Biomedical subjects

H Teraoka

Publications and source records attributed to H Teraoka.

At least 37 records · Page 2Linked to original sources

PubMed1999

A simple immunoradiometric assay for measuring the entire molecules of adrenomedullin in human plasma.

We developed a one-step two-site immunoradiometric assay (IRMA) using two kinds of monoclonal antibodies, which enables us to directly measure the entire molecules of adrenomedullin (AM) (the sum of mature-type AM (abbreviated, m-AM) amidated at the C-terminus and Gly-extended non-amidated AM) in human plasma using a small amount of sample (100 microl) without prior extraction. The detection limit of this assay was 0.5 pmol/l for a 100-microl sample. Intra- and inter-assay precisions were 3.4-7.3% and 5.8-7.6%, respectively. The dilution curves of plasma samples showed good linearity and analytical recovery was 89-118%. The mean total AM in plasma of healthy subjects was 9.00+/-2.13 pmol/l, whereas m-AM was 1.05+/-0.24 pmol/l. This method, together with our previously reported simplified method to specifically measure m-AM (Ohta et al., Clin Chem 1999;45:244-251), allows facile estimation of the plasma concentration of AM-Gly by subtracting m-AM from the total AM measured by the procedure described in this paper. We were able to show that the concentration of total AM in patients with sepsis was markedly higher than that in the healthy controls and that the ratios of m-AM/total AM were significantly different between the controls and patients.

Adrenomedullin↗

PubMed1999

Smooth muscle layer-specific variations in the autonomic innervation of bovine myometrium.

1. To clarify the autonomic innervation regulating longitudinal muscle (LM) and circular muscle (CM) motility in the bovine uterus, functional (nerve stimulation, adrenergic drug responsiveness) and biochemical studies (catecholamine content, radioligand binding) were conducted on parous luteal-phase myometrium. 2. Electrical field stimulation (EFS; 60 V, 0.5-msec duration) caused tetrodotoxin (1 microM)-sensitive contractions in a frequency-dependent manner (0.5-20 Hz) in both LM and CM layers. 3. The EFS-induced LM contractions were potentiated by propranolol and conspicuously decreased by phentolamine, yohimbine, idazoxan or guanethidine, but were unaffected by prazosin or atropine. 4. On the other hand, CM contractions were only slightly decreased by phentolamine, idazoxan, yohimbine and guanethidine, but were insensitive to propranolol, prazosin or atropine. 5. The noradrenaline content in LM was about five times higher than that in CM. 6. Noradrenaline, adrenaline, clonidine, xylazine, UK14,304 and phenylephrine caused concentration-dependent contractions of both smooth muscle layers. 7. Clonidine, UK14,304 and xylazine were more potent contractile agents than noradrenaline and phenylephrine. 8. The contractile response to noradrenaline was competitively antagonized by yohimbine, but not by prazosin. 9. Binding studies using [3H]-prazosin and [3H]-rauwolscine revealed that the bovine myometrium contained both alpha1- and alpha2-adrenoceptors, but the alpha2-type receptor was dominant in both LM (94% of alpha-adrenoceptors) and CM (88%) layers. 10. The distribution of alpha-adrenoceptors was muscle layer-specific; that is, the concentration of alpha1-receptors in LM was the same as in CM, but the concentration of alpha2-receptors in LM was 2.6 times higher than that in CM. 11. The results of the present study indicate that there are layer-specific variations in the functional innervation of the parous bovine myometrium (exclusive adrenergic innervation in LM and adrenergic [minor] plus nonadrenergic, noncholinergic innervation [major] in CM), and that alpha2-adrenoceptors, which were responsive to the excitatory response of endogenous and exogenous noradrenaline, were dominant in both muscle layers of the bovine myometrium.

Adrenergic alpha-Agonists↗

PubMed1999

Development of noradrenergic neurons in the zebrafish hindbrain requires BMP, FGF8, and the homeodomain protein soulless/Phox2a.

We report that the zebrafish mutation soulless, in which the development of locus coeruleus (LC) noradrenergic (NA) neurons failed to occur, disrupts the homeodomain protein Phox2a. Phox2a is not only necessary but also sufficient to induce Phox2b+ dopamine-beta-hydroxylase+ and tyrosine hydroxylase+ NA neurons in ectopic locations. Phox2a is first detected in LC progenitors in the dorsal anterior hindbrain, and its expression there is dependent on FGF8 from the mid/hindbrain boundary and on optimal concentrations of BMP signal from the epidermal ectoderm/future dorsal neural plate junction. These findings suggest that Phox2a coordinates the specification of LC in part through the induction of Phox2b and in response to cooperating signals that operate along the mediolateral and anteroposterior axes of the neural plate.

