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Volume 124, Issue 1
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Yasuhisa Fukui, Sayoko Ihara, Satoshi Nagata1998 Volume 124 Issue 1 Pages 1-7
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphatidylinositol (PI)-3 kinase is an enzyme that phosphorylates the D-3 position of PI and its derivatives. It is activated immediately after growth factor or differentiation factor stimulation, suggesting that PI-3 kinase is involved in signal transduction of the stimulation. PI-3 kinase appears to play various important roles including signaling to the nucleus, vesicle transport, and rearrangement of the cytoskeleton since many cell responses which require these events are affected by inhibition or activation of PI-3 kinase. To understand how PI-3 kinase can act in such multiple ways, it is important to identify the factors downstream of PI-3 kinase. In this review, we discuss the factors downstream of PI-3 kinase and the methods used to identify them. Recent studies revealed that some proteins involved in vesicle transport or in rearrangement of the cytoskeleton are regulated by the phospholipids generated by PI-3 kinase, implying the mechanism by which PI-3 kinase regulates these cell responses.View full abstractDownload PDF (2122K) -
Kunihiko Suzuki, Marie T. Vanier, Kinuko Suzuki1998 Volume 124 Issue 1 Pages 8-19
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOnly a limited number of genetic mouse models of abnormal sphingolipid metabolism are known to occur spontaneously. However, recent progress in the combined homologous recombination and embryonic stem cell technology allows inactivation of any genes of choice once they are cloned. Not only is it possible to generate mutant mouse lines that are equivalent to known human genetic disorders but genetic conditions unknown or highly unlikely to occur in humans, such as simultaneous inactivation of more than one gene, can also be created. Most of the human disorders due to genetic defects in sphingolipid catabolism have been duplicated in the mouse. With increasing activity in cloning of the enzymes involved in sphingolipid biosynthesis, genetic mouse models of abnormal sphingolipid biosynthesis are beginning to appear. These models have already provided invaluable insight into the metabolism and physiological functions of sphingolipids and are expected to be utilized extensively for evaluation of the pathogenesis and of treatment approaches of these genetic disorders.View full abstractDownload PDF (1627K) -
Yong-Fu Li, Yasuo Hata, Tomomi Fujii, Tatsuo Kurihara, Nobuyoshi Esaki1998 Volume 124 Issue 1 Pages 20-22
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of a complex prepared by soaking a single crystal of the S175A mutant of L-2-haloacid dehalogenase from Pseudomonas sp. YL in a solution containing a poor substrate, L-2-chloropropionamide, has been determined by X-ray analysis at 2.15 Å resolution with a crystallographic R factor of 19.8%. The present analysis has revealed the structure of the reaction intermediate trapped in the crystal. In the intermediate, the substrate moiety lacking chlorine is covalently bound to the carboxyl group of D10, and adopts the pro-D-configuration at the C2 atom. The amide group of the substrate is hydrogen-bonded with the hydroxy group of S118. The methyl group bound to the C2 atom exists in a hydrophobic pocket which is important for recognition of the alkyl group of the substrate. The guanidino group of R41 has reasonable orientation for halogen-abstraction.View full abstractDownload PDF (336K) -
The Human PTF γ/SNAP43 Gene: Structure, Chromosomal Location, and Identification of a VNTR in 5'-UTRJee-Hye Maeng, Jong-Bok Yoon1998 Volume 124 Issue 1 Pages 23-27
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPTF/SNAPc is a multisubunit complex which specifically recognizes the PSEs of small nuclear RNA genes and activates transcription by RNA polymerase II or III. Here we describe the isolation and characterization of genomic clones encoding the human PTF γ/SNAP43 gene. The gene spans approximately 29 kilobases, and is composed of 9 exons and 8 introns. A major transcription initiation site was identified at the position 58 base pairs upstream of the AUG translation initiator codon on primer extension analysis with HeLa mRNA. The 5' flanking region lacks a typical TATA box but contains many putative binding sites for various transcription factors, such as Sp1, Oct1, NF1, AP1, E2F, and USF. Immediately downstream of the transcription start site, we found a VNTR of a 17-bp sequence rich in (G+C). Four different alleles with two to five copies of the tandem repeat were identified in 10 individuals examined, indicating a high degree of variation at the PTF γ/SNAP43 locus. In addition, the PTF γ/SNAP43 gene was mapped to human chromosome 14q22 by fluorescence in situ hybridization.View full abstractDownload PDF (3065K) -
