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H Sakai

Publications and source records attributed to H Sakai.

At least 415 records · Page 23Linked to original sources
PubMed1997

Identification of differentially expressed mRNAs during rat C6 glial cell differentiation by mRNA fingerprinting using arbitrarily primed PCR (RAP).

Differentiation of glial cells is controlled by a complex program of differential expressions of many genes. To identify differentially expressed genes that are involved in rat C6 glial cell differentiation induced by dibutyryl cyclic AMP and theophylline, mRNA fingerprinting using arbitrarily primed PCR (RAP) was used. Four cDNA fragments, that were differentially expressed during differentiation, were isolated. Sequence analysis revealed that one of them, abundantly expressed during differentiation, was homologous to a hamster calcium-dependent serine protease. Another one was highly similar to rabbit dystrobrevin and the other two clones were identical to rat triose phosphate isomerase and calnexin. The results obtained suggest that the expressions of particular genes were changed and that RAP is a useful method to identify genes which are differentially expressed during glial cell differentiation.

Animals↗
PubMed1997

Identification of a second transcriptional start site for the insecticidal protein gene cryIVA of Bacillus thuringiensis subsp. israelensis.

Expression of cryIVA, one of the insecticidal protein genes of B. thuringiensis subsp. israelensis, is regulated at the transcriptional level. The cryIVA gene is specifically transcribed during the stationary phase of this bacterium. As shown in our previous report [Yoshisue et al. (1993a)], the transcription from the -364 position of the cryIVA gene is conducted by the major promoter P1 that is functional during middle stages of the stationary phase of B. thuringiensis. In the present study, we have identified a second transcriptional start point P2 for the cryIVA gene in addition to P1, the major transcriptional start point. The transcription from P2 of the cryIVA gene occurred later than that from P1, during later stages of stationary phase of B. thuringiensis subsp. israelensis. The -10 and -35 nt sequences upstream from P2 of cryIVA are similar to those of the omega 28-specific promoters of B. thuringiensis genes and of the omega K-specific promoters of B. subtilis genes. It is most likely that the region upstream from P2 of cryIVA contains the nt sequences that determine the omega 28-specific promoter, the second one, for the cryIVA gene.

Amino Acid Sequence↗
PubMed1997

Na+ and Ca2+ channel expression in cultured newt retinal pigment epithelial cells: comparison with neuronal types of ion channels.

We cultured retinal pigment epithelial (RPE) cells dissociated from adult newt eye and analyzed their voltage-gated ion channels during culture using whole-cell patch-clamp techniques. The results were compared with those of retinal neurons under identical experimental conditions. After 6-9 days in culture (early stage), > 60% of RPE cells developed voltage-gated Na+ and Ca2+ channels that were not observed in freshly dissociated RPE cells. The number of cells expressing Na+ channels and Na+ current density were high after 12-15 days in culture (intermediate stage), while the number of Ca2+ channel-expressing cells and Ca2+ current density were high after 20-30 days in culture (late stage). The activation voltage of the Na+ current in the RPE cells was similar to that in neurons. The voltage dependence of Na+ current inactivation was somewhat different between two cell types. The steepness of the inactivation curve tended to be less in cultured RPE cells than in neurons, and the half-inactivation voltage was about-54 mV for the RPE cells and -45 mV for neurons. The Ca2+ current expressed in cultured RPE cells was too small to detect without replacement of external Ca2+ with Ba2+. The Ba2+ current, like Ca2+ current in neurons, was enhanced by Bay-K 8644 and blocked by nicardipine. These results suggest that the RPE cells, like neurons, expressed L-type Ca2+ channels in culture. The possibility that the development of both Na2+ and Ca2+ channels in cultured RPE cells is a manifestation of the transdifferentiation of RPE cells into neurons is discussed.

Animals↗
PubMed1997

Effects of retinoids on the production of tumour necrosis factor-alpha and nitric oxide by lipopolysaccharide-stimulated rat Kupffer cells in vitro: evidence for participation of retinoid X receptor signalling pathway.

