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H Misawa

Publications and source records attributed to H Misawa.

At least 55 records · Page 3Linked to original sources
PubMed2000

The gene of Ca2+-binding protein regucalcin is highly conserved in vertebrate species.

Regucalcin is a Ca2+-binding protein, which plays a regulatory role in liver cell functions related to Ca2+. In this study we have cloned cDNA for regucalcin from rabbit, bovine, chicken and toad livers by using rapid amplification of cDNA ends (RACE) method. The nucleotide and amino acid sequences of them are compared with published human, rat and mouse sequences. Comparison analysis revealed that the nucleotide sequences of regucalcin from seven vertebrate species were highly conserved in their coding region. The overall regucalcin proteins in these species consisted of 299 amino acids, and they had 69.9-91.3% identity. Furthermore, phylogenetic analysis showed that regucalcin in seven species appears to form a single cluster. This study demonstrates a great conservation of the regucalcin genes throughout evolution.

Amino Acid Sequence↗
PubMed2000

Translocation of regucalcin to rat liver nucleus: involvement of nuclear protein kinase and protein phosphatase regulation.

The translocation of regucalcin to the nuclei of normal rat liver was investigated. The existence of endogenous regucalcin in isolated liver nuclei was confirmed by Western blotting using anti-regucalcin antibody. Nuclear translocation of regucalcin was estimated by sodium sulfate-polyacrylamide gel electrophoresis analysis. When isolated liver nuclei were incubated in the presence of exogenous regucalcin (50 microg/ml; 1.5 microM), potent band for regucalcin was found in the nuclei, indicating that the protein is translocated into the nucleus. This translocation was an early event. Nuclear regucalcin translocation was not appreciably changed in the presence of adenosine 5'-triphosphate (2 mM), guanosine 5'-triphosphate (2 mM), calcium chloride (0.1 mM), and the lectin wheat germ agglutinin (50 or 100 microg/ml), suggesting that its translocation is not mediated through nuclear localization signal. Moreover, Ca2+-dependent protein kinase and protein tyrosine phosphatase activities in isolated liver nuclei were significantly increased in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture, and these increases were completely abolished by the addition of regucalcin (50 microg/ml). This study demonstrates that regucalcin is translocated into liver nucleus, and that it can regulate the nuclear function.

Animals↗
PubMed2000

Transcript heterogeneity of the human gene for Ca2+-binding protein regucalcin.

Regucalcin is a Ca2+-binding protein which plays a regulatory role in liver cell functions related to Ca2+. In this study we have cloned and characterized cDNA for regucalcin from human liver and human hepatoma cell line Hep G2 by screening and rapid amplification of cDNA ends (RACE). The nucleotide sequences of the clones revealed that they were identical in their coding region and differed only in their 5' untranslated regions (UTRs). Northern blot analysis showed that regucalcin mRNA in the Hep G2 was longer than that of the liver. The present study demonstrates the existence of transcript heterogeneity of the human gene for regucalcin.

5' Untranslated Regions↗
PubMed2000

[Evaluation of population-based lung cancer screening in Niigata and analysis of interval cases based on comparison lung cancer registry with screening records].

Survival analysis has been used as one way to examine the efficacy of cancer screening. However, this type of study is susceptible to many sources of bias, especially to a length bias. In the city of Sukumo in Japan, a survival analysis considering screening history for the purpose of eliminating the length bias was conducted. The efficacy of lung cancer screening was assessed by comparing those who participated in the screening in the year preceding diagnosis, with those who did not. The 5-year survival rate was 44% in the former and 16% in the latter. We tried to evaluate population-based lung-cancer screening in Niigata using the method as that of Sukumo by comparing lung-cancer registry data with screening records, those who died of lung cancer from 1991 to 1994 in the study area and had the opportunity to be screened were categorized according to the screening history for the year preceding diagnosis. The 5-year survival rate was 41% in those who were screened and 19% in those not screened. In addition, the median survival time of the former was 37 months, significantly longer than the 12 months of the latter. Replication of study results in different populations may suggest the efficacy of lung-cancer screening on an annual basis. We also analyzed the clinical background of 47 interval cases in the search for a future direction for the improvement of the screening method.