Takashi Ohtsuki, Gota Kawai, Kimitsuna Watanabe1998 Volume 124 Issue 1 Pages 28-34
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMost nematode mitochondrial (mt) tRNAs have a TV-replacement loop (TV loop) which replaces the normal T arm and the variable loop in standard tRNAs with a less-structured loop. The tertiary structure of such tRNAs has been discussed theoretically with reference to the crystal structure of yeast tRNAPhe [Wolstenholme et al. (1994) Nucleic Acids Res. 22, 4300-4306] and examined experimentally by chemical and enzymatic probing [Watanabe et al. (1994) J. Biol. Chem. 269, 22902-22906]. The results suggest that most regions of the tRNA other than the TV loop are folded in a similar manner to yeast tRNAPhe. To confirm this notion more clearly, the tertiary structure of Ascaris suum mt tRNAMet was analyzed by NMR using various synthetic tRNAs site-specifically labeled with stable isotopes, which were prepared by a combination of chemical synthesis and enzymatic ligation. Tertiary interactions involving G(L2), G(L3), U(L4), and U8 were observed in the NMR spectra of the labeled tRNAs, but those relating to G(L5) were not. On the basis of these results, a possible tertiary structural model of nematode mitochondrial tRNAMet was constructed.View full abstractDownload PDF (1839K) -
Nam-Soon Kim, Tomoko Yamaguchi, Shingo Sekine, Mihoro Saeki, Shawichi ...1998 Volume 124 Issue 1 Pages 35-39
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDuring large-scale in vitro translation analysis of a human full-length cDNA bank, we found a clone producing a remarkably smaller translation product than that expected from the open reading frame. The cDNA encodes a polyubiquitin, UbC, composed of nine tandem repeats of the ubiquitin unit. The bank contained twelve UbC cDNAs including four full-length ones. Sequencing analysis of these clones showed that UbC cDNAs can be classified into two types, UbC1 and UbC2, in each of which there are six polymorphic nucleotide variations. The present UbC cDNA was in vitro translated in a rabbit reticulocyte or wheat germ extract to produce a free ubiquitin labeled with [35S]methionine. The labeled ubiquitin could be used as a substrate for thiol ester formation with ubiquitinactivating enzyme E1 or ubiquitin-conjugating enzyme E2.View full abstractDownload PDF (1746K) -
Akihiro Michihara, Makoto Sawamura, Yasuo Nara, Katsumi Ikeda, Yukio Y ...1998 Volume 124 Issue 1 Pages 40-44
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpontaneously hypertensive rat (stroke-prone) (SHRSP) has a low serum cholesterol level as compared with the normotensive Wistar Kyoto rat (WKY). We previously indicated that the lower activity of mevalonate pyrophosphate decarboxylase (MPD) was responsible for the reduced cholesterol biosynthesis in the liver of SHRSP [Sawamura et al. (1992) J. Biol. Chem. 267, 6051-6055]. To elucidate the mechanism of the reduced activity, we purified liver MPD from SHRSP treated with cholestyramine and pravastatin in this study. We compared its enzymatic properties with those of the enzyme from WKY, and also measured the amounts of MPD in the crude extract of various tissues in WKY and SHRSP by Western blot analysis. Results indicated that (i) MPD of SHRSP has essentially the same properties as MPD of WKY, except for a difference in the dependency on divalent cations. (ii) The amount, as well as the activity, of MPD in the crude extract of brain and liver was reduced in SHRSP. (iii) There was no difference between SHRSP and WKY, in the ratio of the enzyme activity to the amount of MPD in the crude extract. These data led us to conclude that the lower activity of MPD was caused by the reduced amount of this enzyme in SHRSP.View full abstractDownload PDF (2600K) -
Teisuke Takita, Eriko Akita, Kuniyo Inouye, Ben'ichiro Tonomura1998 Volume 124 Issue 1 Pages 45-50
