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S Wakabayashi

Publications and source records attributed to S Wakabayashi.

At least 37 records · Page 2Linked to original sources
PubMed2000

Intracellular degradation of histidine-rich glycoprotein mutants: tokushima-1 and 2 mutants are degraded by different proteolytic systems.

We reported the first case of a congenital histidine-rich glycoprotein deficiency (HRG Tokushima) in which substitution of Gly85 with Glu (G85E) in the first cystatin domain resulted in intracellular degradation and a low plasma level of HRG [Shigekiyo, T. et al. (1998) Blood 91, 128-133]. Recently, we identified the gene mutation of a second case of HRG deficiency as a Cys223 to Arg (C223R) mutation in the second cystatin domain. To investigate the molecular and cellular bases of these deficiencies, we expressed these HRG mutants in baby hamster kidney (BHK) cells. Pulse-chase experiments in the absence and presence of various proteinase inhibitors revealed that, while wild-type HRG was completely secreted during 4-h chase periods, both the G85E and C223R mutants were only partially secreted and primarily degraded within the cells. The intracellular degradation of the C223R mutant was almost completely inhibited in the presence of a proteasome inhibitor, lactacystin, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, resulting in increased secretion of the C223R mutant, and thus implicating the proteasome system in this degradation process. In contrast, the sum of the amounts of the G85E mutant inside and outside the cells decreased during the chase periods even in the presence of the proteasome inhibitor, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, although proteasome-specific inhibitor lactacystin and one of the cysteine protease inhibitors, E-64-d, prevented the intracellular degradation. These results suggested that intracellular degradation of G85E HRG occurred to some extent through a hitherto unknown mechanism. Similar studies involving recombinant mutants in which Gly85 or Cys223 was replaced with several other amino acids revealed that proteins with mutations leading to the destruction of the predicted b-sheet structure of the cystatin domains were eliminated by the intracellular quality control system.

Amino Acid Sequence↗
PubMed2000

11C-labeled KF18446: a potential central nervous system adenosine A2a receptor ligand.

UNLABELLED: To develop PET ligands for mapping central nervous system (CNS) adenosine A2a receptors that are localized in the striatum and are coupled with dopamine receptors, 3 11C-labeled xanthine-type adenosine A2a antagonists, [11C]KF18446 ([7-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthin e), [11C]KF19631 ([7-methyl-11C]-(E)-1,3-diallyl-7-methyl-8-(3,4,5-trimethoxystyryl)xanth ine), and [11C]CSC ([7-methyl-11C]-8-chlorostyrylcaffeine), were compared with [11C]KF17837 ([7-methyl-11C]-(E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylx anthine). METHODS: The regional brain uptake of the tracers, the effect of the coinjected adenosine antagonists on the uptake, and the metabolism were studied in mice. In rats, the regional brain uptake of the tracers was visualized by ex vivo autoradiography (ARG). The A2a receptor binding of antagonist 1 was also measured by in vitro ARG. Imaging of the monkey brain was performed with PET with antagonist 1. RESULTS: In mice, the highest striatal uptake was found for antagonist 1 followed by antagonists 2 and 4. The uptake was inhibited by each of 3 KF compounds and by CSC, but not by an A1 antagonist KF15372. Another selective nonxanthine-type A2a antagonist SCH 58261 significantly decreased the striatal uptake of only antagonist 1, the labeled metabolites of which were less than 20% in the plasma 30 min postinjection, but were negligible in the brain tissue. In ex vivo ARG, antagonist 1 showed the highest striatal uptake and the highest uptake ratio of the striatum to the other brain regions. A high and selective binding of antagonist 1 to the striatum was also confirmed by in vitro ARG. PET with antagonist 1 visualized adenosine A2a receptors in the monkey striatum. CONCLUSION: These results indicate that antagonist 1 ([11C]KF18446) is the most suitable PET ligand for mapping adenosine A2a receptors in the CNS.

Animals↗
PubMed2000

Histidine-rich glycoprotein (HRG) Tokushima 2: novel HRG deficiency, molecular and cellular characterization.

