Biomedical subjects

H Teraoka

Publications and source records attributed to H Teraoka.

At least 55 records · Page 3Linked to original sources

PubMed1996

CPP32/Yama/apopain cleaves the catalytic component of DNA-dependent protein kinase in the holoenzyme.

DNA-dependent protein kinase (DNA-PK) is composed of a 460-kDa catalytic component (p460) and a DNA-binding component Ku protein. Immunoblot analysis after treatment of Jurkat cells with anti-Fas antibody demonstrated the cleavage of p460 concomitantly with an increase in CPP32/Yama/apopain activity. Recombinant CPP32/Yama/apopain specifically cleaved p460 in the DNA-PK preparation that had been purified from Raji cells into 230- and 160-kDa polypeptides, the latter of which was detected in anti-Fas-treated Jurkat cells. The regulatory component Ku protein was not significantly affected by CPP32/Yama/apopain. DNA-PK activity was decreased with the disappearance of p460 in the incubation of DNA-PK with CPP32/Yama/apopain. These results suggest that the catalytic component of DNA-PK is one of the target proteins for CPP32/Yama/apopain in Fas-mediated apoptosis.

Amino Acid Sequence↗

PubMed1996

Characterization of DNA ligase from the fungus Coprinus cinereus.

DNA ligase was highly purified from the fungus Coprinus cinereus at the miotic recombination stage, pachytene. The pachytene DNA ligase showed three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimated by the [32P]AMP-labeling assay. These three polypeptides were susceptible to reaction with an mAb against a 16-amino-acid sequence in human DNA ligase I, which is conserved in C-terminal regions of mammalian, vaccinia virus and yeast DNA ligases. Since rapidly purified preparations from fresh pachytene cells exhibited a single polypeptide of DNA ligase with a molecular mass of 88 kDa, the smaller polypeptides seemed to be limited-degradation products of the 88-kDa polypeptide during the isolation and purification procedures. K(m) values for ATP and (dT)20 hybridized with (dA)n were 1.5 microM and 90 nM, respectively. This enzyme was capable of joining (dT)20.(rA)n and (rA)12-18 (dT)n as well as (dT)20.(dA)n and able to ligate blunt-ended DNA in the presence of poly(ethylene glycol) 6000. DNA ligases were also partially purified from zygotene cells at the meiotic pairing stage and mitotic mycelium cells. In their molecular mass, immuno-reactivity, K(m) value and substrate specificity, they were indistinguishable from pachytene DNA ligase. These results suggest that the fungus C. cinereus at the pachytene stage contains DNA ligase with a molecular mass of 88 kDa as a main or a single species, which is quite similar to DNA ligases from the zygotene and mycelium cells in molecular and catalytic properties.

Amino Acid Sequence↗

PubMed1996

DNA-dependent protein kinase inhibitor (OK-1035) suppresses p21 expression in HCT116 cells containing wild-type p53 induced by adriamycin.

The tumor growth suppressor p21 has been shown to be induced by wild-type p53 (wt-p53) and to be a potent inhibitor of cyclin-dependent kinases and PCNA/DNA polymerase delta. Although wt-p53 is reported to be phosphorylated by several protein kinases, the function and significance of the phosphorylation of wt-p53 are not yet fully understood. Using OK-1035, a selective inhibitor of DNA-dependent protein kinase (DNA-PK), we demonstrated the importance of the phosphorylation of wt-p53 by DNA-PK in the DNA damage-mediated expression of the p21 gene. Treatment of HCT116, a human colon carcinoma cell line, with adriamycin induced the expression of wt-p53 and p21. By addition of OK-1035 to this culture, the induction of p21 protein was significantly decreased in a dose-dependent manner, whereas wt-p53 induction was not affected. Northern blot analysis revealed that suppression of p21 protein expression by OK-1035 resulted from reduction in the level of p21 mRNA. OK-1035 did not directly affect the binding ability of wt-p53 to its consensus DNA sequence. Our observations support the idea that wt-p53 induces the transcriptional activation of the p21 gene only after it is phosphorylated by DNA-PK.

Amino Acid Sequence↗

PubMed1996

Autoantibodies to DNA-dependent protein kinase. Probes for the catalytic subunit.

