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K Nakashima

Publications and source records attributed to K Nakashima.

At least 127 records · Page 7Linked to original sources
PubMed2000

Cell density-dependent regulation of hepatic development by a gp130-independent pathway.

We previously demonstrated that oncostatin M (OSM) promotes hepatic development in concert with glucocorticoid. The livers from mice deficient for gp130, a signaling subunit of the OSM receptor, displayed reduced expression of hepatic differentiation marker and defective glycogenic function. However, these phenotypes were not completely abolished in gp130(-/-) mice, suggesting that there is an alternative pathway regulating hepatic development in vivo. To test this possibility, we cultured gp130(-/-) fetal hepatic cells and investigated a signal that induces hepatic differentiation. When hepatocytes were forced to interact with each other by inoculating cells at high densities, hepatic differentiation was induced even in the absence of gp130. Moreover, cells stimulated with OSM and/or cultured at a high density possess many other metabolic functions. These observations suggest that fetal hepatic cells acquire multiple characteristics of differentiated hepatocytes in response to the signals generated by cell-cell contacts as well as by OSM.

Ammonia↗
PubMed2000

Leptin as a modulator of sweet taste sensitivities in mice.

Leptin acts as a potent inhibitory factor against obesity by regulating energy expenditure, food intake, and adiposity. The obese diabetic db/db mouse, which has defects in leptin receptor, displays enhanced neural responses and elevated behavioral preference to sweet stimuli. Here, we show the effects of leptin on the peripheral taste system. An administration of leptin into lean mice suppressed responses of peripheral taste nerves (chorda tympani and glossopharyngeal) to sweet substances (sucrose and saccharin) without affecting responses to sour, salty, and bitter substances. Whole-cell patch-clamp recordings of activities of taste receptor cells isolated from circumvallate papillae (innervated by the glossopharyngeal nerve) demonstrated that leptin activated outward K(+) currents, which resulted in hyperpolarization of taste cells. The db/db mouse with impaired leptin receptors showed no such leptin suppression. Taste tissue (circumvallate papilla) of lean mice expressed leptin-receptor mRNA and some of the taste cells exhibited immunoreactivities to antibodies of the leptin receptor. Taken together, these observations suggest that the taste organ is a peripheral target for leptin, and that leptin may be a sweet-sensing modulator (suppressor) that may take part in regulation of food intake. Defects in this leptin suppression system in db/db mice may lead to their enhanced peripheral neural responses and enhanced behavioral preferences for sweet substances.

Animals↗
PubMed2000

A biphasic regulation of receptor mRNA expressions for growth hormone, glucocorticoid and mineralocorticoid in the rat dentate gyrus during acute stress.

Acute stress increases circulating ACTH and glucocorticoid levels. The hippocampus (HIP) is a target of such stress hormones as glucocorticoid and it also expresses receptors for growth hormone (GH), particularly in the dentate gyms (DG). In order to understand the interactions between glucocorticoids and functions of GH in HIP during acute stress, the mRNA levels for GH receptor (GHR), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) were investigated in DG in rats exposed to restraint stress in the water (RSW). Using in situ hybridization histochemistry (ISHH), high level expressions of GHR mRNA were detected in DG. These were down-regulated by 14% after 0.5 h of RSW and then up-regulated by 38% over the initial level after 4 h of RSW. This biphasic enhancement of GHR mRNA expression in DG followed the elevation of plasma glucocorticoid levels and paralleled with biphasic expressions of mRNAs for GR and MR in DG. Although circulating GH levels did not show any correlation with the hippocampal GHR mRNA expression, adrenalectomy (ADX) decreased GHR mRNA expression in DG, and the dexamethasone treatment (DEX; 20 microg/100 microl, i.p.) of ADX rats rapidly increased the GHR mRNA expression in DG. These results have suggested that the GHR mRNA expression in the DG is regulated, at least in part, by glucocorticoids and that GH may be involved in responses of the DG to acute stress.