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmino acid activation reaction of the lysyl-tRNA synthetase [L-lysine: tRNALys ligase (AMP forming); EC 6. 1. 1. 6] from Bacillus stearothermophilus was studied fluorometrically by the stopped-flow method. The addition of L-lysine to the enzyme solution caused quenching of the protein fluorescence and the subsequent addition of ATP restored the quenched fluorescence [Takita et al. (1996) J. Biochem. 119, 680-689; Takita et al. (1997) 121, 244-250]. In the stopped-flow analysis, however, the former fluorescence change (quenching) could not be detected, while the latter change (restoration) was detectable. The L-lysine binding process was suggested to be much faster than the ATP binding process, being completed within the dead-time of the apparatus, ca. 3 ms. The hyperbolic dependence of kapp on the initial ATP concentration suggested that the ATP binding to the enzyme•L-lysine complex followed a two-step mechanism. Two L-lysine analogues that exhibit the qualitatively similar behavior to L-lysine in the fluorometric titration, L-lysine hydroxamate and L-lysine amide, were examined similarly. The two-step process was also suggested for these analogues, and the forward rate constant in the rate-determining step for L-lysine amide (221±7 s-1) was significantly larger than those for L-lysine (45.7±4.6 s-1) and L-lysine hydroxamate (14.5±1.7 s-1) at pH 8.0, 30°C.View full abstractDownload PDF (1864K) -
Kiichi Sato, Akira Okubo, Sunao Yamazaki1998 Volume 124 Issue 1 Pages 51-54
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nitrous oxide reductase from the photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, has been purified. The enzyme is composed of two identical subunits of 66 kDa, and contains four copper atoms per subunit. Copper supplementation of the medium resulted in a 3.5-fold increase in the enzyme yield with doubly enhanced specific activity. The activity of the purified nitrous oxide reductase was completely inhibited by 100 μM zinc ions.View full abstractDownload PDF (1418K) -
Yuichi Saeki, Yoichi Sakakibara, Yoichi Araki, Ken Yanagisawa, Masahit ...1998 Volume 124 Issue 1 Pages 55-64
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mouse liver homogenate was shown to contain enzymatic activities catalyzing the sulfation of 3, 4-dihydroxyphenylalanine (Dopa) and tyrosine isomers with a pH optimum of 8.25. Western blot analysis revealed a 34 kDa protein exhibiting immunologic crossreactivity to antiserum against rat liver SULT1B1 sulfotransferase. By employing the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, a 910-base pair product encoding the putative mouse liver SULT1B1 sulfotransferase was obtained. Using this PCR product as a probe, a cDNA containing the entire open reading frame of the mouse liver SULT1B1 sulfotransferase was cloned from a mouse liver Lambda ZAP cDNA library. The nucleotide sequence indicated it is a new enzyme. The deduced amino acid sequence exhibited 87.6, 72.3, 55.9, 54.2, 52.8, 51.1, and 49.4% identity to the amino acid sequences of the rat liver SULT1B1 sulfotransferase, human thyroid hormone sulfotransferase, mouse phenol sulfotransferase, rat liver phenol sulfotransferase, rat liver hydroxyarylamine sulfotransferase, mouse estrogen sulfotransferase, and rat estrogen sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pcDNA3) harboring the cDNA encoding this new enzyme, a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against the rat liver SULT1B1 sulfotransferase was expressed. The recombinant sulfotransferase exhibited enzymatic activities toward Dopa and tyrosine isomers, as well as dopamine and 3, 3', 5-triiodo-L-thyronine. Northern blot analyses indicated the SULT1B1 sulfotransferase was predominantly expressed in liver, but not in the other ten mouse organs examined. Furthermore, the enzyme was found to be expressed in a developmental stage-dependent manner, being at a very low level in liver samples from 1-day-old mice and then gradually increasing to the maximum level in liver samples from 4-week-old mice.View full abstractDownload PDF (5145K) -
Hiroaki Miyazaki, Yuji Naitoh, Yoshitsugu Nakahashi, Shingo Yanagitani ...1998 Volume 124 Issue 1 Pages 65-71
