Low-dose cisplatin and 5-fluorouracil in combination can repress increased gene expression of cellular resistance determinants to themselves.

The synergistic mechanism of cisplatin (CDDP) and 5-fluorouracil (5-FU) in combination remains unclear, despite its substantial antitumor activity, which has been demonstrated clinically. To clarify the mechanism(s), we determined the sensitivity or resistance factors to either drug in seven gastrointestinal cancer cell lines and then analyzed the altered gene expression after different exposures to CDDP and 5-FU. At the basal gene expression level, glutathione S-transferase pi (GSTpi) expression correlated with the observed resistance to CDDP, whereas dihydropyrimidine dehydrogenase (DPD) and multidrug resistance-associated protein (MRP) expression was related to 5-FU resistance. GSTpi, DPD, and MRP expression increased in response to the respective drug, but they also increased in response to the other drug as well. Additionally, 5-FU revealed a drastically increased thymidylate synthase (TS) gene expression in 5-FU-resistant cells. However, the increasing actions of CDDP and 5-FU on GSTpi, DPD, MRP, and TS expression varied according to the exposure time, concentration, and schedule. A low concentration of CDDP (1 microg/ml, 30 min) followed by 5-FU (0.5 microg/ml, 72 h) was found to cause a less increased expression of DPD, MRP, GSTpi, and TS than either drug alone, thus resulting in synergistic cytotoxicity in 5-FU-resistant COLO201 and CDDP-resistant HCC-48 cells. The sequential combination of CDDP and 5-FU inhibited the growth of human normal renal proximal tubule cells by less than 20%. Low concentrations of CDDP followed by continuous exposure to 5-FU can repress increased gene expression related to both drug resistances, thereby being synergistically cytotoxic in human gastrointestinal cancer cells.

ATP-Binding Cassette Transporters↗

PubMed1999

11p15 translocations involving the NUP98 gene in childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome.

In a survey of childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/MDS) in Japan, we found 11p15 translocations in 5 (6%) of 81 children with t-AML/MDS. t(11;17)(p15;q21), t(11;12)(p15;q13), t(7;11)(p15;p15), inv(11)(p15q22), and add(11)(p15) were each found in one patient. Southern blotting and/or RT-PCR analyses revealed rearrangements of the NUP98 gene in tumor samples of all five patients. Rearrangements of DDX10 were detected in t-AML/MDS cells with inv(11), and rearrangements of HOXA9 were detected in t-AML cells with t(7;11). The 17q21 breakpoint of t(11;17) and the 12q13 breakpoint of t(11;12)(p15;q13) coincided with the loci of the HOXB and HOXC gene families, respectively. Therefore, it is reasonable to speculate that one of the HOXB genes and one of the HOXC genes were fused to NUP98 by t(11;17) and t(11;12), respectively, in t-AML/MDS cells. We propose that NUP98 may be a target gene for t-AML/MDS, and that t-AML/MDS with a fusion of NUP98 and HOX or DDX10 genes may be more frequent in children than in patients of other age groups.

Acute Disease↗

PubMed1998

Potassium channels activated in the endothelium-dependent hyperpolarization in guinea-pig coronary artery.

1. Properties of endothelium-dependent hyperpolarization evoked by acetylcholine (ACh) in smooth muscle of the guinea-pig coronary artery were investigated using conventional microelectrode techniques. 2. ACh hyperpolarized the membrane in an endothelium-dependent manner. The hyperpolarization comprised two components: an initial and a slow hyperpolarization. The former appeared during application of ACh, while the latter occurred after withdrawal of ACh. 3. Indomethacin and f1p4ofenac, inhibitors of the enzyme cyclo-oxygenase, blocked only the slow hyperpolarization, indicating that this potential was produced by endothelial prostanoids. 4. Clotrimazole and SKF 525a, known inhibitors of the enzyme cytochrome P450, inhibited both the initial and the slow hyperpolarizations, suggesting that these chemicals acted as non-selective inhibitors of arachidonic acid metabolism. Inhibition of the lipoxygenase pathway of arachidonic acid metabolism by nordihydroguaiaretic acid had no effect on either component of the hyperpolarization. 5. The slow hyperpolarization was inhibited by 4-aminopyridine (4-AP; 10(4) 10(-3) M) and glibenclamide (10(-6) M). The initial hyperpolarization was greatly inhibited by charybdotoxin (CTX; 5 x 10(-8) M) and partially inhibited by apamin (10(-7) M), but was not inhibited by glibenclamide (10(-5) M). Ba2+ (10(-4) M) depolarized the membrane and increased the amplitude of both components of the ACh-induced hyperpolarization. 6. Hyperpolarizations produced by Y-26763, a K+ channel opener, were inhibited by glibenclamide, but not by 4-AP. 7. The results indicate that the slow hyperpolarization is produced by endothelial prostanoids through activation of 4-AP-sensitive K+ channels (possibly delayed rectifier type). The initial hyperpolarization is produced mainly through activation of CTX-sensitive K+ channels (possibly Ca(2+)-sensitive type).

