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K Kobayashi

Publications and source records attributed to K Kobayashi.

At least 181 records · Page 10Linked to original sources
PubMed2001

Comparative study of glycosyltransferase activities in Caco-2 cells before and after enterocytic differentiation using lectin-affinity high-performance liquid chromatography.

Human colonic adenocarcinoma Caco-2 cells differentiate into enterocytes by induction with sodium butyrate after confluence. Our previous studies have shown that there are high levels of H type 1 blood group antigen and core 2 structure present in O-glycans of the glycoproteins from these differentiated cells and these O-glycans appear to be indispensable for the process of differentiation of the cells (J. Amano and M. Oshima, 1999, J. Biol. Chem. 274, 21209-21216). Here, we have determined the glycosyltransferase activities using lectin-affinity HPLC because the method enabled easy separation and identification of mixtures of isomeric oligosaccharide structures due to the high resolution and reproducibility. The activities of beta 3-galactosyltransferase, alpha 2-fucosyltransferase, which are responsible for H type 1 antigen biosynthesis, and core 2 beta 6-N-acetylglucosaminyltransferase in differentiated Caco-2 cells were higher than those in undifferentiated cells. These results demonstrate that an increase in specific glycosyltransferase activities brought on a change of the O-glycan structures during differentiation.

Animals↗
PubMed2001

Developmental and functional analyses of CD8(+) NK1.1(+) T cells in class-I-restricted TCR transgenic mice.

Using a class-I-restricted T cell receptor (TCR) transgenic mice (Tgm), 2C (Valpha3.1/Vbeta 8.2, specific for L(d) + LSPFPFDL), the development and cytokine production of tg-TCR(+) NKT cells were analyzed. We found that CD8(+) or double negative (DN) NKT cells constituted a major population of NKT cells in the H-2(b/b) 2C Tgm (positive selecting background) or the H-2(b/d) 2C Tgm (negative selecting background), respectively. Virtually no NKT cells were generated in the H-2(k/k) 2C Tgm (neutral selecting background). CD8(+) NKT cells in the H-2(b/b) 2C Tgm expressed CD8alphabeta heterodimers, whereas those in the H-2(b/d) 2C Tgm expressed CD8alphaalpha homodimers. These findings suggest that development of a subpopulation of NKT cells is influenced by the H-2 molecules. Upon stimulation with anti-CD3 mAb, tg-TCR(+) NKT cells generated in the H-2(b/b) and H-2(b/d) backgrounds produced IFN-gamma, but not IL-4.

Animals↗
PubMed2001

A DnaJ protein, apobec-1-binding protein-2, modulates apolipoprotein B mRNA editing.

Mammalian homologues of DnaJ proteins, also known as Hsp40 proteins, are co-chaperonins that complement Hsp70 chaperone function. Using the yeast two-hybrid system, we cloned an apolipoprotein (apo) B mRNA editing complementation protein, called apobec-1-binding protein-2 (ABBP-2), and found that it is a Class II DnaJ homologue. ABBP-2 binds to apobec-1, the mammalian apoB mRNA editase, via its J domain and neighboring G/F domain. It is a ubiquitously expressed protein, and, by transfection analysis of GFP-ABBP-2, we found that the protein is located in both the nucleus and cytosol of transfected cells, with predominance in the nucleus. Down-regulation of ABBP-2 expression in cultured cells inhibits endogenous apobec-1-mediated apoB mRNA editing. Like other Hsp40 proteins, ABBP-2 binds to Hsp70 and has ATPase-stimulating activity. Apobec-1-mediated apoB mRNA editing activity of in vitro tissue extracts requires the presence of Hsp70/ABBP-2. Although exogenously added ATP is not required for editing activity, removal of the endogenous ATP present in these extracts, which disrupts ABBP-2-Hsp70 interaction, completely inhibits editing. ABBP-2 differs from previously described auxiliary proteins (ABBP-1, ACF, and GRY-RBP) in that it does not contain any RNA recognition motifs. Not only is ABBP-2 required for efficient apoB mRNA editing, this newly discovered apobec-1-binding protein may help determine the subcellular distribution and trafficking of apobec-1 via its interaction with the chaperonin Hsp70.

Adenosine Triphosphate↗
PubMed2001

Syntheses and properties of ditelluroxanes and oligochalcogenoxanes: hypervalent oligomers with Te-O apical linkages in the main chain.

