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Volume 135 (2004)
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- Issue 2 Pages 155-
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Volume 135, Issue 2
Displaying 1-15 of 15 articles from this issue
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Yoshinori Moriyama, Akitsugu Yamamoto2004 Volume 135 Issue 2 Pages 155-163
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSVesicular glutamate transporter (VGLUT) is responsible for the active transport of L-glutamate in synaptic vesicles and thus plays an essential role in the glutamatergic chemical transmission in the central nervous system. VGLUT comprises three isoforms, VGLUT 1, 2, and 3, and is a potential marker for the glutamatergic phenotype. Recent studies indicated that VGLUT is also expressed in non-neuronal cells, and localized with various organelles such as synaptic-like microvesicles in the pineal gland, and hormone-containing secretory granules in endocrine cells. L-Glutamate is stored in these organelles, secreted upon various forms of stimulation, and then acts as a paracrine-like modulator. Thus, VGLUTs highlight a novel framework of glutamatergic signaling and reveal its diverse modes of action.View full abstractDownload PDF (3463K) -
Ge-Hong Sun-Wada, Yoichi Kamei, Yoh Wada, Masamitsu Futai2004 Volume 135 Issue 2 Pages 165-169
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe GATA 6 transcription factor, as well as GATA 4 and GATA 5, is expressed in a variety of mammalian tissues including the precardiac mesoderm and endoderm, and gut-related organs. Genetic studies have also implicated GATA factors as important regulators of gut endoderm development. Previously, we identified the promoter and a cardiac-specific enhancer of mouse GATA6 [Sun-Wada et al. (2000) J. Biochem. 127, 703-709], however, little is known about the regulatory elements that govern GATA6 expression in the primitive gut. Here, we identified a distal enhancer of the GATA 6 gene directing expression in the gut by creating transgenic mice. A sequence of approximately 200 bp between -8.0 kb and -7.8 kb contains element (s) that enhance transcription in the gut during embryonic development, when linked to the hsp 68 promoter/lacZ fusion gene. Our results also show that GATA 6 expression is controlled by multiple regulatory regions including cardiac-specific and gut-specific enhancers.View full abstractDownload PDF (2967K) -
Yukiko Okuyama, Jang-hyun Cho, Yuki Nakajima, Ko-ichi Homma, Kazuhisa ...2004 Volume 135 Issue 2 Pages 171-177
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe found that azurocidin, a secretory protein in neutrophils, binds to calreticulin, a multifunctional chaperone of the endoplasmic reticulum. Azurocidin is known to induce cytokine production in monocytes, but the mechanism of monocyte activation by azurocidin remains unknown. On the other hand, an antibacterial peptide, KLKL-LLLLKLK-NH2 (L 5), is known to bind to cell surface calreticulin of human neutrophils, resulting in their activation to produce O2-. Therefore, we examined whether cell surface calreticulin is involved in the activation of human monocytes by azurocidin to produce IL-6. We found that carlreticulin is in fact located on the surface of monocytes and that the IL-6 production stimulated by an azurucidin is inhibited by anti-calreticulin antibody. Possibly, binding between cell surface calreticulin and azurocidin is prerequisite for the activation of monocytes by azurocidin to produce IL-6.View full abstractDownload PDF (4729K) -
Keun-Hong Park, Toshihiro Akaike2004 Volume 135 Issue 2 Pages 179-183
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA derivative of sulfonylurea (SU) that mimicks glibenclamide in chemical structure was synthesized and incorporated into a water-soluble polymeric backbone as a biospecific polymer for stimulating insulin secretion. In this study, a backbone polymer fluorescence-labeled with rodamine-B isothiocyanate was found to be strongly adsorbed onto MIN6 cells, probably due to its specific interaction mediated by SU receptors on the cell membrane. The intensity of fluorescence on the cells was significantly increased by increasing the incubation time and polymer concentration. To verify the specific interaction between the SU (K+ channel closer)-incorporated copolymer and MIN 6 cells, the cells were pretreated with diazoxide, an agonist of the ATP-sensitive K+ channel (K+ channel opener), before adding the polymer to the cell culture medium. This treatment suppressed the interaction between SU and MIN 6 cells. A confocal laser microscopic study confirmed this effect. The results of this study provide evidence that SU-incorporated copolymer stimulates insulin secretion through the specific interactions of SU moieties in the polymer with MIN 6 cells.View full abstractDownload PDF (1853K) -