Acute Disease↗
PubMed2000

Genomic organization, sequence and chromosomal localization of the mouse Tbr2 gene and a comparative study with Tbr1.

Members of the T-box family are known to play critical roles in the embryonic development of most animal species. Recently, we have isolated its new mammalian member, Tbr2, from mouse embryonic brain. We have also shown that the expression patterns of Tbr2 and the closely related Tbr1 appear to be reciprocal in the developing brain; Tbr2 is expressed in mesencephalon and rhombencephalon, but expression of Tbr1 is restricted to telencephalon. To investigate possible structural and functional relationships of Tbr2 and other T-box containing genes, we analyzed genomic organization of the murine Tbr2 gene. The Tbr2 gene is composed of six exons (1353, 155, 122, 159, 62 and 1035bp), and five introns (920, 643, 602, 85 and 2036bp). This exon/intron organization is very similar to that of Tbr1. We also analyzed the 3.9kb sequence of the 5' promoter region flanking the Tbr2 gene and the corresponding region of the Tbr1 gene. The sites for Brn-2 and Tst-1 were found in the promoter of Tbr2 but not Tbr1. On the contrary, there were eight HNF-3beta binding sites in the Tbr1 gene promoter but only three in the Tbr2 promoter. The differential presence of putative binding sites for these brain-specific transcription factors may explain the reciprocal expression of Tbr1 and Tbr2. Furthermore, a single chromosomal locus for mouse Tbr2 was assigned to 9F3 by fluorescence in-situ hybridization 1.

Animals↗
PubMed2000

Plasma homocysteine and MTHFR C677T genotype in levodopa-treated patients with PD.

Plasma homocysteine and cysteine levels were measured in 90 patients with PD with the MTHFR C677T (T/T) genotype. The authors found that the levels of homocysteine-a possible risk factor for vascular disease-were elevated by 60% in levodopa-treated patients with PD, with the most marked elevation occurring in patients with the T/T genotype. Cysteine levels in subjects with PD did not differ from levels in control subjects. In the T/T genotype patients, homocysteine and folate levels were inversely correlated. Increased homocysteine might be related to levodopa, MTHFR genotype, and folate in PD.

Aged↗
PubMed2000

Skin-specific caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18.

To study the pathophysiological roles of overexpressed caspase-1 (CASP1), originally designated as IL-1 beta-converting enzyme, we generated transgenic mice in which human CASP1 is overexpressed in their keratinocytes. The transgenic mice spontaneously developed recalcitrant dermatitis and skin ulcers, characterized by the presence of massive keratinocyte apoptosis. The skin of the mice contained the active form of human CASP1 and expressed mRNA for caspase-activated DNase, an effector endonuclease responsible for DNA fragmentation. Their skin and sera showed elevated levels of mature IL-18 and IL-1 beta, but not of IFN-gamma. The plasma from these animals induced IFN-gamma production by IL-18-responsive NK cells. Administration of heat-killed Propionibacterium acnes, a potent in vivo type 1 cell inducer, caused IFN-gamma-mediated lethal liver injury in the transgenic mice, which was completely inhibited by treatment with neutralizing anti-IL-18 Ab. These results indicated that in vivo overexpression of CASP1 caused spontaneous apoptotic tissue injury and rendered mice highly susceptible to exogenous type 1 cell-inducing condition in collaboration with endogenously accumulated proinflammatory cytokines.

Animals↗
PubMed2000

BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6.

Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-beta (TGF-beta) superfamily, regulates a variety of cell fates and functions. At present, the molecular mechanism by which BMP2 induces apoptosis has not been fully elucidated. Here we propose a BMP2 signaling pathway that mediates apoptosis in mouse hybridoma MH60 cells whose growth is interleukin-6 (IL-6)-dependent. BMP2 dose-dependently induces apoptosis in MH60 cells even in the presence of IL-6. BMP2 has no inhibitory effect on the IL-6-induced tyrosine phosphorylation of STAT3, and the bcl-2 gene expression which is known to be regulated by STAT3, suggesting that BMP2-induced apoptosis is not attributed to alteration of the IL-6-mediated bcl-2 pathway. We demonstrate that BMP2 induces activation of TGF-beta-activated kinase (TAK1) and subsequent phosphorylation of p38 stress-activated protein kinase. In addition, forced expression of kinase-negative TAK1 in MH60 cells blocks BMP2-induced apoptosis. These results indicate that BMP2-induced apoptosis is mediated through the TAK1-p38 pathway in MH60 cells. We also show that MH60-derived transfectants expressing Smad6 are resistant to the apoptotic signal of BMP2. Interestingly, this ectopic expression of Smad6 blocks BMP2-induced TAK1 activation and p38 phosphorylation. Moreover, Smad6 can directly bind to TAK1. These findings suggest that Smad6 is likely to function as a negative regulator of the TAK1 pathway in the BMP2 signaling, in addition to the previously reported Smad pathway.

Animals↗
PubMed2000

Cyclization into hydrindanones using samarium diiodide

Samarium(II) iodide has been employed to promote vinylogous pinacol coupling reaction of aldehyde onto alpha,beta-unsaturated ketones. The diastereoselectivity of 6-endo products was changed by addition of a proton source and/or HMPA and by the reaction temperature. The cyclization reactions described herein provide a general approach to the syntheses of 3,3-dimethylhydrindanes with a cis-relationship between the OH at C-4 and the proton at C-3a with good diastereoselectivity and under mild reaction conditions.

Journal Article↗
PubMed2000

D-beta-hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease.

The heroin analogue 1-methyl-4-phenylpyridinium, MPP(+), both in vitro and in vivo, produces death of dopaminergic substantia nigral cells by inhibiting the mitochondrial NADH dehydrogenase multienzyme complex, producing a syndrome indistinguishable from Parkinson's disease. Similarly, a fragment of amyloid protein, Abeta(1-42), is lethal to hippocampal cells, producing recent memory deficits characteristic of Alzheimer's disease. Here we show that addition of 4 mM d-beta-hydroxybutyrate protected cultured mesencephalic neurons from MPP(+) toxicity and hippocampal neurons from Abeta(1-42) toxicity. Our previous work in heart showed that ketone bodies, normal metabolites, can correct defects in mitochondrial energy generation. The ability of ketone bodies to protect neurons in culture suggests that defects in mitochondrial energy generation contribute to the pathophysiology of both brain diseases. These findings further suggest that ketone bodies may play a therapeutic role in these most common forms of human neurodegeneration.

1-Methyl-4-phenylpyridinium↗
PubMed2000

Two novel first exons in the prolactin receptor gene are transcribed in a tissue-specific and sexual maturation-dependent manner to encode multiple 5'-truncated transcripts in the testis of the chicken.

Cloning and sequencing of the chicken prolactin receptor (PRLR) gene segment from the transmembrane domain to the box 2 motif revealed the presence of the two testis-specific first exons, TSE-1 and TSE-2, encoding the unique 5'-end sequences of the reported and newly identified multiple 5'-truncated PRLR transcripts containing only the cytoplasmic domain in the testis. TSE-1 was located downstream of the exon encoding the transmembrane domain and TSE-2 presented downstream of the exon encoding the box 1 motif. These findings indicate that the box 1-containing 5'-truncated transcripts are generated by the utilization of TSE-1 as the first exon with distinct splicing donor sites to the box 1-containing exon, and that the utilization of TSE-2 as the first exon and its splicing to the box 2-containing exon results in the generation of the box 1-lacking transcript. Three transcription initiation sites for the box 1-containing 5'-truncated transcripts and two transcription initiation sites for the box 1-lacking transcript were detected by the RNase protection assays. Reverse transcription-polymerase chain reaction analysis showed that the expression levels of all these 5'-truncated PRLR transcripts are simultaneously increased during sexual maturation, accompanying the decrease of the amount of the canonical full-length transcript for PRLR.