Acetylcholine↗

PubMed1998

Novel mutations of the endothelin B receptor gene in patients with Hirschsprung's disease and their characterization.

Hirschsprung's disease (HSCR) is a congenital intestinal disease, characterized by the absence of ganglion cells in the distal portion of the intestinal tract. Recently, three susceptibility genes have been identified in HSCR, namely the RET protooncogene, the endothelin B (ETB) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3). To investigate whether mutations in EDNRB could be related with HSCR in non-inbred populations in Japan, we examined alterations of the gene in 31 isolated patients. Three novel mutations were detected as follows: two transversions, A to T and C to A at nucleotides 311 (N104I) and 1170 (S390R), respectively, and a transition, T to C at nucleotide 325 (C109R). To analyze functions of these mutant receptors, they were expressed in Chinese hamster ovary cells. S390R mutation did not change the binding affinities but caused the decreases in the ligand-induced increment of intracellular calcium and in the inhibition of adenylyl cyclase activity, showing the impairment of the intracellular signaling. C109R receptors were proved to be localized near the nuclei as an unusual 44-kDa protein with the extremely low affinity to endothelin-1 (ET-1) and not to be translocated into the plasma membrane. On the other hand, N104I receptors showed almost the same binding affinities and functional properties as those of the wild type. Therefore, we conclude that S390R and C109R mutations could cause HSCR but that N104I mutation might be polymorphous.

Adenylyl Cyclase Inhibitors↗

PubMed1998

Enhanced expression of mitochondrial genes in senescent endothelial cells and fibroblasts.

It has been suggested that some mitochondrial genes are important in cellular senescence. In order to identify the mitochondrial genes that are involved in cellular senescence, we have constructed a cDNA library from senescent human vascular endothelial cells and isolated 86 senescence-specific cDNA clones by differential screening. Among the clones, we identified four distinct mitochondrial genes including NADH dehydrogenase subunit 2 (ND2), ND3, ATPase 6 and 16S ribosomal RNA. We then compared the levels of expression of these genes in young and senescent cells by using two endothelial and two fibroblast cell strains. Northern blot and slot blot hybridization confirmed that the expression levels of ND3, ATPase 6 and 16S rRNA were elevated in senescent cells of all four strains. The expression level of ND2 was also elevated during cellular senescence in three of the four strains. Because mitochondria are actively involved in oxidative phosphorylation and respiratory functions, the altered expression levels of these genes may participate in aging processes.

Adenosine Triphosphatases↗

PubMed1998

The C-terminal silencing domain of Rap1p is essential for the repression of ribosomal protein genes in response to a defect in the secretory pathway.

We have previously shown that a functional secretory pathway is essential for continued ribosome synthesis in Saccharomyces cerevisiae. When a temperature-sensitive mutant defective in the secretory pathway is transferred to the non-permissive temperature, transcription of both rRNA genes and ribosomal protein genes is nearly abolished. In order to define the cis -acting element(s) of ribosomal protein genes sensitive to a defect in the secretory pathway, we have constructed a series of fusion genes containing the CYH2 promoter region, with various deletions, fused to lacZ. Each fusion gene for which transcription is detected is subject to the repression. Rap1p is the transcriptional activator for most ribosomal protein genes, as well as having an important role in silencing in the vicinity of telomeres and at the silent mating-type loci. To assess its role in the repression of transcription by the defect in the secretory pathway, we have introduced rap1 mutations. The replacement of wild-type Rap1p by Rap1p truncated at the C-terminal region caused substantial attenuation of the repression. Furthermore, we have demonstrated that the Rap1p-truncation affects the repression of TCM1 , encoding ribosomal protein L3, which has no Rap1p-binding site in its upstream regulatory region. These results suggest that the repression of transcription of ribosomal protein genes by a secretory defect is mediated through Rap1p, but does not require a Rap1p-binding site within the UAS.

Artificial Gene Fusion↗

PubMed1998

Endothelium is required for 12-hydroperoxyeicosatetraenoic acid-induced vasoconstriction.