Akiko Tadokoro, Hidemi Hayashi, Toshihiko Kishimoto, Yasutaka Makino, ...2004 Volume 135 Issue 2 Pages 185-191
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSEscherichia coli spr (s-uppressor of prc) mutants and nlpI mutants show thermosensitive growth. The thermosensitivity of the spr mutants was suppressed by the nlpI mutations. Expression of the fusion genes encoding hexa-histidine-tagged NlpI (NlpI-His) and purification of the tagged NlpI showed that NIpI-His bound with Prc protease and IbpB chaperone. NlpI-His with the amino acid substitution of G103D did not bind with either of these proteins, while NlpI-His variants (NlpI-284-His, NlpI-Q283-His, and NlpI-G282-His) lacking 10 to 12 residues from the carboxy terminus bound with both proteins. The tagged NlpI lacking 11 amino acid residues from the carboxy terminus was processed by Prc, but that lacking 12 residues was not. The thermosen-sitivity of the nlpI mutant was corrected by the production of the former NlpI variant, but not by production of the latter. Expression of the truncated NlpI that lacked 10 or 11 residues from the carboxy terminus corrected the thermosensitivity of the prc nlpI double mutant, while expression of the full-length NlpI did not. Thus, it was suggested that NlpI was activated by Prc protease processing.View full abstractDownload PDF (3483K) -
Rahman Hafizur, Masato Yano, Tomomi Gotoh, Masataka Mori, Kazutoyo Ter ...2004 Volume 135 Issue 2 Pages 193-200
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSType I DnaJs comprise one type of Hsp70 cochaperones. Previously, we showed that two type I DnaJ cochaperones, DjA1 (HSDJ/Hdj-2/Rdj-1/dj2) and DjA2 (cpr3/DNAJ3/Rdj-2/dj3), are important for mitochondrial protein import and luciferase refolding. Another type I DnaJ homolog, DjA4 (mmDjA4/dj4), is highly expressed in heart and testis, and the coexpression of Hsp70 and DjA4 protects against heat stress-induced cell death. Here, we have studied the chaperone functions of DjA4 by assaying the refolding of chemically or thermally denatured luciferase, suppression of luciferase aggregation, and the ATPase of Hsp70s, and compared these activities with those of DjA2. DjA4 stimulates the hydrolysis of ATP by Hsp70. DjA2, but not DjA4, together with Hsp70 caused denatured luciferase to refold efficiently. Together with Hsp70, both DjA2 and DjA4 are efficient in suppressing luciferase aggregation. bag-1 further stimulates ATP hydrolysis and protein refolding by Hsp70 plus DjA2 but not by Hsp70 plus DjA4. Hsp70-2, a testis-specific Hsp70 family member, behaves very similarly to Hsp70 in all these assays. Thus, Hsp70 and Hsp70-2 have similar activities in vitro, and DjA2 and DjA4 can function as partner cochaperones of Hsp70 and Hsp70-2. However, DjA4 is not functionally equivalent in modulating Hsp70s.View full abstractDownload PDF (3488K) -
Shin-ichiro Yoshimura, Akitsugu Yamamoto, Yoshio Misumi, Miwa Sohda, F ...2004 Volume 135 Issue 2 Pages 201-216
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWhen the ER to Golgi transport is blocked by a GTP-restricted mutant of Sarlp (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM 130 and GRASP 65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM 130 and GRASP 65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the micro-injection of Sar1p (H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM 130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.View full abstractDownload PDF (10398K) -
Shinya Kasamatsu, Ayuko Sato, Taichi Yamamoto, Vincent W. Keng, Hirosh ...2004 Volume 135 Issue 2 Pages 217-223
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe homeodomain-containing protein Hex acts as an activator as well as a repressor of transcription in animals. While its repression domain has been mapped to the amino-terminal region, the activation domain has never been identified. Here, we show that the homeodomain and the acidic carboxyl-terminal region are necessary for full activation of the sodium-dependent bile acid cotransporter gene promoter in a cell type-independent manner, suggesting that the carboxyl-terminal region comprising residues 197 to 271 functions as the activation domain. In addition, we observed that a Hex mutant without this activation domain acts as a dominant-negative mutant as to the transactivating function of Hex.View full abstractDownload PDF (3062K) -
Tomotake Morita, Kaoru Takegawa, Toshiharu Yagi2004 Volume 135 Issue 2 Pages 225-230