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe induction of metallothionein (MT) isoforms (MT-1, -2) by anticancer drugs was investigated in cultured rat hepatoma H4 II E C3 cells. The steady-state expression of MT-1 mRNAs was higher than that of MT-2 mRNAs. During incubation of the cells with various anticancer drugs, namely, adriamycin, epirubicin, cis-diamminedichloroplatinum (II) (CDDP), and cis-diammine (1, 1-cyclobutyldicarboxylato) platinum (II), both MT-1 and MT-2 mRNAs were coordinately inducible: the levels of isoMT mRNA reached a maxim of approximate by 6-fold at 3 h. Immunofluorescent studies revealed that the cytosolic fluorescence in the cells exposed to 1 μM CDDP for 48 h was more intensified than that in the untreated cells. Transfer of antisense oligonucleotides resulted in marked reduction of isoMT mRNA, and upon exposure to 5 μM CDDP for 48 h, the viabilities of these cells dropped to 25.8% of the controls. These results indicate that anticancer drugs are potent inducers of MT isoforms in hepatoma cells and that a decrease in cellular MTs enhances the susceptibility of hepatoma cells to CDDP. Thus, we conclude that endogenous MTs play a role in determining the sensitivity or resistance of cancer cells to clinically important anticancer agents.View full abstractDownload PDF (4102K) -
Kuniyo Inouye, Soo-Bok Lee, Ben'ichiro Tonomura1998 Volume 124 Issue 1 Pages 72-78
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThermolysin is remarkably activated in the presence of high concentrations (1-5M) of neutral salts and its activity is enhanced 15 times by 4M NaCl at pH 7.0 and 25°C [Inouye, K. (1992) J. Biochem. 112, 335-340]. In this study, the effects of nitration and amination of tyrosyl residues in thermolysin on its halophilic properties were examined. Nitration and successive amination inactivate thermolysin progressively as the degree of modification increases. When 16 tyrosyl residues were nitrated, the activity decreased to 10% of that of the native enzyme, whereas it recovered to 30% when they were aminated. The decrease in the activity by the nitration and amination was shown to be brought about only by a decrease in the molecular activity, kcat; the Michaelis constant, Km, was unaltered. When 14 tyrosyl residues of thermolysin were nitrated, the degree of activation by 4M NaCl at pH 7.0 decreased from 15 to 10, and this decreased further to 5 when the pH of the reaction medium was raised to 8.5. However, when the nitrated tyrosyl residues were reduced to aminotyrosyl residues, the degree of activation was restored to that of the native enzyme. The change in the degree of activation by nitration and amination of thermolysin could be due to the change in the ionization of tyrosyl residues, and it was suggested that removing negative charges from tyrosyl residues of thermolysin enhances its halophilicity.View full abstractDownload PDF (4164K) -
Takanori Satoh, Tatsuya Samejima, Machiko Watanabe, Shin-ichi Nogi, Yo ...1998 Volume 124 Issue 1 Pages 79-88
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe genomic DNA encoding the inorganic pyrophosphatase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ATCC27634), was isolated by colony hybridization with a probe designed as a part of gene amplified by the PCR method, which was derived from the partial amino acid sequence of the enzyme. The DNA was cloned into a plasmid vector, pUC118, after digestion with BamHI. The inserted nucleotide fragment was about 1.8 kbp in length and the nucleotide sequence included a 525 by open reading frame. The deduced amino acid sequence was completely identical with that of the enzyme determined by automated Edman analysis of peptide fragments isolated from digests obtained with Staphylococcus aureus V8 protease and Achromobacter protease I, and also from products obtained on chemical cleavage with cyanogen bromide and 70% formic acid. The subunit of this enzyme is composed of 174 amino acid residues with a calculated molecular weight of 19, 084. Then, the gene was overexpressed in Escherichia coli BL21 (DE3) using a plasmid vector, pET15b, system. The recombinant enzyme was fully active, and exhibited higher thermostability than the E. coli enzyme. Amino acid residues located on the surface of the recombinant enzyme were determined by means of limited proteolysis, and the results revealed that the environment of Lys residues is almost the same as the crystal structure reported previously [Teplyakov, A. et al. (1994) Protein Sci. 3, 1098-1107]. Furthermore, the roles of two tryptophan residues were investigated by site-directed mutagenesis, which indicated that they may be responsible for the structural integrity and thermostability.View full abstractDownload PDF (1131K) -