Kupffer cells play important roles in the development of liver injury by producing cytokines and free radicals. In consequence inhibition of these inflammatory mediators will be one of the targets for treating liver diseases. Retinoids modulate a wide variety of functions of monocytes/macrophages. Cellular effects of retinoids are mediated by two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We examined the effects of several kinds of natural and synthetic retinoids on the production of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) by LPS-stimulated rat Kupffer cells in vitro. Of the various retinoids tested, 9-cis-retinoic acid (9-cis-RA) and Ro 13-6307 which are agonists of both RARs and RXRs, suppressed the production of TNF-alpha and NO in a concentration-dependent fashion, whereas three types of RAR-selective agonists, Ro 13-7410, Ro 40-6055 and Ro 19-0645 did not show any effect. Furthermore, the RAR alpha antagonist, Ro 41-5253, did not prevent the effects induced by 9-cis-RA. The results suggest that these effects of 9-cis RA and Ro 13-6307 were induced by the RXRs-dependent signalling pathway.

Animals↗
PubMed1997

Hepatocytic phenotypes induced in sarcomatous cholangiocarcinoma cells treated with 5-azacytidine.

The sarcomatoid cells found in cholangiocarcinoma (CC) or hepatocellular carcinoma (HCC) are not well characterized. In this study, a human sarcomatoid CC cell line, ETK-1, was established from a patient, and then morphological and phenotypical characteristics of the ETK-1 cells were evaluated before and after treatment with differentiation-inducing 5-azacytidine (5-azaCR). Phenotypically, the ETK-1 cells appeared immature. Exposure to 5-azaCR induced morphological transformation; a converted cell line, MEK, was successfully established. The MEK cells expressed such hepatocyte-specific proteins as alpha-fetoprotein, albumin, integrin alpha1, and thrombopoietin, but lost such bile duct-specific proteins as integrin alpha3 and integrin beta4. The histopathology of MEK xenografts resembled that of HCC. The ETK-1 cells appeared to be converted into hepatocytes by exposure to 5-azaCR. On the other hand, ETK-1 xenografts were diagnosed as tubular adenocarcinoma, and the tumor cells had a ductal phenotype. This suggests the possibility that ETK-1 cells can differentiate along a biliary epithelial cell lineage. ETK-1 and MEK will be useful in studying hepatocytic differentiation and the transformation from a biliary epithelial cell to a hepatocytic lineage.

Animals↗
PubMed1997

The significance of peritoneal cytology in uterine cervix and endometrial cancer.

OBJECTIVE: The purpose of this study was to determine the incidence of positive peritoneal cytology and to elucidate the prognostic value of peritoneal cytology in patients with uterine cervix and endometrial cancer. MATERIALS AND METHODS: The incidence of positive peritoneal cytology was investigated in 642 patients including 339 uterine cervix and 303 endometrial cancers. Survival was estimated by the Kaplan-Meier method in a subgroup of 116 stage II cervix and 199 stage I endometrial cancers, and multivariate analysis using Cox's proportional hazards model was used to identify an independent prognostic factor. RESULTS: The incidence of positive peritoneal cytology was found to be 9% in uterine cervix cancer and 15% in endometrial cancer. The incidence was higher in patients with some clinicopathologic status such as advanced stage, lymph node metastasis, ovarian metastasis, and deeper myometrial invasion. The 5-year survival rate for patients with positive or negative peritoneal cytology was 44 or 80% in stage II cervix cancers and 80 or 92% in clinical stage I endometrial cancers, respectively. Multivariate analysis revealed that independent prognostic determinants were pelvic and paraaortic lymph node metastasis and peritoneal cytology in stage II cervix cancer and peritoneal cytology in stage I endometrial cancer. Proper treatment protocol should be scheduled for patients with positive peritoneal cytology.

Endometrial Neoplasms↗
PubMed1997

Messenger RNA fingerprinting analysis using arbitrarily primed PCR (RAP) of genes expressed during rat C6 glioma cell differentiation.

To identify differentially expressed genes involved in rat C6 glioma cell differentiation induced by cyclic AMP, we adopted mRNA fingerprinting using arbitrarily primed polymerase chain reaction (PCR) (RAP). Four complementary DNA (cDNA) fragments differentially expressed during differentiation were isolated, and they appeared to contain coding regions of corresponding mRNAs. RAP can be used to efficiently identify cDNA fragments by comparing nucleotide and deduced amino acid sequences with those in databases, and is thus a useful method to search for and identify important genes involved in complex cellular processes such as glioma cell differentiation.

Animals↗
PubMed1997

Inhibition of interleukin-2 receptor (CD25) expression induced on T cells from children with acute lymphoblastic leukemia.