The proband, a 76-year-old woman, suffered from dural arteriovenous fistula. Her plasma histidine-rich glycoprotein (HRG) level was 50% of the normal level. A low level of plasma HRG was also found in her third daughter. A single nucleotide substitution of T to C was found at nucleotide position 11,438 in exon 6 of the HRG gene from the proband, converting Cys223 to Arg in the second cystatin-like domain. The same mutation was also identified in her third daughter, but not in the other four family members having normal HRG levels or in 50 unrelated healthy Japanese individuals. Expression studies in BHK cells showed that substantial intracellular degradation of the mutant occurred and only about 40% of the recombinant HRG mutant was secreted. These results indicate that congenital HRG deficiency caused by a substitution of Cys223 to Arg is hereditary in this family.

Aged↗
PubMed1999

Structural characterization of the gene for human histidine-rich glycoprotein, reinvestigation of the 5'-terminal region of cDNA and a search for the liver specific promoter in the gene.

Genomic DNA libraries were screened for the human histidine-rich glycoprotein (HRG) gene and a sequence of 15,499 nucleotides was determined. The gene is composed of 7 exons and 6 introns, and all the exon-intron boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Each of cystatin-like domains I and II of HRG is encoded by three exons, exons I to III and exons IV to VI, respectively, like those of other members of the cystatin superfamily. The entire C-terminal half of the molecule is encoded by the largest exon, VII. The first 103 nucleotides of the cDNA sequence reported for human HRG [Koide, T., Foster, D., Yoshitake, S. , and Davie, E.W. (1986) Biochemistry 25, 2220-2225] could not be found in the determined gene sequence. A homology search of this sequence against a database showed the complete matching to a part of the yeast mitochondrial DNA encoding 21S ribosomal RNA. Rapid amplification of cDNA 5' ends (5'-RACE) analysis revealed that the cDNA has multiple 5'-ends and that a possible starting point is nucleotide 104 of the reported cDNA sequence. These results suggest that the first 103 nucleotides of the cDNA sequence reported for human HRG originated from yeast mitochondrial DNA and were incidentally incorporated into the HRG cDNA in the process of the construction of a cDNA library. Various fragments obtained on restriction endonuclease digestion of the 5'-noncoding region of the HRG gene were ligated to the chloramphenicol acetyltransferase (CAT) gene and then transfected into HepG2 and 293 cells to analyze the promoter activity. The sequence between -262 and -21 from the putative translation initiation site supported the expression of CAT in HepG2 cells but not in 293 cells, suggesting that this segment promotes the liver-specific transcription of the human HRG gene.

5' Untranslated Regions↗
PubMed1998

Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation.

We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.

Amino Acid Sequence↗
PubMed1998

HRG Tokushima: molecular and cellular characterization of histidine-rich glycoprotein (HRG) deficiency.

Previously, we found the first congenital deficiency of histidine-rich glycoprotein (HRG) in a Japanese woman with thrombosis. To elucidate the genetic basis of this deficiency, we first performed Southern blot analysis and found no gross deletion or insertion in the proband's HRG gene. We then examined the nucleotide sequences of all seven exons of the proband's HRG gene. A single nucleotide substitution, G to A at nucleotide position 429, which mutates Gly85 to Glu in the first cystatin-like domain, was found in exon 3 in 13 of 22 amplified clones. This mutation generates a unique Taq I site. Exon 3 was amplified from the proband, her family members, and 50 unrelated normal Japanese individuals, and Taq I fragmentation was examined. Fragmentation of exon 3 was observed in one allele of the genes from the proband and the family members who also have decreased plasma levels of HRG. Fifty unrelated normal Japanese individuals had a normal HRG gene, indicating that the G to A mutation is not a common polymorphism. To elucidate the identified mutation as a cause for the secretion defect of HRG in the proband's plasma, we constructed and transiently expressed the recombinant Tokushima-type HRG mutant (Gly85 to Glu) in baby hamster kidney (BHK) cells, and examined an intracellular event of the mutant protein. The results showed that only about 20% of the Tokushima-type HRG was secreted into the culture medium, and intracellular degradation of the mutant was observed. Thus, the present study strongly suggests that the HRG deficiency is caused by intracellular degradation of the Gly85 to Glu mutant of HRG in the proband.

Adult↗
PubMed1998

Na+/Ca2+ exchanger overexpression impairs calcium signaling in fibroblasts: inhibition of the [Ca2+] increase at the cell periphery and retardation of cell adhesion.