The reaction of ditelluroxanes [Ar2Te-O-TeAr]2+[X]2- (2) (Ar = p-tolyl) with a telluroxide 1, a selenoxide 7, or a carboxylate 10 to produce oligochalcogenoxanes with hypervalent Te-O apical linkages in their main chain is described. The 125Te NMR chemical shifts of 2 (2a: X- = CF3SO3-, 2b: X- = CF3CO2-, 2c: X- = CH3CO2-, 2d: X- = Cl-) are shifted downfield with decreasing nucleophilicity of the counteranions. This result reflects both the cationic character and the reactivity of the Te atoms of 2. The reaction of 2a with one, two, three, or four equivalents of telluroxide 1a (Ar = p-tolyl) selectively gave a tritelluroxane 3a, tetratelluroxane 4a, pentatelluroxane 5a, and hexatelluroxane 6a, respectively. In contrast, the reaction of 2b with an excess of 1a produced only tritelluroxane 3b. An equilibrium between the oligotelluroxanes was confirmed by crossover experiments of the reactions of 2a with 4a and of 2a with 1b (Ar = Ph). The selective equilibrium formation of a selenoxaditelluroxane 8 or a bis(selenoxa)ditelluroxane 9 was achieved by the reaction of 2a with one or two equivalents of selenoxide 7, respectively. The association constant of 2a with 7 to form 8 was estimated to be Ka = (2.18+/-0.12) x 10(4) M(-1) in CD3CN at -40 degrees C. The reaction of 2a with two equivalents of carboxylates 10a-d gave a mixture of bis(carboxylate)ditelluroxanes and diaryldicarboxytelluranes 12b-d, respectively, in which the product ratio of these depended upon the electron-withdrawing ability of 10. The reaction of 3a with two equivalents of 10a-d afforded 11a-d in all cases. The present results suggest that the sigma*-n orbital interaction plays an essential role in the reactivity of ditelluroxanes and in the formation of self-assembled oligochalcogenoxanes, and that a hypervalent bond via a sigma*-n orbital interaction is viable as a new supramolecular synthon for molecular assembly.

Journal Article↗
PubMed2001

Monocyte-dependent cell death of T lymphocyte subsets in chronic hepatitis C.

A high incidence of viral persistence and progression to chronic hepatitis are characteristic features of hepatitis C virus (HCV) infection. Since T lymphocytes play a critical role in virus clearance, their abnormalities in undergoing apoptosis may cause the insufficient antiviral immune responses leading to persistent viral infection. Recent reports have suggested that monocyte-dependent cell death (MDCD) is a mechanism of T lymphocyte apoptosis in chronic viral infection. The current study was designed to monitor apoptosis of CD4+ and CD8+ T lymphocyte subsets in co-culture with CD14+ monocytes under the apoptotic stimuli in patients with chronic hepatitis C. Cell mortalities of CD4+ and CD8+ T lymphocyte subsets from patients exhibiting various degrees of chronic hepatitis C were increased to a greater extent when incubated with CD14+ monocyte subset in medium containing phorbol 12-myristate 13-acetate. The current observations demonstrate that MDCD of CD4+ and CD8+ T lymphocyte subsets may provide a cellular basis for immune dysfunction which results in insufficient viral clearance and progression of liver disease in chronic HCV infection.

Apoptosis↗
PubMed2001

Induction of SPARC by VEGF in human vascular endothelial cells.

SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.

Cells, Cultured↗
PubMed2001

Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria.

The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle.

Amino Acid Transport Systems, Acidic↗
PubMed2001

Rapid calmodulin-dependent interdomain electron transfer in neuronal nitric-oxide synthase measured by pulse radiolysis.

Electron transfer within rat neuronal nitric-oxide synthase (nNOS) was investigated by pulse radiolysis. Radiolytically generated 1-methyl-3-carbamoyl pyridinium (MCP) radical was found to react predominantly with the heme of the enzyme with a second-order rate constant for heme reduction of 3 x 10(8) m(-1) s(-1). In the calmodulin (CaM)-bound enzyme a subsequent first-order phase was observed which had a rate constant of 1.2 x 10(3) s(-1). In the absence of CaM, this phase was absent. Kinetic difference spectra for nNOS reduction indicated that the second phase consisted of heme reoxidation accompanied by formation of a neutral flavin semiquinone, suggesting that it is heme to flavin electron transfer. Experiments with the heme proximal surface mutant, K423E, had no second phase, confirming that the mutation blocks interdomain electron transfer. With the autoinhibitory loop deletion mutant, Delta40, the slow phase was observed even in the absence of CaM consistent with the role of the loop in impeding interdomain electron transfer. The rate of heme to FMN electron transfer observed in the wild-type enzyme is approximately 1000 times faster than the FMN to heme electron transfer rate predicted during catalysis from kinetic modeling, suggesting that the catalytic process is slowed by kinetic gating.