Peripheral blood lymphocytes obtained from children with acute lymphoblastic leukemia (ALL) at onset were studied for the expression of interleukin-2 (IL-2) receptor alpha-chain (CD25) by two-color flow-cytometric analysis. Stimulated with anti-CD3 monoclonal antibody (mAb) alone. CD25 expression was significantly suppressed in CD4+ T cells from 27 of 48 (56.3%) cases and in CD8+ T cells from 29 of 48 (60.4%) cases. When stimulated with anti-CD3 mAb plus phorbol 12-myristate 13-acetate (PMA), CD25 expression was clearly restored in certain cases of ALL. When PMA plus ionomycin were used for stimulation of T cells. CD25 was inducible in a majority of cases. Interestingly CD25 expression upon anti-CD3 mAb stimulation was recovered after complete remission had been achieved. These observations suggest the presence in ALL children at onset of an in vitro defect in the signal transduction pathway of the T-cell-receptor/CD3 complex, resulting in inefficient CD25 expression. However, immune-staining analysis indicated that protein kinase C was normally translocated from the cytosol fraction to the cell membrane fraction. The mobilization of cytoplasmic free calcium is also normal.

Antibodies, Monoclonal↗
PubMed1997

cAMP-mediated c-fos expression in pressure-overloaded acceleration of protein synthesis in adult rat heart.

OBJECTIVES: The aim was to determine whether proto-oncogene c-fos expression and acceleration of protein synthesis by acute pressure overload to the heart were coupled with a cAMP- and protein-kinase-A-dependent system in adult rat heart. METHODS: Isolated adult rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. In the pressure-overloaded group, aortic pressure was raised from 60 to 120 mmHg for the time indicated. Agents that increase cAMP were added to the perfusate at an aortic pressure of 60 mmHg. Furthermore, a selective protein kinase A inhibitor (H-89) or a selective protein kinase C inhibitor (calphostin C) was administered before the elevation of aortic pressure or the addition of the agents. cAMP content or rates of protein synthesis were measured by RIA or the incorporation of [14C]phenylalanine into total heart protein, respectively. c-fos mRNA expression was determined by Northern blot analysis. RESULTS: Elevation of aortic pressure in beating hearts and arrested hearts increased cAMP content at 2 min of perfusion by 36 and 41%, induced c-fos mRNA expression at 30-60 min of perfusion by 4.8- and 2.0-fold, and accelerated rates of protein synthesis during the 2nd hour of perfusion by 39 and 41% over control levels, respectively. Glucagon, forskolin or IBMX mimicked increases in these parameters by elevated aortic pressure. H-89 prevented these changes by elevated pressure overload or exposure to forskolin or IBMX in arrested hearts. On the other hand, calphostin C prevented the pressure-induced increases in c-fos expression and protein synthesis rates in arrested hearts. CONCLUSIONS: These results suggest that c-fos expression induced by acute pressure overload may be coupled with increased cAMP content and protein kinase A activity in addition to increased protein kinase C activity in adult rat heart.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Cholestyramine loading test to assess hepatic reserve for bile acid synthesis in patients with chronic liver diseases.

BACKGROUND & AIMS: Serum 7alpha-hydroxycholesterol level reflects hepatic bile acid synthesis in humans. The aim of this study was to examine the maximal bile acid synthesis in patients with chronic liver diseases. METHODS: Cholestyramine (12 g/day) was administered for 3 days to patients with compensated liver cirrhosis (n = 7), patients with chronic hepatitis (n = 10), and control subjects (n = 9), and serum total 7alpha-hydroxycholesterol level was measured by gas-liquid chromatography-mass spectrometry. RESULTS: A preliminary study showed that cholestyramine increased the serum value to the maximum by the third day. Before cholestyramine treatment, there were no significant differences in serum 7alpha-hydroxycholesterol levels among the groups. Three days of cholestyramine treatment increased the serum levels 5.71 +/- 2.90-fold in the controls, 3.25 +/- 0.85-fold in patients with chronic hepatitis, and 1.70 +/- 0.78-fold in cirrhotic patients. Cirrhotic patients had significantly lower serum 7alpha-hydroxycholesterol levels after the treatment compared with other groups. Serum 7alpha-hydroxycholesterol levels stimulated by cholestyramine significantly correlated with serum albumin levels and indocyanine green retention rate. CONCLUSIONS: Three days of cholestyramine loading increased the bile acid synthesis to its maximal level. The cholestyramine test showed that patients with chronic hepatitis had enough hepatic reserve for bile acid synthesis; however, patients with compensated liver cirrhosis had significantly decreased capacity.