Adult↗
PubMed1999

Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles.

Classical neurotransmitters such as gamma-aminobutyric acid and glutamate are released from synaptic nerve terminals by exocytosis of synaptic vesicles. PC12 cells also have SSVs capable of storing acetylcholine (ACh). A novel method to examine the effect of transient transfection of any gene of interest on the exocytosis of SSVs was developed. The transfection of choline acetyltransferase (ChAT) into PC12 cells which have lost ACh synthesizing activity resulted in the accumulation of a substantial amount of ACh. Synthesized ACh was released in Ca(2+)-dependent manner. Release was thought to occur by an exocytosis of SSVs because: (1) release was abolished by treating the cells with vesamicol, a specific inhibitor of the vesicular ACh transporter (VAChT) localizing specifically in SSVs; and (2) the release was further increased by cotransfecting rat VAChT with the ChAT. By means of this method, we showed that overexpression of complexin I or II with ChAT markedly suppressed high-K(+)-dependent ACh release of SSVs.

Acetylcholine↗
PubMed1999

Decrease in protein kinase and phosphatase activities in the liver nuclei of rats exposed to carbon tetrachloride.

The alteration in protein kinase and phosphatase activities in the liver nuclei of rats administered carbon tetrachloride (CCl(4)) was investigated. Rats received a single oral administration of CCl(4) (1 ml/100 g body wt of 5, 10, and 25% CCl(4) in corn oil), and 5, 24, and 48 h later they were euthanized by bleeding. The administration of CCl(4) (10 and 25%) caused a significant decrease in protein kinase activity in the liver nuclei. The enzyme activity in the liver nuclei from normal and CCl(4)-administered rats was significantly increased by the addition of Ca(2+) (0.5 mM) and calmodulin (10 microg/ml) in the reaction mixture, suggesting that Ca(2+)/calmodulin-dependent protein kinase activation is not suppressed by CCl(4) treatment. Liver nuclear phosphatase activity toward phosphotyrosine, but not phosphoserine and phosphothreonine, was markedly decreased by CCl(4) (5, 10, and 25%) administration. This decrease was seen 5 h after CCl(4) administration. The presence of vanadate (10(-4) M) in the reaction mixture caused a significant decrease in phosphotyrosine phosphatase activity in the liver nuclei from normal and CCl(4)-administered rats, whereas the enzyme activity was not decreased by okadaic acid (10(-5) M) or sodium fluoride (10(-3) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing phosphotyrosine phosphatase activity was seen in the liver nuclei of CCl(4)-administered rats, suggesting that regucalcin-sensitive phosphatase activity is decreased by CCl(4) administration. The present study demonstrates that CCl(4) administration induces a decrease in protein kinase and tyrosine phosphatase activities, which are involved in signaling factors in the liver nuclei of rats.

Animals↗
PubMed1999

The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop.

The Janus family of protein tyrosine kinases (JAKs) regulate cellular processes involved in cell growth, differentiation and transformation through their association with cytokine receptors. However, compared with other kinases, little is known about cellular regulators of the JAKs. We have recently identified a JAK-binding protein (JAB) that inhibits JAK signaling in cells. In the studies presented here we demonstrate that JAB specifically binds to the tyrosine residue (Y1007) in the activation loop of JAK2, whose phosphorylation is required for activation of kinase activity. Binding to the phosphorylated activation loop requires the JAB SH2 domain and an additional N-terminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB-related proteins. An additional N-terminal 12-amino-acid region (kinase inhibitory region) of JAB also contributes to high-affinity binding to the JAK2 tyrosine kinase domain and is required for inhibition of JAK2 signaling and kinase activity. Our studies define a novel type of regulation of tyrosine kinases and might provide a basis for the design of specific tyrosine kinase inhibitors.