Amino Acid Sequence↗

PubMed1999

Ku in the cytoplasm associates with CD40 in human B cells and translocates into the nucleus following incubation with IL-4 and anti-CD40 mAb.

CD40 plays a critical role in survival, growth, differentiation, and class switching of B lymphocytes. Although Ku is required for immunoglobulin class switching, how CD40 signal transduction is coupled to Ku is still unknown. Here, we show that CD40 directly interacts with Ku through the membrane-proximal region of cytoplasmic CD40. Ku was confined to the cytoplasm in human primary B cells, and the engagement of CD40 on the B cells cultured in the presence of IL-4 resulted in the dissociation of Ku from CD40, translocation of Ku into the nucleus, and increase in the activity of DNA-dependent protein kinase. These findings indicate that Ku is involved in the CD40 signal transduction pathway and may play an important role in the CD40-mediated events.

Antibodies, Monoclonal↗

PubMed1999

Preoperative clinical radioimmunodetection of pancreatic cancer by 111 In-labeled chimeric monoclonal antibody Nd2.

The present study was carried out with the purpose of evaluating the clinical usefulness of radioimmunodetection (RAID) with 111In-labeled murine/human chimeric monoclonal antibody, Nd2 (c-Nd2) in patients with pancreatic cancer. Nineteen patients suspected to have pancreatic cancer were administered intravenously 74 MBq/2 mg 111In-labeled c-Nd2 in 100 ml of saline containing 2% albumin over 30 min. A scintigram was obtained on the 3rd day after infusion by using single photon emission computed tomography (SPECT) imaging. Of the 14 patients finally diagnosed as having pancreatic cancer on the basis of surgical specimens or progress of disease, specific focal uptake at the site of the tumor was detected in 12 (true positive cases), representing a sensitivity of 85.7% (12/14), and liver metastasis was found in one case with metastasis. Of the 5 patients diagnosed with tumor-forming pancreatitis (TFP), 4 patients demonstrated true negative imaging, but one patient whose tumor demonstrated interesting findings in histology and immunostaining, showed false positive imaging. Of patients investigated for human anti-chimeric antibody (HACA) response, none showed HACA response, and no allergic reaction was seen in any of the patients administered c-Nd2. These results suggest that RAID with 11In-labeled c-Nd2 is useful for differential preoperative diagnosis between invasive pancreatic cancer and TFP.

Aged↗

PubMed1999

One-step direct assay for mature-type adrenomedullin with monoclonal antibodies.

Adrenomedullin (AM) is a potent hypotensive peptide. Plasma contains mature-type AM (m-AM), which is amidated at the carboxy terminus, and an intermediate, AM-Gly. We developed a one-step two-site IRMA specific for determining human m-AM with monoclonal antibodies. The detection limit was 0.5 pmol/L, and the working range (CV <15%) was 1-300 pmol/L. Dilution of plasma samples showed good linearity. The recovery of added AM was 91-118%. The intra- and interassay imprecision values (CVs) were 4.4-8.2% and 5.5-8.3%, respectively. The assay had no cross-reactivity with AM-Gly or other peptides similar to AM. The mean (+/- SD) plasma human m-AM concentration of 61 healthy subjects was 1.18 +/- 0.65 pmol/L. In conclusion, our IRMA makes it possible to specifically measure m-AM, using a small amount of plasma sample (0.2 mL) by a one-step overnight assay without prior extraction. Our simplified method would be suitable for clinical studies on AM, especially when large numbers of samples must be processed.

Adrenomedullin↗

PubMed1998

Structure-function studies of human leptin.

To elucidate the structural requirement of human leptin for its functions, the wild-type, mutant-type, C-terminal deletion, and N-terminal deletion were expressed in Escherichia coli and purified in soluble forms. These leptin analogs were intracerebroventrically injected into C57BL/6J ob/ob mice, and their in vivo biological activities were evaluated. The mutant-type leptin lacking a C-terminal disulfide bond reduced food intake at doses of more than 15 pmol/mouse, which was as effective as the wild-type leptin. C-terminal deletion without the loop structure, also significantly, but to a lesser extent, reduced food intake at doses of more than 90 pmol/mouse. However, N-terminal deletions showed no effect on food intake. We also evaluated the effects of the leptin analogs on radiolabeled leptin binding to its receptor in the choroid plexus using autoradiography. An excess of unlabeled mutant-type leptin as well as wild-type leptin led to complete inhibition of binding. C-terminal deletions led to weak inhibitory activity, whereas N-terminal deletions caused no inhibitory activity. These results clearly demonstrate that the N-terminal region of leptin is essential for both its biological and receptor binding activities. The amino acid sequence of the C-terminal loop structure is also important for enhancing these actions, whereas the C-terminal disulfide bond is not needed.