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPyridoxal (PL) reductase encoded by the plr1+ gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN). The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B6, or the excretion of PL as PN from yeast cells. In this study, a PL reductase-disrupted (plr1Δ) strain was constructed and its phenotype was examined. The plr1Δ A cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media. In EMM, the plr1Δ strain became flocculent at the late stationary phase for an unknown reason. The plr1Δ cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1+ gene, which maintained the flow of “PL→PN→PNP→PLP” in the salvage synthesis of PLP. The total vitamin B6 and pyridoxamine 5'-phosphate contents in the plr1Δ cells were significantly lower than those in the wild-type ones. The percentages of the PLP amount as to the other vitamin B6 compounds were similar in the two cell types. The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type. In contrast, PN was much higher in the latter than the former. The plr1Δ cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL. The results showed that PL reductase encoded by the plr1Δ gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis.View full abstractDownload PDF (2261K) -
Toshitsugu Yubisui, Takashige Takabayashi, Fumi Takahashi, Shigeki Fuj ...2004 Volume 135 Issue 2 Pages 231-236
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA cDNA clone for cytochrome b5 was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b5. The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b5 of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b5. The recombinant soluble cytochrome b5 showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b5 upon reduction with NADH and NADH-cytochrome b5 reductase. The recombinant Ciona cytochrome b5 is reduced by NADH-cytochrome b5 reductase with an apparent Km value of 3.3 μM. This value is similar to that of the cytochrome b5 of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b5 mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b5 plays important roles in various metabolic processes during development.View full abstractDownload PDF (3085K) -
Chu-Shih Chen, Rumiko Matsuoka, Shoichi Arai, Yukihiko Momiyama, Haruk ...2004 Volume 135 Issue 2 Pages 237-243
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTo determine the pathogenicities of mutated mtDNAs in patients with respiration defects, the possible involvement of nuclear DNA mutations has to be excluded, since respiratory function is controlled by both nuclear DNA and mtDNA. This was achieved by showing that the mutated mtDNAs and respiration defects were co-transferred from patients to mtDNA-less human cells, and the resultant cybrid clones carrying mutated mtDNAs expressed respiration defects. To decide whether the cybrid clones expressed respiration defects, in this study the lowest limits of normal respiratory function were evaluated by transfer of mtDNAs from 54 normal subjects to mtDNA-less HeLa cells. The resultant cybrid clones showed that 71% respiratory function was the lowest limit of mtDNAs from normal subjects. On the other hand, cybrid clones carrying pathogenic mtDNAs from patients with mitochondrial diseases showed 0-64% respiratory function, suggesting that less than 71% respiratory function in cybrid clones should be a reliable indicator of whether the mutated mtD-NAs of the patients were pathogenic.View full abstractDownload PDF (933K) -
Kiyoshi Kyono, Masahiko Miyashiro, Ikuhiko Taguchi2004 Volume 135 Issue 2 Pages 245-252
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in viral replication, and is expected to be one of the target molecules of anti-HCV drugs. Previously, we established a high-throughput screening system for HCV helicase inhibitors using the Scintillation Proximity Assay (SPA) system [Kyono, K. et al. (1998) Anal. Biochem. 257, 120-126]. Here, we show improvement of the preparation method for the HCV NS3/4A complex. Alteration of the expression region led to an increase in protein expression. The partially purified full-length NS3 protein showed higher NS3 pro-tease activity without the cofactor NS4A peptide than the truncated protease domain with the cofactor peptide, suggesting that this protein formed a complex with NS4A. NS3 further purified to homogeneity, as judged on silver staining, remained in a complex with NS4A. Characterization of the helicase activity of this full NS3/4A complex using the SPA helicase assay system revealed that this enzyme preferred Mn2+, and that the optimal pH was 6.0-6.5. The NS3/4A complex could act on a DNA template but could not unwind the M13DNA/DNA substrate.View full abstractDownload PDF (3589K) -
Jalal A. Al jamal2004 Volume 135 Issue 2 Pages 253-258