Amino Acid Sequence↗
PubMed1999

Constitutive expression of mRNA for the same choline acetyltransferase as that in the nervous system, an acetylcholine-synthesizing enzyme, in human leukemic T-cell lines.

Both muscarinic and nicotinic acetylcholine (ACh) receptors are known to be present on the surface of lymphocytes. We have shown that variable amounts of ACh are detectable in the blood of various mammals including humans, and a major portion of blood ACh is localized in circulating mononuclear leukocytes in humans. In order to investigate which types of blood cell are the source of ACh in human blood, expression of mRNA for choline acetyltransferase (ChAT, EC 2.3.1.6), which catalyzes ACh synthesis, was analyzed using human leukemic cell lines as models of lymphocytes and the reverse transcription-polymerase chain reaction (RT-PCR) method. We observed that mRNA for the same ChAT as that in the nervous system is expressed constitutively in all the T-cell lines tested, but not in B-, pre-lymphoma or monocytic cell lines. Furthermore, only T-cell lines showed high ACh-synthesizing activities and intracellular ACh contents. These results suggest that the major portion of ACh in the circulating blood originates from T-lymphocytes.

Acetylcholine↗
PubMed1999

The influence of lung cancer mass screening on surgical results.

BACKGROUND: After the introduction of the mass screening program for lung cancer, the number of patients detected by mass screening increased as well as the number of early staged patients. Therefore, we examined the influence of lung cancer mass screening on surgical results. METHODS: A total of 1177 primary lung cancer cases, who underwent surgery from 1963 to 1992, were retrospectively reviewed. They were grouped according to the changes in the mass screening system: the first period (1963-1977) before lung cancer screening started, the second period (1978-1986) when mass screening was conducted by the local government, and the third period (1987-1992) after the launching of the national screening program. RESULTS: The rate of cases detected by mass screening increased over time and the 5-year survival rate improved significantly, from 33.7% in the first period, to 51.8% in the second period and finally, to 58.4% in the third period. The improvement is attributable to a relative increase of rate of stage I cases and better stage I survival rate. Specifically, in stage I cases, improvement resulted from a relative increase of stage IA in peripheral type and roentgenographically occult lung cancer cases and from better survival rate of these two groups. CONCLUSION: As lung cancer screening has come into widespread use, detection of peripheral small-sized lung cancer and roentgenographically occult lung cancer have increased and consequently, surgical results have improved.

Adenocarcinoma↗
PubMed1999

Muscle venous PO2 and VO2 are linearly related in repetitive tetanic contractions of canine muscle during hypoxic hypoxia.

1. It has previously been shown that perfusion with high O2-affinity-erythrocytes decreases venous PO2 (PVO2) and decreases O2 uptake (VO2) in contracting muscle at the same O2 delivery (arterial O2 concentration x flow). A linear VO2-PVO2 relationship has been obtained with a VO2-axis intercept, suggesting that, during this type of hypoxia, VO2 is composed of a PVO2-dependent and -independent VO2. However, the VO2-PVO2 relation during hypoxic hypoxia has not been examined. 2. To clarify this relation, PVO2 and VO2 have been measured in contracting gastrocnemius (1 Hz trains of 0.2 s isometric tetani) under both normoxic and hypoxic conditions during 5 min of stimulation. 3. Venous O2 changes proportionally with O2 delivery. Each VO2-PVO2 relation was linear, with the mean described by the equation VO2 = 5.06 + 0.41 x PVO2 (n = 6, r = 0.81, P < 0.05). The VO2-axis intercept was significantly different from zero (P < 0.05). 4. These results were similar to those obtained during hypoxia induced by high O2-affinity-erythrocytes. We conclude that there is a linear relationship between PVO2 and VO2 above the VO2-axis intercept, regardless of the type of hypoxia.

Animals↗
PubMed1999

Identification and transgenic analysis of a murine promoter that targets cholinergic neuron expression.

Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using beta-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.

5' Untranslated Regions↗
PubMed1999

Energy expenditure by Ba(2+) contracture in rat ventricular slices derives from cross-bridge cycling.