Animals↗

PubMed1998

Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks.

DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

Animals↗

PubMed1998

High mobility group proteins 1 and 2 can function as DNA-binding regulatory components for DNA-dependent protein kinase in vitro.

The DNA-dependent protein kinase (DNA-PK) holoenzyme consists of a 470-kDa catalytic subunit (DNA-PKcs), a DNA-binding regulatory component known as Ku protein, and double-stranded DNA (dsDNA) with ends. We previously reported that the activity of DNA-PK in vitro is stimulated by non-histone chromosomal high mobility group proteins (HMG) 1 and 2 comprising two similar repeats, termed domains A and B, and an acidic C-terminal. Here we demonstrate that in vitro HMG1 and 2 can completely replace Ku protein as the DNA-binding regulatory component of DNA-PK. DNA-PKcs and Ku protein were separately purified from Raji nuclear extracts, and reconstituted into the DNA-PK holoenzyme in the presence of dsDNA. DNA-PKcs alone catalyzed DNA-dependent phosphorylation at a very low but significant level, and HMG1 and 2 markedly stimulated the phosphorylation of alpha-casein and a specific peptide substrate in a DNA-dependent manner. The HMG2-domains (A+B) polypeptide devoid of the C-terminal acidic region was more effective for DNA-PKcs stimulation than the full-length HMG2, and HMG2-domain A and -domain B polypeptides. Anti(Ku protein) antibodies inhibited the DNA-dependent phosphorylation activity of the DNA-PKcs:Ku protein complex, but not that of DNA-PKcs alone or when it was complexed with HMG1 or 2. These results demonstrate that HMG1 and 2 can function as the DNA-binding regulatory component for DNA-PKcs in vitro, and imply that a conformational change of dsDNA, which is elicited by regulatory components, is important for the stimulation of DNA-PK activity of DNA-PKcs.

Amino Acid Sequence↗

PubMed1998

Endothelial nitric oxide synthase gene is positively associated with essential hypertension.

Essential hypertension has a genetic basis. Accumulating evidence, including findings of elevation of arterial blood pressure in mice lacking the endothelial nitric oxide synthase (eNOS) gene, strongly suggests that alteration in NO metabolism is implicated in hypertension. There are, however, no reports indicating that polymorphism in the eNOS gene is associated with essential hypertension. We have identified a missense variant, Glu298Asp, in exon 7 of the eNOS gene and demonstrated that it is associated with both coronary spastic angina and myocardial infarction. To explore the genetic involvement of the eNOS gene in essential hypertension, we examined the possible association between essential hypertension and several polymorphisms including the Glu298Asp variant, variable number tandem repeats in intron 4 (eNOS4b/4a), and two polymorphisms in introns 18 and 23. We performed a large-scale study of genetic association using two independent populations from Kyoto (n=458; 240 normotensive versus 218 hypertensive subjects) and Kumamoto (n=421; 223 normotensive versus 187 hypertensive subjects), Japan. In both groups, a new coding variant, Glu298Asp, showed a strong association with essential hypertension (Kyoto: odds ratio, 2.3 [95% confidence interval, 1.4 to 3.9]; Kumamoto: odds ratio, 2.4 [95% confidence interval, 1.4 to 4.0]). The allele frequencies of 298Asp in hypertensive subjects were significantly higher than those in normotensive subjects in both groups (Kyoto: 0.103 versus 0.050, P<0.0017; Kumamoto: 0.120 versus 0.058, P<0.0013, respectively). No such disequilibrium between genotypes was significantly associated with any other polymorphisms we examined; the Glu298Asp variant was also not linked to any other polymorphisms. In conclusion, the Glu298Asp missense variant was significantly associated with essential hypertension, which suggests that it is a genetic susceptibility factor for essential hypertension.

Adult↗

PubMed1998

Development of a sensitive ELISA for human leptin, using monoclonal antibodies.

