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Volume 118 (1995)
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- Issue 4 Pages 665-
- Issue 3 Pages 463-
- Issue 2 Pages 251-
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Volume 118, Issue 2
Displaying 1-32 of 32 articles from this issue
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Jun Ohkawa, Tetsuhiko Koguma, Takeo Kohda, Kazunari Taira1995 Volume 118 Issue 2 Pages 251-258
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hammerhead ribozyme belongs to the class of molecules known as antisense RNAs. However, because of short extra sequences that form the so-called catalytic loop, it can act as an enzyme. Since the catalytic domain captures magnesium ions and magnesium ions can cleave phosphodiester bonds, hammerhead ribozymes are recognized as metalloenzymes. In general, the cleavage of phosphodiester bonds involves acid/base catalysis, with proton transfer occurring in the transition state. When the possibility of such a proton-transfer process was examined by measuring solvent isotope effects, it became apparent that no proton transfer occurs in the transition state during reactions catalyzed by a hammerhead ribozyme. It is likely, therefore, that hammerhead ribozymes exploit the general doublemetal-ion mechanism of catalysis, with Mg2+ ions coordinating directly with the attacking and leaving oxygen moieties. Since the hammerhead ribozyme is one of the smallest RNA enzymes known and has potential as an antiviral agent, thus ribozyme has been extensively investigated for applications in vivo. Ribozymes are described that have possible utility as agents against HIV-1.View full abstractDownload PDF (2243K) -
Akira Nagayoshi, Norio Matsuki, Hiroshi Saito, Kazuko Kaneko, Nobuko S ...1995 Volume 118 Issue 2 Pages 259-264
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously shown that fatty liver was easily induced in suncus by starvation and that the plasma level of apolipoprotein B (apoB) was very low. We also previously reported that a defect in the assembling process of apo B-containing lipoprotein (very low density lipoprotein, VLDL) may be one of the reasons for the low level of plasma apo B and for induction of fatty liver by starvation in suncus. We also found that hepatic aryl coenzyme A cholesterol acyltransferase (ACAT) activity is very low in the animals, resulting in decreased cholesteryl ester contents in the liver. A deficiency of cholesteryl ester in suncus liver may be one of the reasons for the defect in the assembling process of VLDL. In this study, we investigated the effect of cholesterol-feeding, which induces an increase in triglyceride and cholesteryl ester of the liver as a consequence of the induction of both intestinal and hepatic ACAT activities, on the secretion of VLDL. Although the basal ACAT activity of intestinal mucosa was high, cholesterol-feeding did not induce either an increase in plasma lipid or an increase in intestinal ACAT activities in suncus. The hepatic secretion rate of VLDL was estimated by treatment with Triton WR1339, which is well known to inhibit the catabolism of VLDL. Cholesterol-feeding caused a slight increase in hepatic triglyceride and cholesteryl ester but no increase either in the secretion rate of VLDL or in hepatic ACAT activity. This evidence suggests that either the secretion rate of VLDL is not dependent on absolute hepatic contents of cholesteryl ester, or it is dependent on the level of cholesteryl ester which is produced by hepatic ACAT.View full abstractDownload PDF (763K) -
Takao Matsuba, Masayuki Yamada, Hideo Suzuki, Akira Kanai, Osamu Isoza ...1995 Volume 118 Issue 2 Pages 265-270
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStarting with a previously isolated cDNA for human thyrotropin receptor (TSHR), we established a transformed myeloma cell line, SP56, which expresses human TSHR on its cell surface. Binding analysis showed that SP56 bears 1.1×105 TSHR per cell with a Kd of 2.2×10-10M. Using the purified cellular membrane, we established a TSH binding inhibition immunoglobulin (TBII) assay for autoantibodies against TSHR. We compared it with the TBII assay utilizing porcine thyroid membranes expressing porcine TSHR, which has been widely used for TBII assay, by using 96 serum samples from patients with autoimmune thyroid disease and normal individuals. Our TBII assay was more sensitive than the one using porcine TSHR: of 38 sera of patients which were judged negative for autoantibodies to TSHR (TBII value below 10%) by the latter assay, 28 were positive (above 20%) in our assay. By using a perfusion culture system, we obtained as many as 3×1010 SP56 cells, from which 3, 450mg protein of the membrane could be purified; this is sufcient for 15, 000 assays. The results indicate that the membrane of the myeloma cell line SP56 is more suitable for use in the TBII assay than the porcine thyroid membrane, in terms of sensitivity to autoantibodies against TSHR in human sera.View full abstractDownload PDF (695K) -
Yoshiko Miyazaki, Shiro Oka, Seiko Yamaguchi, Seiko Mizuno, Ikuya Yano1995 Volume 118 Issue 2 Pages 271-277