We examined the Ca2+ handling property and cell function of CCL39 fibroblasts highly overexpressing the cardiac isoform (NCX1) of Na+/ Ca2+ exchanger. In NCX1 transfectants in 146 mM Na+, ionomycin, alpha-thrombin or thapsigargin only produced a small transient increase in [Ca2+]i compared to the large increase seen in control cells, although resting [Ca2+]i was not significantly different between these cells. In Na+-free medium, in contrast, the [Ca2+]i responses in NCX1 transfectants and control cells stimulated with these agents were not different, indicating that the Ca2+ content of the intracellular store(s) does not decrease on NCX1 transfection. The expression levels of the endoplasmic reticulum and plasma membrane Ca2+-ATPases, and thrombin- or serum-stimulated cell growth were not altered in NCX1 transfectants. The latter finding suggests that Ca2+ signaling in the nucleus is not impaired appreciably. On fluorescence imaging and confocal microscopy, we found that [Ca2+] did not increase in the peripheral cytoplasm of these cells treated with alpha-thrombin in Na+-containing medium. In these NCX1 transfectants, activation of the plasma membrane Ca2+-activated K+ channels by thrombin or ionomycin was markedly suppressed, and the integrin-mediated adhesion to substrate was significantly delayed compared with control cells. NCX1-overexpressing CCL39 cells thus seem to be a good model with which we can study the Ca2+-regulated membrane processes under physiologically relevant conditions.

Animals↗
PubMed1998

Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger.

To assess the role of Ca2+ in regulation of the Na+/H+ exchanger (NHE1),we used CCL-39 fibroblasts overexpressing the Na+/Ca2+ exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca2+ rise and long-lasting cytoplasmic alkalinization (60-80% inhibition) induced by alpha-thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant with the calmodulin (CaM)-binding domain deleted (amino acids 637-656), suggesting that the effect of NCX1 transfection involves Ca2+-CaM binding. Expression of NCX1 only slightly inhibited platelet-derived growth factor BB-induced alkalinization and did not affect hyperosmolarity- or phorbol 12-myristate 13-acetate-induced alkalinization. Downregulation of protein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely in NCX1-transfected cells, suggesting that the thrombin effect is mediated exclusively via Ca2+ and PKC. On the other hand, deletion mutant study revealed that PKC-dependent regulation occurs through a small cytoplasmic segment (amino aids 566-595). These data suggest that a mechanism involving direct Ca2+-CaM binding lasts for a relatively long period after agonist stimulation, despite apparent short-lived Ca2+ mobilization, and further support our previous conclusion that Ca2+- and PKC-dependent mechanisms are mediated through distinct segments of the NHE1 cytoplasmic domain.

Animals↗
PubMed1998

Posthyperventilatory steal response in chronic cerebral hemodynamic stress: a positron emission tomography study.

BACKGROUND AND PURPOSE: The alteration of regional cerebral blood flow (CBF) during and after hyperventilation was measured using positron emission tomography (PET) to determine the circulatory response induced by daily respiratory changes in the cerebral area under chronic hemodynamic stress. METHODS: Three normal volunteers and 12 patients with an obstruction of major cerebral arteries underwent PET measurements of the CBF after an injection of H2(15)O: (1) in the resting condition, (2) during hyperventilation (HV scan), (3) 1 to 3 minutes after hyperventilation (post-HV scan), (4) during the inhalation of 5% CO2, and (5) after an injection of acetazolamide. Eleven patients also underwent a 15O gas study to measure CBF, oxygen extraction fraction (OEF), and cerebral blood volume (CBV). RESULTS: (1) In 9 patients, the CBF value in the post-HV scan was lower than that in the HV scan in 1 or more regions in the area of the obstructed arteries, although the PaCO2 level during the post-HV scan was higher than that during the HV scan in all patients. All control regions in the patients and in the normal volunteers showed an elevated CBF in the post-HV scan compared with the HV scan. (2) The negative post-HV response (posthyperventilatory steal) was prominent in 4 patients with moyamoya vessels and in another 5 patients with atherosclerotic disease who had PET evidence of hemodynamic stress (elevated CBV or OEF). (3) The regional pre- to post-HV change in CBF was significantly correlated with the CBF responses to acetazolamide and CO2. CONCLUSIONS: Vasodilatation after the termination of hyperventilation in the normal areas induces a steal response in the cerebral area suffering from hemodynamic stress and may cause profound hypoperfusion in everyday situations. This phenomenon may be important to our understanding of the clinical symptoms and the natural course of chronic cerebral occlusive disease bearing hemodynamic stress.

Acetazolamide↗
PubMed1998

Evaluation of carbon-11-labeled KF17837: a potential CNS adenosine A2a receptor ligand.