DNA-dependent protein kinase (DNA-PK) is an important nuclear enzyme which consists of a catalytic subunit known as DNA-PKcs and a regulatory component identified as the Ku autoantigen. In the present study, we surveyed 312 patients in a search for this specificity. 10 sera immunoprecipitated a large polypeptide which exactly comigrated with DNA-PKcs in SDS-PAGE. Immunoblot analysis demonstrated that this polypeptide was recognizable by a rabbit antiserum specific for DNA-PKcs. Although the patient sera did not bind to biochemically purified DNA-PKcs in immunoblots or ELISA, they were able to deplete DNA-PK catalytic activity from extracts of HeLa cells in a dose-dependent manner. We conclude that these antibodies should be useful probes for studies which aim to define the role of DNA-PK in cells. Since six sera simultaneously contained antibodies to the Ku protein, these studies suggest that relatively intact forms of DNA-PK complex act as autoantigenic particles in selected patients.

Antigens, Nuclear↗

PubMed1996

A stable Escherichia coli-mycobacteria shuttle vector 'pSO246' in Mycobacterium bovis BCG.

The most widely used plasmid vector system in mycobacteria is based on pAL5000 from Mycobacterium fortuitum. The derivatives of the pAL5000-based shuttle vectors between Escherichia coli and mycobacteria, which we have utilized to secrete recombinant antigens, were generated. The stability of the vectors was assessed in Mycobacterium bovis BCG (BCG). The plasmid vector pSO246 was stable in BCG for at least 50 generations.

Bacterial Proteins↗

PubMed1996

Reversible G1 arrest induced by dimethyl sulfoxide in human lymphoid cell lines: dimethyl sulfoxide inhibits IL-6-induced differentiation of SKW6-CL4 into IgM-secreting plasma cells.

We have previously found that dimethyl sulfoxide (DMSO), a known inducer of differentiation in several kinds of myeloid cells, arrests proliferation of human lymphoid cells including Raji and Akata Burkitt's lymphoma cells at the G1 phase. We investigated whether DMSO affects cell proliferation and differentiation of the lymphoid cell line SKW6-CL4, which is capable of differentiating terminally into IgM-producing cells. As in the case of Raji, Akata, and Molt-4, the proliferation of SKW6-CL4 was reversibly arrested at the G1 phase by treatment with 2% DMSO for 5 days even in the presence of interleukin-6 (IL-6). DMSO inhibited spontaneous IgM secretion as well as IL-6-induced IgM production in SKW6-CL4 at a concentration lower than that affecting cell proliferation. Of the cell-surface differentiation markers CD10, CD20, CD21, and CD23, the expression of CD20 was suppressed by DMSO treatment, and partial restoration of the expression was observed 24 to 48 h after release from DMSO. The level of IL-6 receptor protein was not affected by DMSO treatment. These results indicate that DMSO not only arrests the cell cycle of a human lymphoid cell line SKW6-CL4 at the G1 phase but also inhibits the differentiation into IgM-secreting cells at a concentration lower than that affecting cell proliferation and that DMSO overcomes the effect of IL-6 on terminal differentiation of SKW6-CL4. As a whole, proliferation of human lymphoblastoid cell lines was revealed to be reversibly arrested at the G1 phase by DMSO, which is known to induce differentiation in several myeloid cells, without inducing cell differentiation.

Antigens, CD↗

PubMed1996

Inositol 1,4,5-trisphosphate- and caffeine-sensitive Ca(2+)-storing organelle in bovine adrenal chromaffin cells.

The uptake and release properties of Ca2+ by several subcellular fractions of the bovine adrenal medulla were investigated. Investigation by the 45Ca2+ tracer method showed that permeabilized cells and the fractions of mitochondria (MT) and microsomes (MC) caused ATP-dependent Ca2+ uptake in a Ca2+ concentration-dependent manner (pCa 8-4), whereas permeabilized cells and the fractions of secretory granules (SG) were able to accumulate a significant amount of Ca2+ even in the absence of ATP, which was completed by the addition of hexokinase and glucose. In these organelle fractions, Ca2+ uptake in the presence of ATP at pCa 7 and pCa 5.8 was well-correlated with the activity of the NADPH cytochrome c reductase (marker enzyme for the endoplasmic reticulum) and cytochrome c oxidase (marker enzyme for mitochondria), respectively. As detected by Fura-2 ratiometry, both inositol 1,4,5-trisphosphate (IP3) and caffeine caused concentration-dependent Ca2+ releases from permeabilized cells and MC, but not from MT and SG. In an ATP-depleted condition, homogenates still took up a significant amount of Ca2+ but was not able to respond to IP3 and caffeine. These results suggest that the endoplasmic reticulum is a major Ca(2+)-storing organelle, which releases Ca2+ in response to IP3 and caffeine in bovine adrenal chromaffin cells.