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSphingomonas paucimobilis, a Gram-negative opportunistic pathogen, is actively phagocytosed by human peripheral polymorphonuclear leukocytes (PMN) in vitro. However, when live or killed cells were delipidated, the phagocytic rate was clearly decreased. Therefore, we have investigated the physiological role of membrane lipids in phagocytic processes. S. paucimobilis type strain 2395 produces four classes of acidic glycosphingolipids (GL-1, GL-2, GL-3, and GL-4) with the common components of glucuronic acid, 2-hydroxy myristic acid and d18:0 or d21:1 long-chain base. The effect of acidic glycosphingolipids on phagocytosis by PMN using killed Staphylococcus aureus cells coated with glucuronosyl ceramide (GL-1) or ceramide tetrahexoside (GL-4) was also examined. The rate of phagosome-lysosome fusion by PMN was determined by counting acridine orangestained bacteria under a fluorescence microscope. Both phagocytosis and phagosome-lysosome fusion by PMN of glycosphingolipid-coated bacteria were stimulated markedly in a dose-dependent manner. It was noted that GL-1 or GL-4 stimulated phagosome-lysosome fusion dramatically, but synthetic lipid A did not. Superoxide anion release from PMN was enhanced significantly by the coating with synthetic lipid A at higher concentration, but only slightly with GL-1 or GL-4. Glucuronic acid was an inhibitor of phagocytosis of GL-1-coated S. aureus by PMN. The effect of acidic glycosphingolipids obtained from mammalian tissue on phagocytosis was also compared with that of bacterial glycosphingolipids. Ganglioside GM3 and sulfatide showed a marked stimulative activity for phagocytosis by PMN, while the neutral glycosphingolipids did not. Thus, bacterial acidic glycosphingolipid and mammalian acidic glycosphingolipid promote phagocytosis and phagosome-lysosome fusion by PMN.View full abstractDownload PDF (3085K) -
Kenji Nagata, Tetsuya Furuike, Shin-Ichiro Nishimura1995 Volume 118 Issue 2 Pages 278-284
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRadical copolymerization of a polymerizable dansyl derivative, N-2-propenyl-(5-dimethylamino)-1-naphthalene sulfonamide, with sugar monomers and acrylamide proceeded smoothly in aqueous solution in the presence of ammonium persulfate and N, N, N', N'-tetramethylethylenediamine and afforded a novel type of water-soluble glycopolymers having fluorescent side-chains. Fluorescence emission spectra of these polymeric sugarligands by excitation at 340nm revealed maxima at 448 and 528nm. When the glycopolymer carrying galactose residues was saturated with Ricinus communis agglutinin (RCA60), the fluorescence emission maxima at 448 and 528nm were not shifted significantly, although the fluorescence intensities were decreased by 20 and 14%, respectively. Polymeric sugar-cluster effects drastically enhanced the association constants of galactose residues with RCA60 in the order of 108M-1. The significance for efficient binding of galactose density on the glycopolymer was also demonstrated by using glycopolymers with different degrees of galactose branching.View full abstractDownload PDF (583K) -
Yoshihiro Deyashiki, Yoshiyuki Tamada, Yoshiyuki Miyabe, Masayuki Naka ...1995 Volume 118 Issue 2 Pages 285-290
Published: 1995
Released on J-STAGE: November 18, 2008
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Nobuo Nakanishi, Satoshi Onozawa, Reiko Matsumoto, Hiroyuki Hasegawa, ...1995 Volume 118 Issue 2 Pages 291-296
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVesicular amine transport is crucial for activity of monoaminergic neurons since translocation of cytosolic amine to storage vesicles is required for both exocytotic release and reuptake of the neurotransmitter. We developed a convenient assay system for vesicular amine transport based on permeabilizing the plasma membrane of pheochromocytoma PC12 cells with digitonin at concentrations of 100 to 150μM. Serotonin (5HT) is a better substrate than either epinephrine or norepinephrine in this assay system. In the presence of 2mM ATP, 5HT uptake by the permeabilized cells increased linearly for at least 30min at 25°C, whereas without addition of exogenous ATP, 5HT uptake reached an apparent plateau after 20min incubation. Reserpine (500nM) completely blocked the ATP-dependent 5HT uptake in this system whereas nomifensine (10μM) had no effect, indicating specificity for vesicular transport. Treatment of intact PC12 cells with AMP dose-dependently decreased 5HT uptake with an EC50 of 20-30μM as measured after cell permeabilization. Treatment of the intact PC12 cells with forskolin and phorbol 12-myristate 13-acetate prior to permeabilization also down-regulated vesicular 5HT transport, whereas the addition of either of these agents into the reaction mixture for the amine uptake by already permeabilized cells did not alter the veiscular uptake activity. Thus, the system can be applied to studies on regulation of the vesicular amine transport by physiological signaling molecules, intracellular messengers, and drugs.View full abstractDownload PDF (2081K) -
Shinne-Ren Lin, Shu-Hwa Chi, Long-Sen Chang, Kou-Wha Kuo, Chun-Chang C ...1995 Volume 118 Issue 2 Pages 297-301
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cationic groups of arginine and lysine residues in Oh-4, a novel α-neurotoxin from king cobra (Ophiophagus hannah) venom were subjected to modification with p-hydroxyphenylglyoxal (HPG) and trinitrobenzene sulfonate (TNBS), respectively. Monoderivatization of Arg-35, resulted in a drastic loss in neurotoxicity to 25% of the native toxin. The activity was decreased to a greater extent with the derivative extensively modified on Arg-35, -9, and -37. The Arg-35-modified derivative retained about a half of the antigenicity of the native toxin, and extensive modification on Arg-9 and Arg-37 caused a further decrease in the antigenicity of the toxin molecule. Selective trinitrophenylation (TNP-) of Lys-51 caused losses of neurotoxicity and antigenicity by 77 and 83%, respectively. These results indicate that Arg-35 and Lys-51 in Oh-4 have important roles in the neurotoxicity. In contrast to the Arg residues at 9, 35, and 37, Lys-51 plays a more critical role in the antigenicity.View full abstractDownload PDF (524K) -