UNLABELLED: The 11C-labeled KF17837 ([7-methyl-11C](E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxa nthine) was evaluated as a PET ligand for mapping adenosine A2a receptors in the central nervous system (CNS). METHODS: The regional brain distribution of [11C]KF17837 and the effect of adenosine antagonists on the distribution were measured in mice by the tissue sampling method. In rats, the regional brain uptake of [11C]KF17837 and the effect of carrier KF17837 was visualized by autoradiography. Imaging of the monkey brain with [11C]KF17837 was performed by PET. RESULTS: In mice, a high uptake of [11C]KF17837 was found in the striatum in which A2a receptors were highly enriched. The uptake was decreased by co-injection of carrier KF17837 or a xanthine-type A2a antagonist CSC but not by nonxanthine-type A2a antagonists ZM 241385 or SCH 58261, or an A1 antagonist KF15372. In the rat brain, [11C]KF17837 was accumulated higher in the striatum than in other brain regions, and the uptake was blocked by co-injection of carrier KF17837. In a monkey PET study, a high striatal uptake of radioactivity was observed. CONCLUSION: Carbon-11-KF17837 binds to adenosine A2a receptors in the striatum. However, the presence of an unknown but specific binding site for xanthine-type compounds also was suggested in the other brain regions. The results also suggested that the in vivo receptor-binding sites of xanthine-type ligands are slightly different from those of nonxanthine-type A2a antagonists.

Animals↗
PubMed1997

Calmodulin-binding autoinhibitory domain controls "pH-sensing" in the Na+/H+ exchanger NHE1 through sequence-specific interaction.

The calmodulin (CaM)-binding domain reduces the affinity of the Na+/H+ exchanger NHE1 for intracellular H+ by exerting an autoinhibitory function in quiescent cells. We replaced this domain (aa 637-656) with homologous segments from other NHE isoforms (NHE2 and 4) or functionally similar regions from other sources (Na+/Ca2+ exchanger, CaM-dependent protein kinase II, plasma membrane Ca2+-pump, or CaM-binding peptide Trp3). The NHE-1-, NHE2-, and NHE4-segments bound CaM with Kds of 16, 130, and 27 nM, respectively. These chimeric molecules were expressed in the exchanger-deficient cell PS120. NHE1 with incorporated NHE2-segment was activated in response to Ca2+-mobilizing agents ionomycin and thrombin resulting in an alkaline shift of the intracellular pH (pHi)-dependence of 22Na+ uptake, as was the case with the intact rat NHE2. In contrast, incorporation of the NHE4-segment or other CaM-binding segments induced a constitutive alkaline shift of pHi-dependence with concomitant abolishment of Ca2+-dependent activation, indicating that these segments could not function as an autoinhibitory domain in NHE1. Detailed analyses revealed that Leu639, Lys651 and Tyr652, conserved in the NHE1- and NHE2-segments, but not in the NHE4-segment, are important for the autoinhibition. Furthermore, 125I-labeled CaM-binding peptide from NHE1 was efficiently crosslinked to the NHE1 protein, suggesting that the inhibitory domain physically interacts with part(s) of the molecule. Together, these findings support the notion that the reduction of H+ affinity in Na+/H+ exchange occurs through a mechanism involving a highly sequence-specific interaction of the inhibitory domain with its putative acceptor in NHE1.

Amino Acid Sequence↗
PubMed1997

Brain incorporation of [1-11C]arachidonate in normocapnic and hypercapnic monkeys, measured with positron emission tomography.

Positron emission tomography (PET) was used to determine brain incorporation coefficients k* of [1-11C]arachidonate in isoflurane-anesthetized rhesus monkeys, as well as cerebral blood flow (CBF) using [15O]water. Intravenously injected [1-11C]arachidonate disappeared from plasma with a half-life of 1.1 min, whereas brain radioactivity reached a steady-state by 10 min. Mean values of k* were the same whether calculated by a single-time point method at 20 min after injection began, or by least-squares fitting of an equation for total brain radioactivity to data at all time points. k* equalled 1.1-1.2 x 10(-4) ml x s(-1) x g(-1) in gray matter and was unaffected by a 2.6-fold increase in CBF caused by hypercapnia. These results indicate that brain incorporation of [1-11C]arachidonate can be quantified in the primate using PET, and that incorporation is flow-independent.