The pharmacological effects of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) were examined using isolated canine basilar artery segments and isometric tension recording. 12-HPETE produced transient contraction of the artery segment while arachidonic acid or 12-hydroxyeicosatetraenoic acid (12-HETE) had a much lower potency. 12-HPETE-induced contraction which showed a requirement of a functional endothelium and a rapid insensitivity to re-administered 12-HPETE, was completely inhibited by the potassium channel blocker, glibenclamide. Other hydroperoxides did cross-desensitize the 12-HPETE-induced contraction, however, arachidonic acid or 12-HETE did not affect markedly. Here, we present that 12-HPETE is involved in the regulation of vascular tension via its effects on the endothelium.

Animals↗

PubMed1998

Application of nitrite reductase from Alcaligenes faecalis S-6 for nitrite measurement.

The enzymatic reaction of nitrite reductase (NIR) from Alcaligenes faecalis S-6 was applied to the measurement of nitrite. NIR was immobilized on the surface of a gold electrode using filter paper and a dialysis membrane, and used as a working electrode in a three-electrode system. Amperometric methods were applied using NIR and the electron mediator 1-methoxy PMS (1-methoxy-5-methylphenazinium methylsulfate). The decrease in cathodic current showed a correlation to nitrite concentration over the range 0-1 mg/l. Measurements using a batch-flow type system gave a lower detection limit of 0.01 mg/l. This is sufficient for the detection of nitrite in natural waters.

Alcaligenes↗

PubMed1998

Overexpression of high-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1 in recombinant Rhodococcus cells.

High-molecular-mass nitrile hydratase (H-NHase, 530 kDa) is a cobalt-containing enzyme produced by Rhodococcus rhodochrous J1. For efficient production of H-NHase in R. rhodochrous ATCC12674, several plasmids were constructed. The enzyme was produced in the recombinant Rhodococcus cells only in the presence of an upstream region (approximately 4 kb) of the H-NHase gene under the control of the promoter for the amidase-NHase gene cluster from Rhodococcus sp. N-774. Although H-NHase was produced as a soluble protein in the cells, the protein did not show NHase activity. However, when the recombinant R. rhodochrous ATCC12674 cells were cultured in the presence of amide compounds, such as crotonamide and methacrylamide, markedly high NHase activity was detected, Gel-filtration chromatography revealed that the NHases produced by the cells grown in the presence and absence of the amide compounds had a molecular mass of more than 500 kDa and 50-80 kDa respectively. These results suggest that the amide compounds are essential for subunit assembly to form an enzymatically active multimer. By the use of the recombinant expression system, NHase activity 1.7 times higher than that of the original strain, R. rhodochrous J1, was achieved.

Dose-Response Relationship, Drug↗

PubMed1998

Crystallization and preliminary X-ray diffraction studies of a rice cysteine proteinase inhibitor, Oryzacystatin-I.

Oryzacystatin-I from rice seeds was overexpressed in Escherichia coli, purified, and crystallized by the sitting-drop vapor diffusion method. Crystals obtained with 2-methyl-2,4-pentanediol as a precipitant exhibited space group I4122, with unit cell parameters of a = b = 100.0 A, c = 54.2 A, and diffracted up to 2.8 A resolution at 100 K. The crystals have one molecule per asymmetric unit.

Base Sequence↗

PubMed1998

Designing the hydrophobic core of Thermus flavus malate dehydrogenase based on side-chain packing.

We have developed a computational method for the de novo design of hydrophobic cores of proteins and tested it experimentally. The method is composed of a pair of programs, (i) to optimize side-chain conformations using an updated rotamer library for potential hydrophobic residues, based on the backbone structure of the protein of interest, and (ii) to estimate changes in Gibbs free energies between the folded and unfolded structures of the optimized sequence. Using these programs, we have engineered several variants of Thermus flavus malate dehydrogenase. To quantitate the stability change in each variant, the circular dichroism spectra of the proteins were measured as a function of guanidine hydrochloride concentration and deltadeltaG(H2O) values of the proteins were determined by extrapolation of the experimental data. However, variants with double replacements showed different denaturation cooperativity from that of the wild type and therefore it was difficult to simply compare the theoretical and experimental stability of each variant using calculated deltadeltaG and experimental deltadeltaG(H2O) values. When the calculated deltadeltaG values were compared with those at 3.5 M guanidine hydrochloride, which was the transition midpoint obtained from the denaturation curve of the wild type, good correlation was observed.

Circular Dichroism↗

PubMed1998

Altered expression of type 2 CRH receptor mRNA in the VMH by glucocorticoids and starvation.