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSVoltage-dependent anion-selective channel proteins (VDACs) are pore-forming proteins found in the outer mitochondria) membrane of all eukaryotes and in brain postsynaptic membranes. VDACs regulate anion fluxes of a series of metabolites including ATP, thus regulating mitochondrial metabolic functions. Hexokinase binds to porin. The mitochondrially bound hexokinase can greatly increase the rate of aerobic glycolysis. The activities of hexokinase and protein levels of mitochondrial porin were determined in brains of hypothyroid rabbits and in hypothyroid rabbits administered with thyroxine. Proteins were separated by electrophoresis, and the proteins of interest were quantified. Western blotting analysis revealed a significant decrease (-50%) in the relative amount of porin in the hypothyroid compared with euthyroid rabbits. The changes in the developmental pattern of hexokinase activity in the brain of hypothyroid rabbits and the effect of T 4 on this enzyme activity have been investigated. Hypothyroid rabbits showed lower activity than their corresponding agematched normal neonates. Administration of thyroxine to the hypothyroid neonates at birth abolished the effects of methimazole [1-methyl-2-mercaptoimidazole (AM)]. These findings apparently indicate that the synthesis of the pore-forming protein and the hexokinase enzymes are under thyroid control during the fetal and the early postnatal period.View full abstractDownload PDF (1942K) -
Ayuko Sato, Vincent W. Keng, Taichi Yamamoto, Shinya Kasamatsu, Tomoko ...2004 Volume 135 Issue 2 Pages 259-268
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHex is one of the homeobox genes suggested to be important for hematopoietic cell differentiation. However, its biological function and mechanism of transcriptional regulation in hematopoietic cells remain elusive. We have identified the regulatory region necessary for transcription of the mouse Hex gene in K 562 leukemia cells through transient reporter assays involving various deletion mutants. This region, comprising +775 to +1177 in the first intron, had enhancer-like properties and showed high activity in other hematopoietic cell lines such as U 937, HEL, and RAW 264.7, but little activity in other Hex-expressing cell lines such as MH1C1 and H4IIE hepatoma cells, suggesting that this region functions as a hematopoietic cell-specific enhancerlike element. Binding site mutation of hematopoietic transcription factors, such as GATAs and c-Myb present in the enhancer-like element, significantly decreased the luciferase reporter gene expression in K562 cells. Electrophoretic mobility shift assays showed that GATA-1, GATA-2, or c-Myb actually binds to three of these putative binding sites, and also suggested that several unidentified factors might interact with the enhancer-like element. Overexpression of GATA-1, GATA-2, or c-Myb stimu-lated the enhancer-like activity via these three binding sites. Thus, we conclude that Hex expression in hematopoietic cells is mainly regulated by GATA-1, GATA-2, and c-Myb via this intronic enhancer-like element.View full abstractDownload PDF (2542K) -
Yasushi Kamisaka, Naomi Noda, Masakazu Yamaoka2004 Volume 135 Issue 2 Pages 269-276
Published: February 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe studied the regulation of lipid body biogenesis in the oleaginous fungus Mortierella ramanniana var. angulispora by investigating culture conditions to modulate lipid body size, which we found was affected by the carbon-to-nitrogen ratio (C/N ratio) in the culture medium. Increasing the nitrogen source or decreasing the C/N ratio from 38 to 9 induced the appearance of lipid bodies with diameters less than 2-3 μm, which are usually found at a C/N ratio of 38 in this fungus. To determine factors regulating lipid body size, we compared lipid body fractions from fungal cells cul-tured at different C/N ratios. We found some differences in polypeptide profiles between lipid body fractions from fungal cells cultured at different C/N ratios for 2 days when the lipid bodies were enlarged at a C/N ratio of 38. We then compared the phosphorylation of lipid body proteins, since protein phosphorylation plays a pivotal role in various aspects of signal transduction. In vitro phosphorylation in the lipid body fraction indicated that protein kinase activity toward endogenous and exogenous substrates such as histone HIS, VIIS, and myelin basic protein increased in the lipid body fraction at a C/N ratio of 9. Further analysis by in-gel protein kinase assay indicated the presence of at least three activated protein kinases with molecular masses of 75, 72, and 42 kDa, which were also autophosphorylated. These results indicate the presence of nutrient-regulated protein kinases and increased phosphorylation in lipid bodies, which correlate with the appearance of smaller lipid bodies in this fungus. Further studies to characterize these protein kinases at the molecular level should provide new insights into the link between nutrient sensing and lipid storage.View full abstractDownload PDF (4485K)
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