Adrenal Glands↗

PubMed1996

Nitrite/nitrate (NOX) and type II phospholipase A2, leukotriene B4, and platelet-activating factor levels in patients with septic shock.

We measured serum concentrations of nitrite/nitrate (NOX), type II phospholipase A2 (PLA2), leukotriene B4 (LTB4), and platelet-activating factor (PAF) in patients with sepsis. These findings were compared between patients with and without septic shock. Serum concentrations of NOX, type II PLA2, LTB4, and PAF acetylhydrolase (PAF-AH) were significantly higher in the group with septic shock (P < 0.0001; P = 0.0060; P = 0.0052; P = 0.0052), indicating the severity of the disease. There were significant correlations between the serum NOX level and serum levels of type II PLA2, LTB4, and PAF-AH (r = 0.6890, P < 0.0001; r = 0.3755, P = 0.0409; r = 0.5095, P = 0.0040, respectively). It is speculated that LTB4 and PAF, both produced with type II PLA2, interact with each other and are involved in the deterioration of pathologic features associated with sepsis. Furthermore, nitric oxide (NO) and eicosanoids interact to play an important role in vascular dilatation during septic shock.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1995

OK-1035, a selective inhibitor of DNA-dependent protein kinase.

Screening for inhibitors of DNA-dependent protein kinase (DNA-PK) revealed 3-cyano-5-(4-pyridyl)-6-hydrazonomethyl-2-pyridone, designated OK-1035, to be a potent and selective inhibitor. When a synthetic peptide was used as a substrate, OK-1035 caused 50% inhibition of DNA-PK activity at 8 microM, a concentration more than 50 times lower than those required against seven other protein kinases tested. OK-1035 inhibited the phosphorylation by DNA-PK of consensus peptide as well as that of recombinant human wild type-p53. Kinetic studies indicated that OK-1035 inhibited DNA-PK activity in an ATP-competitive manner.

Amino Acid Sequence↗

PubMed1995

The human pancreatitis-associated protein (PAP)-encoding gene generates multiple transcripts through alternative use of 5' exons.

The nucleotide (nt) sequence of the human cDNA encoding PAP, a pancreatic secretory protein induced during acute pancreatitis, was found to be identical with that of a gene activated in human primary hepatocellular cancer, designated HIP. To obtain insight into the expression of PAP/HIP, we characterized the gene organization, especially focusing on the 5'-flanking region, and found that it spans about 3 kb and is composed of six exon. Exon 1 encodes the 5'-noncoding sequence and exon 2 consists of three miniexons, 2a, 2b and 2c; the common exon 2c encodes the sequence including the start codon. Analysis by RT-PCR revealed the presence of at least three different types 5'-ends of human PAP/HIP transcripts which were derived from alternative use of 5'-exons. Although all three types of transcripts were expressed in both normal small intestine and pancreas, their gene expression was increased ectopically in gastric cancer, hepatocellular cancer and pancreatic acinar cell carcinoma. Furthermore, significant differences among the transcript types were detected between normal and tumor tissues, and especially between gastric and hepatocellular cancers, suggesting that PAP/HIP expression may vary with differences in 5'-alternative splicing.

Alternative Splicing↗

PubMed1995

Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells.

The biological action of adrenomedullin, a novel hypotensive peptide, on bovine aortic endothelial cells, was examined. The specific binding of adrenomedullin to these cells was observed, and adrenomedullin was found to induce intracellular cAMP accumulation in a dose-dependent manner. EC50 for the cAMP accumulation was about 100 times lower than the apparent IC50 for the binding assay. Adrenomedullin also induced increase of intracellular free Ca2+ in endothelial cells in a dose-dependent manner. The Ca2+ response to adrenomedullin was biphasic with an initial transient increase due to the release from thapsigargin-sensitive intracellular Ca2+ storage and a prolonged increase by influx through the ion channel on the plasma membrane. This intracellular free Ca2+ increase resulted from phospholipase C activation and inositol 1,4,5-trisphosphate formation, and seemed to cause nitric oxide synthase activation by monitoring intracellular cGMP accumulation. Both cAMP accumulation and Ca2+ increased responses to adrenomedullin were mediated by cholera toxin-sensitive G protein, but the two signal transduction pathways were independent. Thus, the results suggest that adrenomedullin elicits the hypotensive effect through at least two mechanisms, a direct action on vascular smooth muscle cells to increase intracellular cAMP and an action on endothelial cells to stimulate nitric oxide release, with both leading to vascular relaxation.