Masaya Fujita, Yoshito Sadaie1998 Volume 124 Issue 1 Pages 89-97
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe σH of Bacillus subtilis directs transcription of a large number of early sporulation genes, whereas the principal σ factor, σA, is essential for the transcription of the genes for vegetative growth and early sporulation. We have purified σA and σH proteins, and characterized their properties. The genes encoding σA or σH were separately cloned into an expression vector under the control of T7 promoter. Both proteins were overproduced in Escherichia coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride. Antigenicities and N-terminal amino acid sequences of the overproduced proteins were used to identify both proteins. Unlike σA protein, σH protein showed a DNA-binding ability. To compare the promoter selectivity of the σA protein with that of the σH protein, transcription in vitro of 16 promoters was performed using RNA polymerase holoenzymes reconstituted from a purified core enzyme with either σH or σA. These holoenzymes correctly recognized each of the cognate promoters; σH-RNA polymerase recognized σH promoters but not σA promoters, and vice versa. A competition experiment for core RNA polymerase using σA and σH revealed that σA had a stronger affinity. We propose that the predicted replacement of a σ subunit in a holoenzyme from σA to σH in vivo at late logarithmic growth phase may require an additional factor, or the modification of a core enzyme or σ factor.View full abstractDownload PDF (5630K) -
Masaya Fujita, Yoshito Sadaie1998 Volume 124 Issue 1 Pages 98-104
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThrough mainly in vivo studies, the initiation of sporulation in Bacillus subtilis has been shown to depend on the phosphorylation of the SpoOA transcription factor mediated by the multicomponent phosphorelay via KinAB (C), SpoOF, SpoOB, and SpoOA in this order. RNA polymerase containing σA (EσA) or σH (EσH) transcribes the genes of the phosphorelay components. Phosphorylated SpoOA is also involved in their expression and is required for the induction of σH by repressing its repressor gene abrB. We have examined the effects of phosphorylated SpoOA (SpoOA-P) and AbrB on in vitro transcription of the genes involved in the SpoOA phosphorylation and initiation of sporulation. SpoOA-P repressed EσA-dependent transcription of the kinC and EσH-dependent transcription of spoOA and kinA. EσH-dependent transcription of spoOF was stimulated by SpoOA-P at low concentrations but was repressed by higher amounts of SpoOA-P. On the other hand, AbrB repressed EσA-dependent transcription of spoOH (σH gene), kinC, and abrB, although its effect was not strong. With the present results providing in vitro evidence for the roles of SpoOA-P and AbrB as transcriptional regulators, and other results described in the literature, the positive and negative feedback loops controlling the temporal expression of early sporulation genes are discussed.View full abstractDownload PDF (2932K) -
Isamu Matsunaga, Maki Yamada, Emi Kusunose, Tokuhiko Miki, Kosuke Ichi ...1998 Volume 124 Issue 1 Pages 105-110
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlthough fatty acid α-hydroxylase (FAAH) activity has been detected in various species, FAAH has not been sufficiently characterized. In this report, we describe the properties of FAAH highly purified from Sphingomonas paucimobilis. The FAAH was purified by about 5, 200-fold. Blotting analysis with a specific antibody against the FAAH showed that its apparent molecular mass was approximately 43 kDa. FAAH showed α-hydroxylation activity in the presence of H2O2, but little if any activity with cumene hydroperoxide, t-butyl hydroperoxide, or t-butyl peroxybenzonate. The Km value for H2O2 was 72 μM. Highly purified FAAH oxidized various non-esterified saturated and unsaturated fatty acids including myristic acid, but not myristoyl-CoA. Potassium cyanide and sodium azide inhibited the FAAH activity in a concentration-dependent manner. Other respiratory chain inhibitors such as rotenone and antimycin A did not inhibit the activity. Among cytochrome P450 inhibitors, SKF-525A markedly inhibited the activity at the concentration of 2mM, but CO did not. Imidazole, an inhibitor of plant α-oxidation, showed no inhibitory effect at 1mM.View full abstractDownload PDF (1730K) -