Hitoshi Shimoi, Yuzuru Iimura, Takaji Obata1995 Volume 118 Issue 2 Pages 302-311
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA yeast cell wall glycoprotein with a molecular weight of 40, 000, named gp40, was solubilized from SDS-extracted cell wall of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Based on its amino acid sequence, we cloned and sequenced the gene encoding the precursor of gp40, named CWP1; cell wall protein gene. The DNA sequence of the CWP1 gene was identical to YKL443, an open reading frame identified in a genome sequencing program for yeast chromosome XI. This gene encoded a serine-rich protein of 239 amino acids with a molecular weight of 24, 267. The presence of hydrophobic sequences in the N- and C-termini of the CWP1 protein suggests that it is secreted as a glycosylphosphatidylinositol-anchored protein and is subsequently integrated into the cell wall. Since a gene disruption experiment showed no growth defect, the CWP1 gene is not essential for growth. Mutant CWP1 protein deficient in the C-terminal hydrophobic sequence was secreted into the culture medium, not anchored to the cell wall, thereby indicating that this hydrophobic sequence plays a crucial role in anchoring to the cell wall. Homology between the CWP1 protein and TIP1 family of cold shock proteins suggests that they belong to a new family of cell wall proteins.View full abstractDownload PDF (4845K) -
Yutaro Azuma, Yoshiaki Onishi, Yutaka Sato, Harutoshi Kizaki1995 Volume 118 Issue 2 Pages 312-318
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe studied the effects of protein tyrosine kinase inhibitors with different modes of action on topoisomerase activity and cell death in CTLL-2 cells, whose growth is IL-2-dependent. The Flavonoids genistein, biochanin A, and apigenin inhibited topoisomerase II to the same extent as etoposide, a specific inhibitor of the enzyme. Methyl 2, 5-dihydroxycinnamate (2, 5-MeC) also inhibited topoisomerase II, but was less potent than genistein. Herbimycin A and staurosporine did not inhibit topoisomerase II. None of the inhibitors of protein tyrosine kinases examined inhibited topoisomerase I activity. All the inhibitors induced cell death with internucleosomal DNA fragmentation in the presence of IL-2. Genistein, biochanin A, and apigenin induced DNA fragmentation and cell death early in the incubation period and did not alter the profiles of phosphotyrosine proteins in either the lysate or pelleted fractions, indicating that the early cell death was induced by the inhibition of topoisomerase II activity rather than by the inhibition of protein tyrosine kinase activity. 2, 5-MeC similarly induced early cell death and DNA fragmentation, but to a lesser extent than genistein presumably due to the inhibition of topoisomerase II activity. Herbimycin A induced a slow increase in DNA fragmentation and cell death, accompanied by a decrease in phosphotyrosine proteins in the pelleted fraction, suggesting that the inhibition of protein tyrosine phosphorylation, presumably of the nuclear proteins, is related to cell death and DNA fragmentation. Staurosporine-induced DNA fragmentation appeared to be due to mechanism(s) other than the inhibition of topoisomerases and protein tyrosine kinases, since it neither altered the profiles of phosphotyrosine proteins nor inhibited topoisomerase activity.View full abstractDownload PDF (4248K) -
Tsutomu Nakamura, Issei Yoshioka, Mamoru Takahashi, Hiroyuki Toh, Kats ...1995 Volume 118 Issue 2 Pages 319-324
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ppc gene, which encodes Phosphoenolpyruvate carboxylase (PEPC) of an extreme thermophile, Thermus sp., was cloned and sequenced. The ppc gene had a high G+C content (69.2%). An open reading frame for a 857-amino-acid polypeptide was found in the gene. The calculated molecular mass was 95, 632. The amino acid sequence of Thermus PEPC was 31-37% identical and 52-57% similar to those of 17 PEPCs from mesophilic organisms. No Cys residue was found in the polypeptide, demonstrating that this residue is not essential for the catalytic activity of PEPC. The cloned gene was expressed in Escherichia coli and thermostable PEPC was obtained.View full abstractDownload PDF (3351K) -
Haruko Oda, Toshihiko Murayama, Yasuyuki Nomura1995 Volume 118 Issue 2 Pages 325-331
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe influence of activation of protein kinase C (PKC) and cyclic AMP on noradrenaline (NA) release in the neurosecretory rat pheochromocytoma PC12 cell line was investigated. External ATP induced [3H]NA release from prelabeled PC12 cells, in the presence of extracellular CaCl2. The potency order of ATP analogs was adenosine 5'-O-(γ-thiotriphosphate) _??_ ATP>2-methylthio ATP>2', 3'-O-(4-benzoyl) benzoyl ATP. αβ-Methylene ATP, βγ-methylene ATP, and 8-bromo ATP were inactive. Neither ADP, GTP, nor ITP was active. The addition of phorbol 12-myristate 13-acetate (PMA) or agents elevating the cyclic AMP content, such as vasoactive intestinal peptide (VIP) or an adenosine analog, also stimulated [3H]NA release. Not only high K+- but also ATP-stimulated [3H]NA release was enhanced by co-addition with PMA or agents elevating the cyclic AMP content. PMA and VIP had no effect on the cytosolic free Ca2+ concentration ( [Ca2+]i) or on the ATP-stimulated [Ca2+]i, rise, although both stimulatory effects on [3H]NA release were dependent on extracellular CaCl2. The addition of PMA stimulated [3H]NA release dose-dependently, and enhanced 300μM (maximal dose) ATP-stimulated [3H]NA release without changing the affinity for ATP. The effect of PMA was inhibited by PKC inhibitors such as calphostin C and in PKC-depleted cells, and potentiated by elevation of cyclic AMP. These data suggest that the process of ATP-stimulated NA release, not ATP-stimulated Ca2+ influx, is regulated by the dual, PKC- and cyclic AMP-dependent mechanisms, positively and independently. Treatment with pertussis toxin had no effect on the ATP-stimulated [Ca2+]i, rise or [3H] NA release. The possibility of involvement of GTP-binding proteins in NA secretion from PC12 cells is discussed.View full abstractDownload PDF (869K) -