Animals↗
PubMed1997

Characterization of the 5'-flanking regions of plasminogen-related genes A and B.

We examined the promoter activity of two homologous plasminogen-related genes (PRGA and PRGB) employing HepG2 cells. The 5'-flanking regions of these genes were sequenced first, then inserted into the upstream region of the CAT gene in an expression vector. CAT assays revealed that the promoter activity of PRGA was 3-fold that of plasminogen, while the activity of PRGB was 5-fold. Deletion analysis of these genes demonstrated that a region between -283 and +153 bp relative to the transcription initiation site was essential for their expression, and that there were regions with either negative or positive effects on expression farther upstream.

Apolipoproteins↗
PubMed1997

Expression and induction by IL-6 of the normal and variant genes for human plasminogen.

We examined the promoter activity of the gene for human plasminogen (PLG) employing its 1.1 kb fragment of the 5'-flanking region inserted in front of a reporter gene. Deletion analysis revealed that a region surrounding the transcription start site was essential for the PLG expression. Since the PLG gene has three sequences for the interleukin-6 (IL-6) responsive element, we examined the effect of IL-6 on the PLG expression. IL-6 stimulation of PLG resulted in a 2.5-fold increase in its transcription. This is also true for the PLG gene of a case with dysplasminogenemia. Although the patient's gene had six mutations in the 5'-flanking region, its promoter activity was 1.8-fold that of normal PLG.

Carcinoma, Hepatocellular↗
PubMed1997

Identification of cytoplasmic subdomains that control pH-sensing of the Na+/H+ exchanger (NHE1): pH-maintenance, ATP-sensitive, and flexible loop domains.

To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pHi)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we generated a set of deletion mutants of carboxyl-terminated cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We evaluated pHi-sensitivity of these mutants by measuring the resting pHi in cells placed in an acidic medium (pH 6.0) and pHi-dependence of 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of at least four subdomains in terms of pHi-sensitivity of the unstimulated NHE1: I, aa 516-590/595; II, aa 596-635; III aa 636-659; and IV, aa 660-815. Subdomains II and IV were silent for pHi-sensitivity. Subdomain I had a pHi-maintenance function, preserving pHi-sensitivity in a physiological range, whereas subdomain III, overlapping with the high affinity calmodulin (CaM)-binding site, exhibited an autoinhibitory function. Deletion of subdomain I abolished the decrease of pHi-sensitivity induced by cell ATP depletion, indicating that domain I plays a crucial role in this phenomenon. Deletion of subdomain III rendered the inhibition by ATP depletion less efficient, suggesting the possible interaction between subdomains I and III. On the other hand, tandem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition by ionomycin or thrombin. However, deletion of subdomain II partially abolished the inhibitory effect of subdomain III. Subdomain II thus seems to function as a mobile "flexible loop," permitting the CaM-binding subdomain III to exert its normal function. These findings, together with our previous data, support a concept that cell ATP, Ca2+, and growth factors regulate NHE1 via a mechanism involving direct or indirect interactions of specific cytoplasmic subdomains with the "H(+)-modifier site.".

Adenosine Triphosphate↗
PubMed1997

Molecular physiology of vertebrate Na+/H+ exchangers.

This review describes recent progress concerning the molecular aspects of the Na+/H+ exchanger. The Na+/H+ exchanger is an important regulator for intracellular pH, cell volume, and transepithelial Na+ transport. It exists in virtually all cells with cell type-dependent pattern of isoform expression, and it is regulated in response to a variety of extracellular stimuli, among them not only agonists such as growth factors and hormones but also mechanical stimuli such as osmotic stress and cell spreading. Thus this transporter is also an excellent model to study the signal transduction. Since the first molecular cloning of the Na+/H+ exchanger, detailed studies revealed many interesting features of this transporter. At present, at least five different isoforms of the Na+/H+ exchanger are known. These isoforms differ in tissue localization, sensitivity of inhibitors, and mode of transcriptional and posttranscriptional regulation, allowing them to participate in different physiological processes. We have only started to understand an intriguing mechanism underlying these functional differences among the exchanger isoforms. Because the Na+/H+ exchanger is relatively simple in terms of its kinetic features, e.g., a simple 1:1 stoichiometry of Na+ and H+ and no input of metabolic energy such as ATP hydrolysis, the study of its structural and mechanistic aspects would also serve as a good model to understand the general mechanism of various ion transporters.

Animals↗