Animals↗
PubMed2000

Structure and tissue distribution of chicken leptin receptor (cOb-R) mRNA.

Chicken leptin receptor (cOb-R) cDNA has been cloned, sequenced and characterized. The predicted cOb-R preprotein was composed of 1148 amino acids showing approximately 60% sequence identity with the long isoform of mammalian leptin receptor, and contained a putative signal peptide, a single transmembrane domain and the conserved box 1, 2 and 3 motifs in the cytoplasmic region. High levels of cOb-R mRNA expression were observed in ovary and brain, and less abundant expression of the mRNA was detected in liver, kidney and intestine in juvenile females and sexually matured hens. The expression levels of cOb-R mRNA did not change during sexual maturation in most tissues, but the mRNA level in the intestine was higher in matured hens than in juveniles. Estrogen treatment was found to enhance the Ob-R mRNA expression in the intestine, but not in other tissues.

Amino Acid Sequence↗
PubMed2000

Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan.

Conventional chromatographic analysis showed that phospholipase A(2) (PLA(2)) isoenzymes of the venom of Trimeresurus flavoviridis (Habu snake) of Okinawa island are profoundly different in composition from those of T. flavoviridis of Amami-Oshima and Tokunoshima islands. The most striking feature was that myotoxic [Lys(49)]PLA(2) isoenzymes, called BPI and BPII, which are expressed abundantly in the venoms of Amami-Oshima and Tokunoshima T. flavoviridis, are missing from the venom of Okinawa T. flavoviridis. Northern blot analysis of Okinawa T. flavoviridis venom-gland mRNA species showed the absence of BPI and BPII mRNA species. Analysis by single-stranded conformational polymorphism-PCR of venom-gland mRNA species of T. flavoviridis from three islands, with reference to five DNA species each encoding different PLA(2) isoenzymes from Tokunoshima T. flavoviridis venom gland, also suggested that BPI and BPII mRNA species are not expressed in Okinawa T. flavoviridis venom gland. In contrast, genomic Southern blot analysis with a variety of probes showed that only the bands corresponding to the upstream and downstream regions of the genes for BPI and/or BPII can be detected in Okinawa T. flavoviridis. These results suggested that the genes for BPI and BPII in Okinawa T. flavoviridis genome had been inactivated to form pseudogenes. Differently from Amami-Oshima and Tokunoshima T. flavovirdis genomic DNAs, PCR amplification of the segments of BPI and BPII genes between the 5' moiety of second exon and the middle portion of second intron failed for Okinawa T. flavoviridis genomic DNAs. In sequence analysis of the two segments involving polymorphism between BPI and BPII genes, which are located in first exon and third exon, respectively, only one base was detected at the polymorphic positions for pseudogene in Okinawa T. flavoviridis genome. Based on these facts, it became evident for pseudogene that the upstream region of BPI gene down to the 5' moiety of second exon and the downstream region of BPII gene starting from the middle portion of second intron are in a linked form with a possible insertion. Such observations suggest that venom-gland genes for PLA(2) isoenzymes in T. flavoviridis snakes isolated for one to two million years have evolved independently. Their evolution is regional and seems, from several lines of consideration and observation, to be adaptive to the environment.

Animals↗
PubMed2000

Oxidative stress-related proteins A170 and heme oxygenase-1 are differently induced in the rat cerebellum under kainate-mediated excitotoxicity.

A170 and heme oxygenase-1 (HO-1) are characterized as oxidative stress-inducible proteins whose induction depends upon common transcription factor via antioxidant responsive element. We investigated the expression of A170 and HO-1 in the cerebellum after kainate administration. In situ hybridization showed constitutive expression of A170 and HO-1 mRNA in Purkinje cell layer; mild induction of A170 or HO-1 was detected, respectively, 8 or 24 h after kainate administration. Immunohistochemical studies also demonstrated that constitutive expression and the induction of A170 protein in Purkinje cells; the induction of HO-1 protein was detected in Bergmann glia but not in Purkinje cells. Thus, the transcription factors involved in the induction of A170 might be different from those in the induction of HO-1 under kainate-mediated excitotoxicity. The existence of cell type-specific stress response was suggested.