Akira Kawada, Kenji Hara, Masataro Hiruma, Hiromitsu Noguchi, Akira Is ...1995 Volume 118 Issue 2 Pages 332-337
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have purified cathepsin L-like proteinase from rat epidermis, determined its NH2-terminal amino acid sequence, and investigated its proteolytic activities on an intermediate filament-associated protein filaggrin and several synthetic substrates. The amino acid sequence of its NH2-terminus was determined to be Val-Pro-Asn-Ser-Leu-Asp-Trp-Arg-Glu-Lys-Gly-Tyr-Val-Thr-Pro-, which differed from that of rat cathepsin L and was not found in the amino acid sequence data bank. The enzyme consisted of a single-chain form with Mr 30, 000. Its hydrolytic properties toward synthetic substrates were similar to those of cathepsin L in other tissues. The enzyme effectively proteolyzed rat epidermal filaggrin into small fragments at pH 4.0-6.0 and was inhibited by a specific cysteine proteinase inhibitor, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)L-leucyl]-agmatin. However, cathepsins D and E from rat epidermis did not hydrolyze filaggrin. This study demonstrated that filaggrin was susceptible to degradation by rat epidermal cathepsin L-like proteinase, suggesting that this proteolytic activity may have relevance to skin differentiation, in which acid proteases are thought to participate.View full abstractDownload PDF (3263K) -
Yuko Nagamine-Natsuka, Shigemi Norioka, Fumio Sakiyama1995 Volume 118 Issue 2 Pages 338-346
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStreptomyces erythraeus produces an extracellular mammalian-type serine protease bearing trypsin-like substrate specificity. The gene encoding the protease was cloned and sequenced as an initial step for investigating its structure-function relationship by site-specific mutagenesis. The cloned gene is composed of an 816-bp open reading frame encoding 272 amino acid residues, suggesting that it is synthesized as a precursor protein containing a 42-residue prepropeptide. In the N-terminal prepropeptide portion, the tract of 30 residues from the initiator methionine has a typical signal sequence for Streptomyces and the remaining 12 residues are thought to comprise a propeptide. The cloned gene was replaced downstream of a strong promoter in a high expression plasmid, pSEV2, and expressed in Streptomyces lividans TK24. The gene product was secreted extracellularly and identified as an inactive precursor which consists of the mature enzyme and the 12-residue N-terminally extended peptide chain. The precursor protein was converted to a fully active mature form by limited proteolysis with α-chymotrypsin at the Phe-(- 1)-Ile- 1 bond. Protein sequence analysis revealed that, except for the C-terminal three residues, recombinant SET is identical with the native enzyme.View full abstractDownload PDF (1937K) -
Kei Amada, Masafumi Yohda, Masafumi Odaka, Isao Endo, Noriyuki Ishii, ...1995 Volume 118 Issue 2 Pages 347-354
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced. The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins. The cloned genes of chaperonin subunits, chaperonin-10 (T. th cpn10) and chaperonin-60 (T. th cpn60), were separately expressed in Escherichia coli cells. The expressed subunits were easily purified from other host proteins including GroEL of E. coli. Since chaperonin from T. thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T. th cpn10, a tetradecamer of T. th cpn60 without T. th cpn10 has not been obtained before. T. th cpn60 tetradecamer tended to dissociate into monomers during storage. T. th cpn10 expressed in E. coli was purified as a stable oligomer, most likely a heptamer. The activity as holo-chaperonin was reconstituted by mixing both subunits. T. th cpn6o tetradecamer itself arrested refolding of other proteins. The monomerized T. th cpn60 was easily purified from T. th cpn6O oligomer by gel permeation chromatography. Thus-obtained T. th cpn60 monomer had an ATP-independent chaperone activity, as shown for T. th cpn60 monomer isolated from authentic holo-chaperonin.View full abstractDownload PDF (2394K) -
Tadanori Tanahashi, Masashi Suzuki, Nobuyuki Itoh, Youji Mitsui1995 Volume 118 Issue 2 Pages 355-363
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe identified a protein with a molecular weight of about 44 kDa in an extract of rat regenerating liver. This protein was undetectable in normal, sham-operated, and completely regenerated liver. We also purified this 44-kDa protein from an extract of rat liver remnant after partial hepatectomy. The partial amino acid sequences of the purified protein were identical to those of γ-actin in non-muscle cells. In addition, anti-pan actin anibody recognized the purified 44-kDa protein, whereas anti-muscle actin and anti-β-actin antibodies did not. Thus, we concluded that the 44-kDa protein was non-muscle γ-actin. An immunohistochemical study revealed that the non-muscle γ-actin accumulated next to the plasma membrane of liver parenchymal cells during regeneration. Moreover, the γ-actin level was augmented in primary cultured rat hepatocytes prior to DNA synthesis. Intracellular γ-actin in cultured hepatocytes was distributed across the entire basal plane after stimulation with hepatocyte mitogens. This change in the distribution of γ-actin correlated with the cell spreading that occurred during the G1/S phase transition. These findings indicated that γ-actin plays specific roles in the growth of liver parenchymal cells during liver regeneration.View full abstractDownload PDF (6689K) -