Adrenomedullin↗

PubMed1995

An improved method for large-scale purification of recombinant human glucagon.

Glucagon was expressed in Escherichia coli as a fusion protein including the glucagon sequence [Ishizaki et al. (1992), Appl. Microbiol. Biotechnol. 36, 483-486]. The high-level expression of a protein in E. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawa et al. (1992), J. Protein Chem. 11, 517-525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon.

Amino Acid Sequence↗

PubMed1995

Changes in gene expression of pancreatitis-associated protein and pancreatic secretory trypsin inhibitors in experimental pancreatitis produced by pancreatic duct occlusion in rats: comparison with gene expression of cholecystokinin and secretin.

Pancreatic duct occlusion is known to produce a sustained increase in the plasma cholecystokinin (CCK) concentration and to affect the tissue content of CCK in the rat. The tissue content of CCK is correlated with regenerative changes in the pancreas after pancreatic duct occlusion. In the present study, we examined the changes in mRNA levels of pancreatic secretory trypsin inhibitors (PSTIs), pancreatitis-associated protein (PAP), and amylase in the pancreas in comparison with changes in CCK and secretin mRNA levels in the intestine and the histological changes produced by pancreatic duct ligation. Rats with an internal bile fistula and with obstruction of pancreatic flow were prepared and were sacrificed 1, 3, 7, 10, 14, and 28 days later. Then mRNA levels of CCK, secretin, PSTIs, PAP, and amylase were determined by slot-blot analysis. The CCK mRNA level gradually increased to a peak on day 10, was slightly lower on day 14, and returned to the control level on day 28. The level of secretin mRNA did not change. The mRNA levels of PSTIs increased significantly on day 3 after occlusion. PAP mRNA was detectable on days 1 and 3, being maximal on day 1. The mRNA level of amylase was markedly decreased on days 1 and 3, then remained lower than the control level. Histological examination showed acute inflammatory changes in the pancreas on days 1 and 3 and regenerative changes from day 7. These results suggest that a change in gene expression of PAP reflects acute inflammatory changes in the pancreas most sensitively.

Acute-Phase Proteins↗

PubMed1995

Regulation of gene expression of pancreatic secretory trypsin inhibitor-61 and -56 by bile and pancreatic juice in rats.

The rat possesses two pancreatic secretory trypsin inhibitors (PSTI-61 and -56). PSTI-61 has been known to stimulate cholecystokinin (CCK) release, whereas PSTI-56 did not. Both PSTIs are synthesized in the pancreatic acinar cells. CCK has a trophic effect on pancreatic acinar cells, and the exclusion of bile-pancreatic juice from the intestine has been known to be a most potent stimulator of CCK release. In the present study, we examined whether the mRNA levels of PSTI-61 and -56 produced by bile-pancreatic juice diversion were different from each other and compared the changes in CCK mRNA levels in the small intestinal mucosa and the plasma and intestinal CCK concentrations. Male Wistar rats were prepared with internal fistula and bile-pancreatic juice was excluded from the proximal intestine, being introduced into the distal ileum. Rats were sacrificed 1, 3, and 7 days after the operation. The concentrations of plasma and intestinal CCK and the levels of mRNA of CCK in the intestinal mucosa and PSTIs in the pancreas were significantly increased by bile-pancreatic juice diversion. The increase in the mRNA level of PSTI-61 was significantly higher than that of PSTI-56. Administration of CCK antagonist inhibited these changes but administration of CCK agonist could not fully reproduce these changes. These studies suggest that bile-pancreatic juice regulates gene expression of CCK and PSTIs and that the regulatory mechanisms of gene expression of PSTI-61 and -56 may be different.

Animals↗

PubMed1995

Binding and functional characterization of alpha-2 adrenoceptors in isolated swine myometrium.