In the rat, high-dose corticosterone (Cort) administration, the hypercortisolism of starvation, and adrenalectomy are all associated with decreased food intake and weight loss. We report here a study of the effects of high-dose Cort administration, starvation, and adrenalectomy on two peripheral hormones known to influence food intake and energy use, insulin and leptin. We also studied the impact of these interventions on the levels of type 2 corticotropin-releasing hormone receptor (CRHR-2) mRNA in the hypothalamic paraventricular nucleus (PVN) and ventromedial hypothalamus (VMH). The VMH is classically referred to as the satiety center because electrical stimulation of the VMH leads to inhibition of food intake, whereas CRHR-2 are thought to transduce the profound anorexogenic effects of CRH or its related peptide urocortin. Starvation and adrenalectomy each lowered plasma insulin and leptin levels and were associated with decrements in CRHR-2 mRNA levels in the VMH. Cort administration increased plasma leptin levels profoundly, as well as plasma insulin levels and the levels of VMH CRHR-2 mRNA. Under all experimental conditions, a positive correlation was seen between plasma leptin levels and VMH CRHR-2 mRNA. These data suggest that decreased food intake and weight loss after high-dose Cort administration at least partially depend on the profound impact of Cort on plasma leptin secretion in the rat; they suggest, moreover, an additional mechanism for the satiety-inducing effects of leptin, namely increasing CRHR-2 in the VMH. The concordance of a fall in plasma insulin and leptin levels with the fall in VMH CRHR-2 mRNA levels further supports the idea that compensatory responses during starvation and adrenalectomy include not only the disinhibiting effects of reduced insulin and leptin levels on appetite through already-described mechanisms but also via an effect of leptin on VMH CRHR-2. Neither Cort administration, starvation, nor adrenalectomy influenced the levels of CRHR-2 mRNA in the PVN, suggesting that these receptors are differentially regulated in different hypothalamic regions.

Adrenalectomy↗

PubMed1998

Severe diabetic scleredema with extension to the extremities and effective treatment using prostaglandin E1.

We report a 49-year-old woman with severe diabetic scleredema (DS). The patient had non-insulin-dependent diabetes mellitus (NIDDM) for 9 years and noticed thickened skin on her back 3 years previously. Her DS rapidly extended to her back and extremities with pain and immobility. Her symptoms of DS improved dramatically after establishing strict glycemic control and intravenous administration of prostaglandin E1 (PGE1). However, the histological findings of her skin biopsy did not change even after the treatment for 12 weeks, and her symptoms worsened again after discontinuation of glycemic control and PGE1 treatment. The causes of DS have been considered to be metabolic abnormalities associated with hyperglycemia and hypoxia in the skin due to diabetic microangiopathy. PGE1 was an effective treatment for DS in our patient. Strict control of hyperglycemia and PGE1 treatment may be sufficient to manage DS, although a very long treatment period is necessary.

Acupuncture Analgesia↗

PubMed1998

Basic studies on hydroxy apatite cement: I. Setting reaction.

Self-setting cements, alpha D-Cement and alpha DT-Cement, were prepared. They consisted of only the calcium phosphates alpha-TCP, TTCP and DCPA. These cements reacted and hardened in a moist environment at 37 degrees C. The powder X-ray diffraction patterns were taken to examine the conversion of their reactions as a function of time. The cements reacted and produced hydroxyapatite. The optimum powder/liquid ratio of alpha D-Cement was 2.0 and that of alpha DT-Cement was 1.8. The initial setting time of alpha D-Cement was 87.5 m and that of alpha DT-Cement was 107.5 m. The component and the product of these cements are calcium phosphates which are the putative minerals in teeth and bones. Therefore, these cements are useful for oral surgery as bone-filling materials.

Bone Substitutes↗

PubMed1998

[Characteristics and progress of treatment for gastric cancers detected by mass screening].

We devised an X-ray examination to detect early gastric cancer, especially small gastric cancer, during the period between 1996 and 1997. As a result, the rate of gastric cancer detection by mass screening was 0.50%; and the rate of early gastric cancer detection was 79%. 1. Characteristics of gastric cancer detected by mass screening: Characteristics of gastric cancer were depressive-type undifferentiated cancer less than 4.0 cm diameter in work-place screening and depressive-type differentiated cancer less than 4.0 cm in area screening; 2. gastric cancer less than 2.0 cm in diameter was 75 (68%) of the 110 cases (total detected gastric cancer). 2) We have to consider that gastric cancer differs by sex and age. 3) EMR accounted for 39 (35%) of the 110 cases in treatment. This method resulted in a higher gastric cancer detection rate and served to maintain QOL.

Endoscopy↗