Calmodulin↗
PubMed2001

Age-dependent and tissue-specific CAG repeat instability occurs in mouse knock-in for a mutant Huntington's disease gene.

Huntington's disease (HD) is a neurodegenerative disorder characterized by the expansion of CAG repeats in exon 1 of the HD gene. To clarify the instability of expanded CAG repeats in HD patients, an HD model mouse has been generated by gene replacement with human exon 1 of the HD gene with expansion to 77 CAG repeats. Chimeric proteins composed of human mutated exon 1 and mouse huntingtin are expressed ubiquitously in brain and peripheral tissues. One or two CAG repeat expansion was found in litters from paternal transmission, whereas contraction of CAG repeat in litters was observed through maternal transmission. Elderly mice show greater CAG repeat instability than younger mice, and a unique case was observed of an expanded 97 CAG repeat mouse. Somatic CAG repeat instability is particularly pronounced in the liver, kidney, stomach, and brain but not in the cerebellum of 100-week-old mice. The same results of expanded CAG repeat instability as observed in this HD model mouse were confirmed in the human brain of HD patients. Glial fibrillary acidic protein (GFAP)-positive cells have been found to be increased in the substantia nigra (SN), globus pallidus (GP), and striatum (St) in the brains of 40-week-old affected mice, although without neuronal cell death. The CAG repeat instability and increase in GFAP-positive cells in this mouse model appear to mirror the abnormalities in HD patients. The HD model mouse may therefore have advantages for investigations of molecular mechanisms underlying instability of CAG repeats.

Age Factors↗
PubMed2001

Development and characterization of monoclonal antibodies against canine trypsin.

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28kDa by SDS-PAGE. Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.

Animals↗
PubMed2001

Ligand-switching intermediates for the CO-sensing transcriptional activator CooA measured by pulse radiolysis.

CooA is a heme-containing and CO-sensing transcriptional activator whose activity is regulated by CO. The protoheme that acts as a CO sensor in CooA shows unique properties for its coordination structure. The Cys75 axial ligand of the ferric heme is replaced by His77 upon the reduction of the heme iron and vice versa. In this work, the ligand-switching process induced by the reduction of the heme was investigated by the technique of pulse radiolysis. Hydrated electron reduced the heme iron in ferric CooA within 1 micros to form the first intermediate with the Soret peak at 440 nm, suggesting that a six-coordinate ferrous heme with a thiolate axial ligand was formed initially. The first intermediate was converted into the second intermediate with the time constant of 40 micros (k = 2.5 x 10(4) x s(-1)). In the second intermediate, the thiolate from Cys75 was thought to be protonated and/or the Fe-S bond was thought to be elongated. The second intermediate was converted into the final reduced form with the time constant of 2.9 ms (k = 3.5 x 10(2) x s(-1)) for wild-type CooA. The ligand exchange between Cys75 and His77 took place during the conversion of the second intermediate into the final reduced form.

Bacterial Proteins↗
PubMed2001

Prolonged Jun N-terminal kinase (JNK) activation and the upregulation of p53 and p21(WAF1/CIP1) preceded apoptosis in hepatocytes after partial hepatectomy and cisplatin.

Cisplatin induced apoptosis in regenerating liver after partial hepatectomy (PH). Apoptosis was determined by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was obvious at 4 h and peaked at 8 h after PH. The activity of Jun N-terminal kinase (JNK) transiently increased at 1 h after PH. However, in cisplatin-injected rats, the JNK activity increased at 30 min and the increased level was maintained up to 4 h after PH. The in vivo activation of JNK was confirmed by the increased level of the phosphorylated c-Jun protein. Western blot analysis showed that the phosphorylated c-Jun level increased at 1 h and reached more than 30-fold the control level at 2 h after PH with cisplatin. The c-jun mRNA levels also markedly increased at 1 h after PH with cisplatin. The protein level of p53 increased after 1 h on cisplatin injection, but no significant change in the mRNA level was observed. The rise in the p53 protein level was followed by the upregulation of p21(WAF1/CIP1) mRNA and protein levels. These results suggested that the enhanced and sustained JNK activation and the upregulation of p53 and p21(WAF1/CIP1) were involved in hepatocyte apoptosis induced by PH with cisplatin.