To clarify the energy-expenditure mechanism during Ba(2+) contracture of mechanically unloaded rat left ventricular (LV) slices, we measured myocardial O(2) consumption (VO(2)) of quiescent slices in Ca(2+)-free Tyrode solution and VO(2) during Ba(2+) contracture by substituting Ca(2+) with Ba(2+). We then investigated the effects of cyclopiazonic acid (CPA) and 2,3-butanedione monoxime (BDM) on the Ba(2+) contracture VO(2). The Ca(2+)-free VO(2) corresponds to that of basal metabolism (2.32 +/- 0.53 ml O(2). min(-1). 100 g LV(-1)). Ba(2+) increased the VO(2) in a dose-dependent manner (from 0.3 to 3.0 mmol/l) from 110 to 150% of basal metabolic VO(2). Blockade of the sarcoplasmic reticulum (SR) Ca(2+) pump by CPA (10 micromol/l) did not at all decrease the Ba(2+)-activated VO(2). BDM (5 mmol/l), which specifically inhibits cross-bridge cycling, reduced the Ba(2+)activated VO(2) almost to basal metabolic VO(2). These energetic results revealed that the Ba(2+)-activated VO(2) was used for the cross-bridge cycling but not for the Ca(2+) handling by the SR Ca(2+) pump.

Animals↗
PubMed1999

New index for oxygen cost of contractility from curved end-systolic pressure-volume relations in cross-circulated rat hearts.

We have already reported the linear oxygen consumption per beat (VO(2))-systolic pressure-volume area (PVA) relation from the curved left ventricular (LV) end-systolic pressure-volume relation (ESPVR) in the cross-circulated rat heart. The VO(2) intercept (PVA-independent VO(2)) is primarily composed of VO(2) for Ca(2+) handling in excitation-contraction (E-C) coupling and basal metabolism. The aim of the present study was to obtain the oxygen cost of LV contractility that indicates VO(2) for Ca(2+) handling in E-C coupling per unit LV contractility change in the rat heart. Oxygen cost of LV contractility is obtainable as a slope of a linear relation between PVA-independent VO(2) and LV contractility. We obtained a composite VO(2)-PVA relation line at a mid-range LV volume (mLVV) under gradually enhanced LV contractility by stepwise increased Ca(2+) infusion and thus the gradually increased PVA-independent VO(2) values. As a LV contractility index, we could not use E(max) (ESP-V ratio; ESP/ESV) for the linear ESPVR because of the curved ESPVR in the rat LV. A PVA at a mLVV (PVA(mLVV)) has been proposed as a good index for assessing rat LV mechanoenergetics. Since the experimentally obtained PVA(mLVV) was not triangular due to the curved ESPVR, we propose an equivalent ESP-V ratio at a mLVV, (eESP/ESV)(mLVV), as a LV contractility index. This index was calculated as an ESP-V ratio of the specific virtual triangular PVA(mLVV) that is energetically equivalent to the real PVA(mLVV). The present approach enabled us to obtain a linear relation between PVA-independent VO(2) and (eESP/ESV)(mLVV) and the oxygen cost of LV contractility as the slope of this relation.

Animals↗
PubMed1998

Cloning and characterization of a novel class II phosphoinositide 3-kinase containing C2 domain.

Phosphoinositide 3-kinases (PI3Ks) have been shown to play critical roles in cell growth, differentiation, survival, and vesicular transport. Class II PI3Ks have been recently identified in mouse and human (PI3K-C2 alpha/m-p170/m-cpk and HsC2-PI3K) and in Drosophila (PI3K 68D/cpk) which contain C2 domain at the C-terminus. However, their physiological function is largely unknown. We report here cloning and characterization of murine PI3K-C2 gamma, a novel class II PI3K. The catalytic domain as well as C2 domain are highly conserved in the Class II PI3K family, while the N-terminal regions of these proteins share little similarity. Unlike other Class II PI3Ks, PI3K-C2 gamma exclusively expressed in the liver, and a N-terminal truncated form was found in lung and a certain hematopoietic cell line. Specific antiserum against PI3K-C2 gamma precipitated PI3K activity from the membrane fraction of mouse liver but not from heart. Recombinant PI3K-C2 gamma exhibited a restricted lipid substrate specificity; it phosphorylated phosphatidylinositol (PtdIns) and PtdIns4P but not PtdIns(4,5)P2. Deletion mutations revealed that both the N-terminal region and the C2 domain were critical for enzymatic activity. The murine PI3K-C2 gamma gene locus was mapped to the distal region of mouse chromosome 6 in a region of homology with human chromosome 12p, which is distinct from the position of HsC2-PI3K. Cloning and biochemical characterization of the third member of class II PI3Ks provide a new insight into the function of this subfamily of PI3Ks.