Aged↗
PubMed1997

Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay.

A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotinylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67,700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, showing a sensitivity of 100% and a specificity of 96%.

Animals↗
PubMed1997

Differential expression of Rho family GTP-binding proteins and protein kinase C isozymes during C6 glial cell differentiation.

The differential expression of Rho family of low molecular weight GTP-binding proteins and protein kinase C (PKC) isozymes were examined during differentiation of rat C6 glial cells to astrocytic phenotypes induced by dibutyryl cAMP (dbcAMP)/theophylline. The cells showed rapid and distinct morphological changes, resembling stellate astrocytes at 12 h after the treatment. The treated cells had a round cell body that extended several long processes each with a beaded appearance. In addition to morphological changes, Western blot analysis revealed that S-100 protein, known as a glial cell differentiation marker, increased and reached the maximal level (approximately 6-fold increase) at 24 h following the addition of dbcAMP. In the control experiments with cells cultured in the absence of serum but also without dbcAMP/theophylline, morphological changes were marginal and apparent increases of S-100 protein were not observed by Western blotting. In response to dbcAMP/theophylline treatment, RhoA showed increases in the mRNA level followed by the protein level, as inferred by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Rac1 and Cdc42 proteins were undetectable by Western blot analyses. In PKC isozymes, increases were observed in PKC beta 1, epsilon, and zeta by RT-PCR, and in beta 1 and epsilon by Western blotting. Among them, PKC epsilon showed the most distinct changes. Its mRNA level transiently increased from 3 to 6 h and then decreased even below the basal level at 18 h after the treatment. In contrast, Western blot analysis revealed that PKC epsilon gradually increased time-dependently to 24 h (approximately 6-fold increase), and remained elevated until 48 h. These results suggested that RhoA and PKC epsilon, and probably also PKC beta 1 and PKC zeta, were closely implicated in C6 cell differentiation.

Animals↗
PubMed1997

Glomerular Fc alphaR expression and disease activity in IgA nephropathy.

In this study, we examined the receptors for the Fc portion of immunoglobulin A (IgA) (Fc alphaR) in the glomeruli as well as circulating polymorphonuclear leukocytes and monocytes at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) assay and at the protein level by an immunohistochemistry/flow cytometry technique using a specific anti-Fc alphaR monoclonal antibody (My 43). Glomeruli were isolated from biopsy specimens of renal tissues from IgA nephropathy (IgAN; 20 cases) and non-IgA mesangial proliferative glomerulonephritis (PGN; 13 cases) patients, and from normal renal tissue specimens obtained from kidneys removed because of malignancies (five cases) applying the microdissection method. There was a relative increase in Fc alphaR in the circulating phagocytes from IgAN patients compared with those from PGN and healthy controls. Fc alphaR expression was present in approximately 40% of glomeruli samples from IgAN patients at the message levels. Fc alphaR-positive specimens were also strongly positive for expression of tumor necrosis factor-alpha, interleukin-1, and interleukin-6 mRNA. Specimens from PGN patients and healthy controls did not show any detectable Fc alphaR message. Serum IgA levels and severity of hematuria were significantly higher in patients with positive Fc alphaR expression. A message for Fc alphaR was detected in the tissues that were more damaged histologically. Our data suggest that there is some in vivo induction of glomerular Fc alphaR expression, possibly mediated by a synergistic stimulus from IgA and inflammatory cytokines, and the expressed receptor is likely to be involved in the disease process of IgAN.

Antigens, CD↗
PubMed1997

Clinical trial of external beam-radiotherapy combined with daily administration of low-dose cisplatin for supratentorial glioblastoma multiforme--a pilot study.