Animals↗
PubMed2000

Synthesis and purification of a deleted human growth hormone, hGH delta 135-146: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities.

Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity. In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system. The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro. The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay. In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH. In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations. By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH. These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.

Animals↗
PubMed2000

Three-dimensional modeling of and ligand docking to vitamin D receptor ligand binding domain.

The ligand binding domain of the human vitamin D receptor (VDR) was modeled based on the crystal structure of the retinoic acid receptor. The ligand binding pocket of our VDR model is spacious at the helix 11 site and confined at the beta-turn site. The ligand 1alpha, 25-dihydroxyvitamin D(3) was assumed to be anchored in the ligand binding pocket with its side chain heading to helix 11 (site 2) and the A-ring toward the beta-turn (site 1). Three residues forming hydrogen bonds with the functionally important 1alpha- and 25-hydroxyl groups of 1alpha,25-dihydroxyvitamin D(3) were identified and confirmed by mutational analysis: the 1alpha-hydroxyl group is forming pincer-type hydrogen bonds with S237 and R274 and the 25-hydroxyl group is interacting with H397. Docking potential for various ligands to the VDR model was examined, and the results are in good agreement with our previous three-dimensional structure-function theory.

Amino Acid Sequence↗
PubMed2000

Detection in the liquid phase applying chemiluminescence.

Several chemiluminescence-based reactions are applicable to the determination of various bio-pharmaceutically important analytes, and they can be applied for monitoring chemiluminescence emission using flow injection, liquid chromatographic and capillary electrophoretic analysis, as well as for the development of chemiluminescence-based sensors or in immunoassays. As in general the emission intensity is linearly proportional to the concentration of any of the reagents, the technique allows the analysis of different species involved in the light-producing reaction, amongst which are the chemiluminescent reagent, oxidants, inhibitors, cofactors, catalysts, some fluorophore, etc. The present overview illustrates some important applications of the last decade on this rather unfamiliar luminescence technique to detectional challenges in the liquid phase. The required instrumentation is limited as no external light source is needed. Also, the technique opens perspectives for increasing detection sensitivity in miniaturized flowing streams. On the other hand, several drawbacks still limit full application, eg dependence of the emission signal upon a number of environmental factors forcing the analyst to make a compromise between separating and measuring conditions, a lack of selectivity in specific cases, the critical detection of the signal at strictly defined periods, especially in the case of sharp emission vs time profiles, and the development of detection devices in capillary electrophoresis.

Chemistry Techniques, Analytical↗
PubMed2000

Quantification of methamphetamine, amphetamine and enantiomers by semi-micro column HPLC with fluorescence detection; applications on abusers' single hair analyses.

Achiral and chiral semi-micro column high-performance liquid chromatographic methods with fluorescence detection to determine methamphetamine and amphetamine in human hair are described. These compounds were extracted into 5% trifluoroacetic acid (TFA) in methanol, derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride and separated either on a 250 x 1.5 mm i.d. octadecyl-silane (ODS) or a 150 x 2 mm i.d. OD-RH column. Linear calibration curves extending over a wide range of concentration that covers the practical samples were obtained for amphetamine, methamphetamine and their enantiomers (r = 0.999). Resolution values for amphetamine and methamphetamine enantiomers were 3.4 and 1.1, respectively. Intra- and inter-day variations of both the methods were not larger than 8.9% expressed as relative standard deviations (n >/= 5). The limits of detection at a signal-to-noise ratio of 3 obtained by both the methods were in the range of 1.0-4.7 fmol/5 microL injection with the achiral method being more sensitive. Abusers' hair samples were analyzed by the two methods and only the S(+)-enantiomers were found in eight Japanese abusers' hair samples. The achiral method was used to study the concentrations of these compounds in single black and white hair strands of abusers.

Amphetamine↗