Animals↗
PubMed2001

Intramolecular electron transfer from c heme to d1 heme in bacterial cytochrome cd1 nitrite reductase occurs over the same distances at very different rates depending on the source of the enzyme.

Intramolecular electron transfer over 12 A from heme c to heme d(1) was investigated in cytochrome cd(1) nitrite reductase from Pseudomonas aeruginosa, following reduction of the c heme by pulse radiolysis. The rate constant for the transfer is relatively slow, k = 3 s(-1). The present observations contrast with a corresponding rate of electron transfer, 1.4 x 10(3) s(-1), measured for cytochrome cd(1) from Paracoccus pantotrophus, though the relative positions of the two heme groups are the same in both enzymes. The rate of intramolecular electron transfer within the enzyme from P. aeruginosa was accelerated 10(4)-fold (1.4 x 10(4) s(-1)) by the binding of cyanide to the d(1) heme. A coordination change at the d(1) heme upon its reduction is suggested to be a major factor in determining the slow rate of electron transfer in the P. aeruginosa enzyme in the absence of cyanide.

Cytochrome c Group↗
PubMed2001

Stage-specific effects of a thiazolidinedione on proliferation, differentiation and PPARgamma mRNA expression in 3T3-L1 adipocytes.

To clarify the target phase of thiazolidinediones, which are ligands for peroxisome proliferator-activated receptor (PPAR)gamma, during adipocyte differentiation, the effects of a thiazolidinedione, pioglitazone, on every stage during the course of adipocyte differentiation were investigated. Pioglitazone did not affect the cellular protein content and [3H]thymidine incorporation into preconfluent 3T3-L1 preadipocytes. Induction of differentiation of confluent 3T3-L1 preadipocytes with insulin, dexamethasone and isomethylbutylxanthine for 48 h resulted in 30% inhibition of [3H]thymidine incorporation into the cells and 354% increase in cellular protein content. Pioglitazone at 1 microM accelerated the increase in cellular protein content by 33% and the inhibition in the [3H]thymidine incorporation by 12%. Pioglitazone, when added from the start of the induction stage, dose-dependently enhanced cellular triglyceride accumulation, and both basal and insulin-stimulated glucose transporting activity producing only a slight increase in the ratio of insulin stimulation to basal glucose transporting activity. In mature adipocytes, however, pioglitazone did not enhance either of the transporting activities. PPARgamma messenger RNA (mRNA) levels estimated by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) increased during the course of adipocyte differentiation. Although pioglitazone dose-dependently up-regulated PPARgamma mRNA levels in postconfluent preadipocytes without induction, it down-regulated them in mature adipocytes. Thus, a PPARgamma agonist, pioglitazone, arrested the growth, and increased protein content and PPARgamma mRNA levels in postconfluent preadipocytes, followed by commitment and hypertrophy of 3T3-L1 cells without changing insulin sensitivity, whereas it failed to stimulate glucose transporting activities and down-regulated PPARgamma mRNA expression in mature adipocytes.

3T3 Cells↗
PubMed2001

Solar 8B and hep neutrino measurements from 1258 days of Super-Kamiokande data.

Solar neutrino measurements from 1258 days of data from the Super-Kamiokande detector are presented. The measurements are based on recoil electrons in the energy range 5.0-20.0 MeV. The measured solar neutrino flux is 2.32+/-0.03(stat)+0.08-0.07(syst)x10(6) cm(-2) x s(-1), which is 45.1+/-0.5(stat)+1.6-1.4(syst)% of that predicted by the BP2000 SSM. The day vs night flux asymmetry (Phi(n)-Phi(d))/Phi(average) is 0.033+/-0.022(stat)+0.013-0.012(syst). The recoil electron energy spectrum is consistent with no spectral distortion. For the hep neutrino flux, we set a 90% C.L. upper limit of 40x10(3) cm(-2) x s(-1), which is 4.3 times the BP2000 SSM prediction.

Journal Article↗
PubMed2001

Constraints on neutrino oscillations using 1258 days of Super-Kamiokande solar neutrino data.

We report the result of a search for neutrino oscillations using precise measurements of the recoil electron energy spectrum and zenith angle variations of the solar neutrino flux from 1258 days of neutrino-electron scattering data in Super-Kamiokande. The absence of significant zenith angle variation and spectrum distortion places strong constraints on neutrino mixing and mass difference in a flux-independent way. Using the Super-Kamiokande flux measurement in addition, two allowed regions at large mixing are found.

Journal Article↗