Takako Kitani, Sachiko Okuno, Hitoshi Fujisawa1995 Volume 118 Issue 2 Pages 364-370
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalmodulin-dependent protein kinase IV (CaM-kinase IV) is thought to play crucial roles in the functioning of Ca2+ in the central nervous system and immune system, and the regulation of its activity is therefore very important. Recombinant CaM-kinase IV is invaluable for studies of its regulatory mechanism, because of its large-amount availability and ready site-specific mutagenesis. In the present study, rat CaM-kinase IV was expressed in Sf9 cells and Escherichia coli, and the kinetic properties were examined with syntide-2 and peptide-γ as substrates. The recombinγant enzymes were produced highly efficiently, comprising as much as about 15% of the total protein in Sf9 cells and 9% in E. coli. The brain enzyme shows two Km values for syntide-2 in the presence of Ca2+/calmodulin, but the recombinant enzymes showed normal kinetic behavior. The brain enzyme and Sf9 enzyme showed Km values for peptide-γ of 53 and 82μM, respectively, but the Km of the E. coli enzyme was as high as 1.7mM, in the presence of Ca2+/calmodulin. Thus, the three enzymes differed in their kinetic properties, but all the three were markedly activated upon incubation with CaM-kinase IV kinase under the Ca2+/calmodulin-dependent protein phosphorylation conditions.View full abstractDownload PDF (2133K) -
Kei-ichi Uemura, Tamotsu Taketomi1995 Volume 118 Issue 2 Pages 371-375
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe roles of protein kinases and calmodulin in regulating neurite outgrowth in murine neuroblastoma NS-20Y cells were investigated by testing the effect of various inhibitors on the neuritogenesis induced by serum deprivation. The percentage of cells with neurites was low (1-3%) in medium containing 10% serum, but reached about 50-60% when the cells were cultured for 24h in serum-free medium. W-7 (10μM), calmidazolium (0.3μM), and trifluoperazine (0.1μM), drugs reported to inhibit calmodulin-dependent events, reduced neurite outgrowth. On the other hand, H-7 (inhibitor of protein kinase C and cyclic AMPdependent protein kinase) and H-89 (inhibitor of cyclic AMP-dependent protein kinase) were ineffective. Genistein (inhibitor of protein tyrosine kinase) and wortmannin (inhibitor of phosphatidylinositol 3-kinase) did not affect the number of cells with neurites. Activation of protein kinases, which is blocked by these inhibitors, does not appear to be essential to the extension and maintenance of neurites. KN-62 and KN-93 (inhibitors of Ca2+/calmodulin-dependent protein kinase II) were also tested but did not inhibit neurite outgrowth. These results suggest that a calmodulin-dependent process, other than the activation of Ca2+/calmodulin-dependent protein kinase II, is involved in the neuritogenesis in murine neuroblastoma NS-20Y cells in serum-free medium.View full abstractDownload PDF (1885K) -
Khalid Mahmud Lodhi, Hajime Sakaguchi, Shigehisa Hirose, Hiromi Hagiwa ...1995 Volume 118 Issue 2 Pages 376-379
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEndothelin (ET) receptors were identified in trout tissues that included the gill, heart, liver, kidney, and intestine. Since both the presence and the physiological function of endothelin1 (ET-1) in trout gills have been determined, we examined the localization and characterization of ET receptors in the gills. Ligand specificity of the receptors, determined by using members of the endothelin family, revealed that endothelin receptors specific for ET-1 were predominant in the gills, but the binding sites of such receptors had low affinity for BQ-123, which is selective for mammalian endothelia A receptors (ETA). We propose that this subtype of receptor be called a fish-type ETA receptor (ETAF) to distinguish it from the mammalian ETA receptor. Affinity labeling of binding sites on gill membranes with 125I-labeled ET-1 and the cross-linking reagent 1, 5-difluoro-2, 4-dinitrobenzene revealed one major protein with an Mr of 58, 000, similar to that found in mammals. Autoradiographic studies demonstrated a high density of endothelin-binding sites in the lamellar sinusoid, which is involved in gas exchange in gills. No binding was detected in the chondrocytes or cartilaginous matrix. Our observations suggest that the endothelin and ET receptor system found in mammals might play a physiological role in the regulation of metabolic functions in fish.View full abstractDownload PDF (1890K) -
Yasuharu Ninomiya, Masahiro Okada, Naoe Kotomura, Katsuyuki Suzuki1995 Volume 118 Issue 2 Pages 380-389