This trial tested the assumed efficacy and safety of external beam-radiotherapy combined with daily administration of low dose cisplatin (CDDP) (ERCLC therapy) for patients with glioblastoma multiforme (GBM). Thirty adult patients with supratentorial GBM received daily postoperative treatment with low dose intravenous CDDP (4-6 mg/m2) administered 30 minutes before external irradiation. In 10 patients, intraoperative radiotherapy (IORT) following surgery was given prior to ERCLC therapy. Tumor response on MRI, interval to tumor progression, survival, and toxicities were analyzed. None of the patients showed a tumor response to ERCIC therapy. Overall, the median time to tumor progression was 6 months with a 1-year tumor progression-free rate of 26.7% and a 2-year rate of 0%. The median survival time was 15 months with a 1-year survival rate of 69.9% and a 2-year rate of 31.5%. The survival rate of patients with IORT was better than that of those without IORT, however, there was no significant difference. Anorexia associated with nausea occurred in 70% and general fatigue in 10.0%. Leukopenia and thrombocytopenia occurred in 26.7% and 33.3%, respectively. However, none of the patients had to be withdrawn from therapy due to these toxicities. Other toxicities were not observed. This clinical study showed that daily administration of low dose CDDP did not enhance tumor response to irradiation for GBM on MRI. Regarding toxicity, however, ERCLC therapy was well tolerated. Although this trial did not provide sufficient data to determine whether ERCLC therapy was effective for GBM due to the small number of patients, additional clinical trials of this therapy may be warranted because that the survival rate in this study was equal to the better results recently reported for newly diagnosed GBM.

Adult↗
PubMed1997

Autoimmune pancreatitis as a new clinical entity. Three cases of autoimmune pancreatitis with effective steroid therapy.

The most common forms of chronic pancreatitis are related to alcohol ingestion, whereas the entity of non-alcohol-associated (idiopathic) pancreatitis is poorly understood. Autoimmunity has been suggested as a possible etiologic factor of idiopathic chronic pancreatitis. A total of 362 Japanese patients underwent endoscopic retrograde pancreatography (ERP) for suspected pancreatic disease, and 161 were diagnosed with chronic pancreatitis. Among them, we found three cases (1.86% incidence) of unique chronic pancreatitis, in which ERP revealed diffuse narrowing of the main pancreatic duct with an irregular wall. We diagnosed these three patients as having pancreatitis associated with an autoimmune mechanism morphologically and biochemically and started them on steroid therapy. The characteristics of the these three patients were as follows: hypergammaglobulinemia, eosinophilia, ultrasonography showing hypoehoic diffuse swelling in the pancreas (sausage-like appearance), ERP showing diffuse narrowing of the main pancreatic duct with irregular like thumbprint-like marks, reversible exocrine insufficiency, and positive anti-carbonic anhydrase II antibody. After one month of the treatment with steroids, pancreatitis dramatically improved morphologically and enzymatically. Here we describe these cases of the suspected autoimmune chronic pancreatitis. We must recognize the concept and the features of autoimmune pancreatitis in order to avoid unnecessary surgery as pancreatic cancer.

Aged↗
PubMed1997

Phenotypic characterization of cytokine expression in patients with IgA nephropathy.

To identify the cytokines that play a relevant role in the pathogenesis of IgA nephropathy, we analyzed and compared the gene expression of proinflammatory cytokines, immuno-regulatory cytokines, and growth factors in peripheral blood mononuclear cells (PBMC). Expression of IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-gamma, TGF-beta, TNF-alpha, and PDGF was examined in 28 patients with IgA nephropathy (IgAN), 20 patients with non-IgA mesangial proliferative glomerulonephritis (mesPGN), and 19 healthy controls. Compared with healthy controls, a significant number of IgAN and mesPGN patients showed increased expression of IL-1 beta, IL-4, IL-10, IL-12, and IFN-gamma. The cytokine profile of renal tissue of 10 IgAN and 5 mesPGN biopsies was simultaneously analyzed and compared with that of PBMC. The proinflammatory IL-1 alpha and growth factor PDGF-B were expressed more in renal tissues than in PBMC. Furthermore, the renal profile of IL-alpha, IFN-gamma, and TNF-alpha expression was associated with the expression of IFN-gamma in PBMC. The serum level of IFN-gamma of IgAN correlated significantly (P = 0.0003) with that of IL-12, suggesting a potential role for cross-stimulation. More importantly, expression of IFN-gamma in PBMC and the elevated serum level correlated with the decline in glomerular filtration rate (P = 0.0012) and severity of renal histopathologic grade. To elucidate the role of leukocytes in renal cytokine expression, surface markers of T cells (CD3), monocytes (CD14), natural killer cells (CD16), and B cells (CD19) were also examined in renal tissues. The prominent renal expression of CD3, CD14, and CD16 implicates the leukocytes as the major source of proinflammatory cytokines in IgAN. Collectively, these findings indicate that IFN-gamma plays a prominent role in an interactive network of cytokines that contribute to the pathogenesis and progression of IgA nephropathy.

Adult↗