1. Alpha-adrenoceptors in the uterus of cyclic mature pigs were investigated for their binding characteristics as shown by a radioligand study employing [3H]-prazosin and [3H]-rauwolscine, and the alpha-adrenoceptors were studied for their functional roles on motility as shown mechanically in vitro. 2. Noradrenaline (10 nM-1 microM) caused a tetrodotoxin-resistant contraction in the cornual longitudinal muscle. The response was decreased by yohimbine (10 nM-1 microM) but not by prazosin (1 microM). 3. Clonidine, xylazine, phenylephrine and methoxamine also elicited a concentration-dependent contraction of the cornual longitudinal muscle. The ranking according to the potency was clonidine > xylazine >> phenylephrine > or = methoxamine. On the other hand, these alpha-adrenoceptor agonists did not elicit contraction of the cornual circular muscle even at 100 microM. 4. Noradrenergic contraction induced by transmural stimulation was significantly decreased by yohimbine (10-100 nM) but not by prazosin (1 microM). 5. The swine myometrium contained both alpha-1 (13%) and alpha-2 (87%) adrenoceptors. The distribution of alpha-adrenoceptors was muscle layer-specific, i.e. the number of both types of adrenoceptors in the longitudinal muscle was four times higher than that in the circular muscle. 6. These results show that although swine myometrium contains both alpha-1 and alpha-2 adrenoceptors, the alpha-2 type is predominant and largely responsible for mediating the excitatory response to both exogenous and endogenous noradrenaline in the cornual longitudinal muscle.

Adrenergic alpha-1 Receptor Agonists↗

PubMed1995

Plasma levels of type II phospholipase A2 and cytokines in patients with sepsis.

Plasma levels of type II phospholipase A2 (type II PLA2), cytokines and endotoxin were determined in patients with sepsis to investigate their interrelations and their role in the patient's prognosis. Type II PLA2 was measured by radioimmunoassay, tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and IL-8 were each measured by enzyme-linked immunosorbent assay (ELISA). Endotoxin was determined by a method based on an endotoxin-specific synthetic substrate. Plasma levels of type II PLA2 were significantly higher in the patients who died of sepsis than in those who survived the illness. There was a significant correlation between type II PLA2 and TNF-alpha and IL-6. Type II PLA2, TNF-alpha, IL-6, and IL-8 may be useful as indices of disease severity. The results suggest that TNF-alpha and IL-6 stimulate the production of type II PLA2 in the plasma of patients with sepsis.

Adult↗

PubMed1994

Molecular properties, substrate specificity and regulation of DNA-dependent protein kinase from Raji Burkitt's lymphoma cells.

A double-stranded DNA-dependent protein serine/threonine kinase (DNA-PK) was purified from a nuclear extract of Raji Burkitt's lymphoma cells by a three-step column-chromatographic procedure. The main silver-stained band visualized after SDS/PAGE corresponded to an autophosphorylated polypeptide of about 350-kDa that represents the catalytic component. The existence of Ku DNA-binding protein as a regulatory component in the purified enzyme was revealed by Western blot/enzyme immunoassay and direct inhibition test with anti-Ku sera from the autoimmune patients. The DNA-PK catalyzed phosphorylation of synthetic peptides corresponding to Myc and RB proteins in a DNA-dependent manner, indicating that DNA-PK may recognize a second core-sequence motif Pro-Ser/Thr- in addition to the putative consensus sequences of -Ser/Thr-Gln. The level of enzyme activity was significantly higher in DMSO-induced G0/G1-arrested Raji cells as well as in the cells after release from DMSO than in the log-phase cells.

Amino Acid Sequence↗

PubMed1994

Stimulation of DNA-dependent protein kinase activity by high mobility group proteins 1 and 2.

After incubation of high mobility group (HMG) proteins 1 and 2 with DNA-dependent protein kinase (DNA-PK) and [gamma-32P]ATP in the presence of double-stranded DNA, not only phosphorylation of HMG proteins but also enhancement of autophosphorylation of the catalytic polypeptide of 350 kDa in DNA-PK was observed. DNA-PK activity determined with a synthetic peptide and alpha-casein as substrates was stimulated several-fold by HMG1, HMG2, and the DNA-binding domains. The stimulation was decreased at higher concentrations of HMG proteins, and DNA-PK activity was inhibited by histone H1. Electrophoretic mobility shift analysis suggests that HMG proteins facilitate the binding of DNA-PK to DNA.

Adenosine Triphosphate↗