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnalysis was made on the genomic structure, functions, and expression of the mouse ELP gene, which codes for the embryonal long terminal repeat binding protein. Extensive screening of the cDNA library of embryonal carcinoma cells (EC cells) identified four isoforms of ELP: ELP1 (the original ELP isolate), ELP2, ELP3, and Ad4BP/SF1. Analysis of the genomic sequences revealed that these ELP isoforms were generated by alternative promoter usage and differential splicing. The mRNAs of isoforms initiated at four transcription start sites distributed on three exons. Sequence analysis of the four isoforms identified three polypeptides. The N-terminal portion of ELP1 and ELP2 was longer than ELP3, and Ad4BP/SF1 by 77 aa. The DNA-binding domain and region II were shared by all four isoforms. The C-terminal portion shared by ELP2, ELP3, and Ad4BP/SF1 was 131 as in length, and that specific to ELP1 was 57 as in length. The ELP3 and Ad4BP/SF1 isoforms were identical for the coding sequence, but the two differed at the 5' noncoding region. Region II and III domains of nuclear receptors were thought to be involved in ligandbinding and transcriptional activation. ELP1, which lacked region III, functioned as a repressor. The isoforms carrying intact region II and region III functioned as transactivators. Expression of the four isoforms was studied in mouse tissues and in tissue culture cells by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Complex patterns of expression of these isoforms were observed in various tissues. All four ELP isoforms were expressed only in EC cells.View full abstractDownload PDF (5291K) -
Toshimitsu Takagi, Kenkoh Muroya, Minako Iwama, Tatsuo Shioda, Toshihi ...1995 Volume 118 Issue 2 Pages 390-396
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have developed an in vitro transcription system using purified Sendai virus particles in which viral mRNA synthesis is almost entirely dependent on the addition of cellular proteins (host factors), one of which can be replaced by highly purified cellular tubulin [Mizumoto et al. (1995) J. Biochem. 117, 527-534]. In this study, to elucidate the function of host factors in transcription, we isolated an active initiation complex as a viral ribonucleoprotein, by incubating virus particles with bovine brain extract in the absence of nucleoside triphosphates, followed by ultracentrifugation. RNA products from the isolated initiation complex contained six mRNA species corresponding to all the virusencoded genes, and most of them had a 5'-cap structure as well as a 3'-poly(A) tail. Immunoblotting showed that tubulin was specifically associated in the active complex. These data suggest that cellular tubulin, one of the host factors essential for Sendai virus transcription, interacts with the viral ribonucleoprotein to form an active complex at the initiation step.View full abstractDownload PDF (2901K) -
Naotada Ishihara, Shohei Yamashina, Masao Sakaguchi, Katsuyoshi Mihara ...1995 Volume 118 Issue 2 Pages 397-404
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA conserved region containing three to five proline residues is present just behind the signal-anchor sequence in the amino terminal portion of most microsomal cytochrome P-450s. We have shown that the proline residues are crucial for correct folding in Schizosaccharomyces pombe cells by using mutants of P-450(M1) in which one to three of the proline residues were changed to alanine. To examine the effects of the mutations on the intracellular localization of P-450s, they were expressed in COS-7 cells. They were found to be localized only in the perinuclear loci as patched structures like the Golgi apparatus, while the wild-type P-450(M1) is localized in the reticular structures which are typical for the ER membrane. However, treatment of the cells with Brefeldin A had no effect on the patched structures. Upon co-expression with another ER membrane protein, CD4D, which possesses a double lysine motif, the expressed CD4D was localized not only in the patched structures as the mutated P-450(M1)s, but also in the reticular structures of ER. When the cells were homogenized and then fractionated, the mutated P-450(M1) was recovered mainly in the low-speed precipitate and in the fractions of much higher density than the normal ER membrane. On electron microscopic observation, unusual membranous bodies were observed near the nucleus only when the mutated P-450(M1) was expressed. From these results, we suggest that the P-450(Ml) molecules which failed to take on the correct conformation became aggregated on the ER membrane and were localized in the perinuclear region forming membrane bodies composed of small vesicles derived from the ER membranes in the cells.View full abstractDownload PDF (4983K) -
Holger Notbohm, Adriana Bigi, Norberto Roveri, Josef Hoch, Yahya Acil, ...1995 Volume 118 Issue 2 Pages 405-410
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSmall angle X-ray diffraction and biochemical analyses were carried out on normal palmar aponeurosis and on tissue from patients suffering from Dupuytren contractures (MD). Pathological tissue exhibits a higher overall content of collagen III. Type I collagen extracted from pathological tissue has a melting point of 0.8°C higher than that of nomal collagen. The only chemical differences compared to normal collagen I are 50% overhydrox-ylation of lysyl residues and a reduced amount of diglycosylated hydroxylysine residues. Analysis of the electron density distribution inside the collagen repeating period of MD-samples reveals disordered molecular packing in MD samples compared to in normal collagen. The disorder, which is higher in the gap region, is considerably reduced upon stretching.View full abstractDownload PDF (1843K) -
Hideaki Mizuno, Makoto Morita, Hiroshi Akanuma1995 Volume 118 Issue 2 Pages 411-417