A new, sensitive ELISA for human leptin in plasma and cerebrospinal fluid (CSF) was developed, using monoclonal antibodies. The lower limit of detection of this ELISA was 0.78 pg/assay. Both intra- and interassay imprecision values were <7%. The dilution curves of plasma and CSF showed good linearity, and the recovery was 83.2-95.6%. There was good correlation between plasma leptin concentrations by the ELISA and a commercially available RIA (r = 0.99). Our ELISA is advantageous because it does not require radioisotopes, it produces results in hours rather than days, and more importantly, it improves on the detection limit and plasma interference of the RIA kit. The new ELISA enables measurement of low concentrations of leptin, as are seen in CSF and in plasma of patients with anorexia nervosa.

Adipose Tissue↗

PubMed1997

Phosphorylation of human general transcription factors TATA-binding protein and transcription factor IIB by DNA-dependent protein kinase--synergistic stimulation of RNA polymerase II basal transcription in vitro.

DNA-dependent protein kinase (DNA-PK) has been known to catalyze phosphorylation of a number of regulatory factors involved in DNA replication and transcription such as simian virus 40 T antigen, p53, c-Myc, Sp1, and RNA polymerase II (Pol II). We examined the possibility that DNA-PK phosphorylates the general transcription factors TATA-binding protein (TBP) and transcription factor (TF) IIB, which play key roles in the formation of transcription initiation complex with Pol II. By using a highly purified preparation of DNA-PK from Raji cells, both TBP and TFIIB were shown to be phosphorylated in vitro by DNA-PK. We then investigated the effect of the phosphorylation of these factors on Pol II basal transcription. Stepwise analysis of preinitiation complex formation by electrophoretic mobility shift assay revealed that the phosphorylation of TBP and TFIIB by DNA-PK did not affect the formation of promoter (P)-TBP and P-TBP-TFIIB complexes but synergistically stimulated the formation of P-TBP-TFIIB-TFIIF-Pol II complex. Similarly, combination of the phosphorylated TBP and TFIIB synergistically stimulated Pol II basal transcription from adenovirus major late promoter. These observations suggest that DNA-PK could positively regulate the Pol II basal transcription by phosphorylating TBP and TFIIB.

DNA-Activated Protein Kinase↗

PubMed1997

Inhibitory effects of caffeine on Ca2+ influx and histamine secretion independent of cAMP in rat peritoneal mast cells.

1. Caffeine did not evoke Ca2+ mobilization and histamine secretion. 2. Caffeine, as well as other methylxanthines but not forskolin or 8 bromo-cAMP, inhibited Ca2+ responses from compound 48/80. 3. Evoked histamine secretion was severely reduced by caffeine but not by cAMP analogs. 4. In beta-escin-permeabilized cells, caffeine did not affect resting and IP3-stimulated 45Ca2+ release, but it inhibited Ca(2+)-induced histamine secretion. 5. These results indicate that caffeine inhibits Ca2+ influx and Ca2+ efficacy in the secretory apparatus independent of cAMP, resulting in the inhibition of secretagogs-evoked histamine secretion from rat mast cells.

Animals↗

PubMed1997

Fc receptor-mediated phagocytosis, superoxide production and calcium signaling of beta 2 integrin-deficient bovine neutrophils.

Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ([Ca2+]i) signaling of complement receptor type 3 (CR3)-deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers. The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils. The [Ca2+]i signals in CR3-deficient neutrophils stimulated with Agg-IgG or concanavalin A were different with mean peak [Ca2+]i concentrations of 78% and 41.9%, respectively, of those of control neutrophils. These findings suggest that Fc receptor-mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces.

Animals↗

PubMed1997

Haplotypic diversity of DQA103 and 05 subtypes differing at amino acid residue 160 encoded in the third exon in 2215 Japanese individuals.