Amino Acid Sequence↗
PubMed1998

Prospective echocardiographic analysis of progressive obstruction of the proximal pulmonary artery in congenital heart disease and obstructed pulmonary flow.

BACKGROUND: It is uncertain whether proximal pulmonary artery (PA) obstruction exists soon after birth and whether its progress relates directly to postnatal ductal constriction in congenital heart disease and obstructed pulmonary flow. METHODS: Serial morphometric analyses of the PA branches by echocardiogram were performed in 28 patients (mean age at initial study 2.5 days) until severe constriction of the ductus occurred (mean age 47 days). These patients were divided into 2 groups by subsequent angiographic or postmortem confirmation; 10 with proximal PA obstruction (group 1) and 18 without obstruction (group 2). RESULTS: At the time of initial examination, the mean indexed diameter of the proximal PA on the side of the ductus arteriosus in group 1 was significantly smaller than that on the contralateral side (5.2+/-0.7 versus 9.0+/-0.7 mm/BSA0.5, P < .001) or that in group 2 (8.0+/-0.4 mm/BSA0.5, P < .001). In group 1, 8 patients had a proximal PA index on the ductal side < or = 5.5 mm/BSA0.5, which was less than those of any group 2 patients. After severe constriction of the ductus, the proximal PA index on the ductal side further decreased only in group 1 (P < .01). CONCLUSIONS: These data indicate that unilateral obstructive lesion of branch PA is present shortly after birth and its progression relates directly to ductal constriction. Neonates with branch PA obstruction can be identified on their initial echocardiogram as having a proximal PA index on the ductal side < or = 5.5 mm/BSA0.5.

Arterial Occlusive Diseases↗
PubMed1998

Acetylcholine synthesis and muscarinic receptor subtype mRNA expression in T-lymphocytes.

We used a sensitive and specific radioimmunoassay for acetylcholine (ACh), and detected significant amounts of ACh in the blood of various mammals, including humans. About 60% of human blood ACh was localized in mononuclear leukocytes. Human leukemic T-cell lines, used as T-lymphocyte models, contained both ACh and choline acetyltransferase (ChAT) activity. Furthermore, ChAT mRNA and protein were detected in the T-cell line MOLT-3. Phytohemagglutinin, a T-cell activator, increased both synthesis and release of ACh by MOLT-3 cells. Muscarinic receptor subtype mRNA expression was confirmed in various T-cell lines. These findings indicate that ACh synthesized by ChAT in T-lymphocytes acts on the muscarinic receptors on lymphocytes in autocrine and/or paracrine pathways and suggest that ACh in blood functions as a modulator of T-cell-dependent immune responses.

Acetylcholine↗
PubMed1998

Loss of cholinergic synapses on the spinal motor neurons of amyotrophic lateral sclerosis.

The expression of vesicular acetylcholine transporter (VAChT) was examined immunohistochemically in the cholinergic synapses on the spinal motor neuron of the patient with sporadic amyotrophic lateral sclerosis (SALS). VAChT immunoreactive synapses were depleted on surviving motor neurons in SALS, while synaptophysin immunoreactivity was undiminished on the same neurons. This discrepancy suggests that in SALS, loss of cholinergic input on lower motor neurons is an early event, and may be part of the cause of death of those motor neurons.

Acetylcholine↗