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cyclic polyol, 1, 5-anhydro-D-glucitol (AG), is generally present in animals, although little is known about the metabolic and physiological roles of AG in any type of animal cells. The present metabolic study demonstrated phosphorylation of AG in human chronic myelogenous leukemia cells, K-562. Phosphorylated AG (AGP) was also proved to be present in various rat organs; its level in most organs ranged between 2 and 5 nmol/g wet tissue, which amounted to 5 to 10% of the AG levels in the respective organs. In the spleen and brain, however, the AGP levels were especially high, 13.4 and 8.3 nmol/g, respectively, or 24.4 and 20.6% of the respective AG levels. These data suggest that AGP is an intermediary metabolite related to AG in animal cells.View full abstractDownload PDF (862K) -
Palmira Zubin, Cristina Defagot, Gustavo Parlanti1995 Volume 118 Issue 2 Pages 418-421
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNovel molecular mediation in the sound transductional process is implicitly suggested. We investigated the presence of the cGMP-synthesis enzyme, guanylate cyclase, in Corti's organ and auditory nerve of the rat. The soluble guanylate cyclase activity found was sensitive to changes of sound intensity in the different acoustic media used, suggesting a potential role for the system that involves this enzyme. The guanylate cyclase activity appeared to be inversely related (in the inner ear) with the sound intensity to which the animals were exposed; different behavior was observed for the auditory nerve. The enzymatic activity found in Corti's organ, a direct bio-receptor of sound, represents the first reported enzymatic activity of this type in this tissue, which apparently could be influenced by the intensity of a physical stimulus such as sound. Finally, an adequate ionic environment appears to play a potential role in the expression of the changes observed, indicating that it may function according to the requirements of the biological sensor.View full abstractDownload PDF (1753K) -
Yu Meng, Takuo Yasunaga, Takeyuki Wakabayashi1995 Volume 118 Issue 2 Pages 422-427
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new method was developed for extracting natural actomyosin with full length connectin (titin) and nebulin from rabbit skeletal muscle. To determine the protein components of native thin filaments, thin filaments were isolated from natural actomyosin by sedimentation in a H2O/D2O/sucrose density gradient. Both α-actinin and nebulin were cosedimented with actin filaments, while connectin was not. This shows that the native thin filaments contained α-actinin and nebulin. This indicates that the native thin filaments were more complicated than synthetic filaments reconstituted from actin, tropomyosin, and troponin. It is known that synthetic filaments are less Ca2+ sensitive than native thin filaments. This difference in Ca2+ sensitivity may be due to the differences in components and/or the different structures of native and synthetic filaments. The newly developed methods described here for extracting natural actomyosin and for isolating native thin filaments are useful for addressing these important problems related to the structure and function of native thin filaments.View full abstractDownload PDF (3508K) -
Tsuyoshi Katoh, Toshio Kodama, Akihisa Fukushima, Michio Yazawa, Fumi ...1995 Volume 118 Issue 2 Pages 428-434
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo synthetic peptides having amino acid sequences of the heavy chain segments in the neck region of porcine aorta smooth muscle myosin were used to study the formation of the 10S folded conformation. These peptides, MHC821-835 and MHC835-846, correspond to residues 821-835 and 835-846, respectively, of the chicken gizzard myosin heavy chain. The effects of these peptides on the filament formation of porcine aorta smooth muscle myosin were examined. The filamentous myosin fraction increased on the addition of MHC835-846 at NaCl concentrations lower than 0.25M for both dephosphorylated and phosphorylated myosin, while the filament formation of dephosphorylated myosin was unaffected by the addition of MHC821-835 up to 30μM. The filaments formed in the presence of MHC835-846 were observed by electron microscopy to be morphologically indistinguishable from those formed in the absence of the peptide. Rotary shadowed images of dephosphorylated myosin monomers revealed mostly the 10S form in the absence of MHC835-846, while the 6S form was predominant in the presence of the peptide. The results indicate that MHC835-846 inhibits the formation of the 10S conformation. On cosedimentation assaying with myosin, rod and light meromyosin, MHC835-846 bound to each of them with a stoichiometry of nearly 1 mol/mol heavy chain. Altogether, these results suggest that the region in the myosin heavy chain corresponding to MHC835-846 may be involved in the binding site for the light meromyosin portion of myosin for formation of the 10S conformation.View full abstractDownload PDF (3609K) -
Toshihiko Eki, Fumio Hanaoka, Takeo Kohda, Masayuki Seki, Michio Ui, T ...1995 Volume 118 Issue 2 Pages 435-441
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe established a modified cell-free system for polyomavirus (PyV) DNA replication, which was supplemented with Escherichia coli single-stranded DNA binding protein (SSB). DNA synthesis in this system was enhanced by 1.4- to over 15-fold depending upon the amount of cell extracts contained in the reaction mixture. By supplementing with E. coli SSB, we were able to reduce the amount of cell extracts in the reaction mixture, and to lower the concentrations of creatine phosphate and Tris, rendering this system more resistant to salts than the conventional PyV DNA replication system. The modified system was characterized using mutant cell extracts which had heat-inactivated DNA polymerase α. DNA synthesis in the system was dependent on PyV T antigen, the PyV origin of DNA replication, mutant cell extracts, and DNA polymerase α-primase complex purified from wild-type cells. The DNA polymerase α-primase complex was not replaced by DNA polymerase α, indicating that this system requires a functional DNA polymerase α-primase complex. This system exhibited species-specificity in the requirement for DNA polymerase α-primase; only mouse DNA polymerase α-primase but not human DNA polymerase α-primase functioned in this system.View full abstractDownload PDF (2880K) -