We analyzed the frequencies and haplotypes of DQA1*03 and *05 subtypes, DQA1*03011 or DQA1*0302 and DQA1*0501 or DQA1*0503, respectively, differing only at codon 160 in the non-polymorphic third exon of the DQA1 gene. Of these, 1,862 and 337 individuals selected as DQA1*03- and DQA1*05-positive samples, respectively among 2,215 unrelated Japanese were typed for their nucleotide variation at residue 160 using PCR-SSP. As observed in other populations, all the samples carrying DQA1*03011 (Gene Frequency, GF: 7.8%) were found to share DQB1*0302, whereas those carrying DQA1*0302 (GF: 44.3%) were associated with a variety of DQB1 alleles including DQB1*0302. Both of the DQA1-DQB1 haplotypes with DQA1*03011 and DQA1*0302 carrying DRB1*0406, DQA1*03011-DQB1*0302 and DQA1*0302-DQB1*0302, showed a strong linkage disequilibrium with B62 (p < 0.001, p < 0.05). These results suggested that DQA1*03011 was generated from a single amino acid change at residue 160 in the DQA1*0302-DQB1*0302 haplotype. However, none of the haplotypes with two different DQA1*03 subtypes carrying DRB1*0403,*0405,*0802 and *0901 showed a linkage disequilibrium with any common B-locus antigens, revealing extensive haplotypic diversity of the DQA1*03 group. For example, DRB1*0802 haplotypes showed linkage disequilibria with two different B-locus antigens, B35 and B61 depending on the presence of DQA1*03011 and DQA1*0302, respectively. The GFs of DQA1*0501 and *0503 were 5.1% and 2.7%, respectively. The DQA1*05 associated haplotypes in the DR52-antigen group with DQB1*0301 were divided into two groups, depending on the bimorphism at residue 160. Such a high degree of haplotypic diversity in association with DRB1 and B alleles observed in the DQA1*03 and *05 groups related to amino acid variation at residue 160, which may affect biological function such as the interaction between CD4 and HLA-DQ molecules, seems to reflect selective pressure in the evolutionary process of HLA antigens.

Alleles↗

PubMed1997

Nuclear translocation of the catalytic component of DNA-dependent protein kinase upon growth stimulation in normal human T lymphocytes.

DNA-dependent protein kinase (DNA-PK), consisting of the 470-kDa catalytic component (DNA-PKcs) and the DNA-binding regulatory component Ku protein (p70/p80), catalyzes phosphorylation of a variety of DNA replication/transcription/repair factors in the presence of double-stranded DNA. In the resting states of human peripheral blood mononuclear cells, DNA-PK activity and the protein level of DNA-PKcs were very low in the nuclear extracts, but they were high in the whole cell extracts. Depending upon proliferation of the T lymphocytes, DNA-PK activity and the protein level of DNA-PKcs in the nuclear extracts greatly increased. Immunocytochemical analysis suggested translocation of DNA-PKcs from the cytoplasm to the nucleus upon growth stimulation in the T lymphocytes.

Amino Acid Sequence↗

PubMed1997

Mutations within the reactive-site region of human pancreatic secretory trypsin inhibitor confer alpha-thrombin and factor Xa inhibitory activities.

Mutations were introduced into the reactive-site region of human pancreatic secretory trypsin inhibitor (PSTI) to produce thrombin and/or factor Xa inhibitors. All of five mutants showed trypsin inhibitory activity as strong as wild-type PSTI. Moreover, the Arg (P1), Pro-Arg (P2-P1), and Pro-Arg-Ile-Tyr-Asn (P2-P1-P1'-P2'-P3') (bold letters indicated replaced amino acids compared to the wild type) mutants had additional inhibitory activities toward factor Xa, both thrombin and factor Xa, and thrombin, respectively, at 1 x 10(-5) M.

Antithrombins↗

PubMed1997

Relationship between the chemiluminescent response of bovine neutrophils and changes in intracellular free calcium concentration.

The relationship between luminol dependent chemiluminescent (LDCL) response and changes in intracellular free Ca2+ concentrations in the bovine neutrophils was evaluated. LDCL responses and changes in intracellular Ca2+ concentrations of neutrophils were clearly detected by the stimulation with opsonized zymosan (OPZ), concanavalin A(ConA), heat-aggregated IgG (H-agg.IgG) and phorbol myristate acetate (PMA). Patterns of LDCL responses and intracellular Ca2+ of neutrophils showed characteristic features for each stimulant. PMA was a weak stimulant of the intracellular Ca2+ concentration, whereas it was a strong stimulant of LDCL response. Con A strongly stimulated an increase in the intracellular Ca2+ concentration, but was a weak stimulant of LDCL response. LDCL response of intracellular Ca(2+)-depleted neutrophils treated with ionomycin, stimulated with each stimulant was inhibited markedly without extracellular Ca2+. The sustained phase of intracellular Ca2+ concentrations stimulated with OPZ was inhibited significantly (P < 0.05) by the preincubation with anti-CD18 antibody, whereas the transient phase of intracellular Ca2+ concentrations was not inhibited. These results indicate that LDCL response is regulated at least in part by the elevation of the intracellular Ca2+, and a rise in intracellular Ca2+ concentration, which may be mediated by specific receptors appears to be essential in the LDCL response of bovine neutrophils.

Animals↗