Hiroaki Naraba, Yohsuke Imai, Ichiro Kudo, Yasuhito Nakagawa, Sachiko ...1995 Volume 118 Issue 2 Pages 442-447
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat inflammatory neutrophils induced arachidonic acid release and platelet-activating factor (PAF) production in response to opsonized zymosan (OPZ) dose-dependently. Phospholipase A2 activity also increased dose-dependently, paralleling the increases in arachidonic acid and PAF. The time courses of the activities of phospholipase A2 and acetyltransferase, and the amounts of free arachidonic acid, lyso-PAF, and PAF demonstrated that activation of the enzymes in the remodeling pathway could be required for PAF production in rat neutrophils, which agrees with the documented fact for macrophages. Phospholipase A2 could be a rate-limiting enzyme for PAF production, since an increased lyso-PAF amount or addition of exogenous lyso-PAF reflected the increase in PAF formation in the cells. This phospholipase A2 activity in rat neutrophils could be attributed to cytosolic type phospholipase A2, because the activity was mostly suppressed by a specific antibody to cytosolic phospholipase A2. As previously reported, pretreatment of neutrophils with the acyl-CoA synthetase inhibitor, triacsin C, or the acyltransferase inhibitor, merthiolate, enhanced PAF production as well as arachidonic acid release by the cells in response to OPZ. Triacsin C inhibited arachidonoyl-CoA production and merthiolate suppressed the transacylation of lyso-PAF to 1-alkyl-2-arachidonoyl-GPC. These results suggest that these inhibitors of acylation of lyso-PAF caused accumulation of lyso-PAF, which resulted in enhancement of PAF production when phospholipase A2 and acetyltransferase were activated by OPZ. Thus the activation of cytosolic phospholipase A2 and the acylation of lyso-PAF by such as arachidonic acid could be regulating factors for PAF production in stimulated rat neutrophils.View full abstractDownload PDF (776K) -
Kinue Suzuki, Masako Uchii, Ryushi Nozawa1995 Volume 118 Issue 2 Pages 448-452
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCertain osteoclastic markers (multinucleation and tartrate-resistant acid phosphatase) were induced in human leukemia HL-60 cells by treatment with 10-7M 1, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] for 10 days. However, no formation of pits on a bone substrate by vitamin-treated HL-60 cells was detected. Expression of calcitonin receptors (CTR), another osteoclastic marker, was examined by means of the reverse transcriptase polymerase chain reaction. The human CTR-cDNA (T47D isotype) was amplified from untreated HL-60 cells, but not from cells treated with 1, 25(OH)2D3. The CTR mRNA disappeared within 24h after the treatment. Thus, 1, 25(OH)2D3-differentiated HL-60 cells failed to show two intrinsic characteristics of osteoclasts, pit formation on a bone substrate and expression of CTR. We then examined the expression of CTR on established human leukemia cell lines. The CTR mRNA was expressed in myeloblastic ML-1 and promyelocytic HL-60 leukemia cells but not in more mature macrophage-like cell lines, U-937 and THP-1 cells. Neither B cell leukemia BALL-1, T cell leukemia Jurkat, promegakaryoblastic leukemia Meg-J, nor cervix uteri carcinoma HeLa S3 cells amplified the CTR products. The cDNA of BIN67-isotype CTR, that has an additional 16-amino acid insert in the putative first intracellular loop of T47D-type CTR [Kuestner et al. (1994) Mol. Pharmacol. 46, 246-255], was amplified by neither strain tested. It was suggested that the T47D-type CTR is a novel differentiation antigen of immature myeloid lineage cells.View full abstractDownload PDF (1700K) -
Eikichi Hashimoto, Fumito Takeuchi, Yukie Tanaka, Hirohei Yamamura1995 Volume 118 Issue 2 Pages 453-460
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA common and effective phosphate acceptor protein for casein kinase II and Ca2+-phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from the cytosolic fraction of Xenopus laevis oocytes. Its molecular mass was estimated to be approximately 25, 000 by SDS-polyacrylamide slab gel electrophoresis and gel filtration analyses. About 1 and 2 mol of phosphate were incorporated per mol of this protein with casein kinase II and protein kinase C, respectively, and the phosphorylated amino acid was identified as serine irrespective of the protein kinase employed. The Km values were calculated to be 1 and 0.5μM for this Mr 25, 000 protein with casein kinase II and protein kinase C, respectively. However, this protein was a relatively poor substrate for casein kinase I and did not serve as one for cAMP-dependent protein kinase. The amino acid sequence of its amino-terminal region suggests that this protein is a newly identified substrate for these two protein serine/threonine kinases.View full abstractDownload PDF (3393K) -
1995 Volume 118 Issue 2 Pages 461
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (21K)
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