Valérie Vandamme-Feldhaus, Roland Schauer1998 Volume 124 Issue 1 Pages 111-121
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMicrosomes prepared from bovine submandibular glands incubated with radioactive AcCoA incorporated acid-insoluble radioactivity, which was dependent on time, and the concentrations of AcCoA and proteins, and was inhibited by CoA in a concentrationdependent manner. Under the conditions used, the apparent Km for AcCoA was 1.63 μM with a Vmax of 21.9 pmol/mg protein•min. The radioactivity incorporated was mainly due to the O-acetylation of glycosidically bound Neu5Ac. The primary attachment site of O-acetyl groups was exclusively the hydroxyl at C-7 of Neu5Ac, the presence of an AcCoA:Neu5Ac 7-O-acetyltransferase thus being demonstrated. After longer incubation 9-O-acetylated Neu5Ac also appeared, suggesting the migration of an ester group from C-7 to C-9. This isomerisation was inhibited by heat-inactivation of the microsomal protein, enzymatic isomerisation by a “migrase” thus being suggested. Data are presented which lead to the assumption that this 7-O-acetylation involves at least two reactions: the transport by a translocase of acetyl groups from AcCoA from the cytosol across the Golgi membrane, followed by the enzymatic transfer of these acetyl groups onto sialic acids in the Golgi lumen.View full abstractDownload PDF (6059K) -
Hiroyuki Mori, Hiroyuki Sugiyama, Miyuki Yamanaka, Ken Sato, Mitsuo Ta ...1998 Volume 124 Issue 1 Pages 122-129
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtein translocation across the cytoplasmic membrane of Escherichia coli is accomplished by concerted actions of the translocation ATPase SecA and the membrane-embedded SecE/Y/G complex. SecA interacts with preproteins and undergoes ATP-driven cycles of membrane insertion-deinsertion. To address how SecA interacts functionally with other components in the translocation machinery, we characterized a SecA mutant lacking amino-terminal 8 amino acid residues (SecA N-8). Although the absence of the 8 residues did not grossly affect the interaction of SecA with a preprotein, ATP, or phospholipids, nor did it affect the intrinsic ATPase activity, it gave differential effects on the translocation of different preproteins. It also affected the translocation ATPase activity, the ability of membrane insertion, and the topology inversion of SecG coupled with the membrane insertion-deinsertion of SecA. Most noteworthy, SecA N-8 was pronouncedly defective in the translocation of proton motive force-dependent preproteins, in which SecG might have a role. We propose that the amino-terminal region of SecA is important for the functional interaction with SecG.View full abstractDownload PDF (3895K) -
Tsuyoshi Shimo, Tohru Nakanishi, Yusuke Kimura, Takashi Nishida, Kiyot ...1998 Volume 124 Issue 1 Pages 130-140
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we cloned an mRNA predominantly expressed in hypertrophic chondrocytes by differential display-PCR from a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) that is identical to that of connective tissue growth factor (CTGF). In the present study, we investigated the roles of CTGF in the proliferation and migration of vascular endothelial cells using its antisense oligonucleotide and antisense RNA, because angiogenesis into the hypertrophic zone of cartilage occurs at the final step of endochondral ossification. Immunohistochemical and immunofluorescence techniques revealed that not only hypertrophic chondrocytes but also endothelial cells in the cost-chondral junctions of mouse ribs were stained with an anti-CTGF antibody in vivo. Northern blot analysis revealed that CTGF was strongly expressed in chondrocytic cells as well as bovine aorta endothelial (BAE) cells in culture, but not in other types of cells such as osteoblastic cells. Its expression in BAE cells was greater in the growing phase than in the confluent phase. When one-half of a monolayer of a confluent culture of BAE cells had been peeled off, only the cells proliferating and extending into the vacant area were stained with the anti-CTGF antibody. The addition of an antisense oligonucleotide inhibited the proliferation and extension of the BAE cells into the vacant area. The antisense oligonucleotide also inhibited the proliferation of BAE cells in the rapidly proliferating phase. In a Boyden chamber assay, pretreatment with the antisense oligonucleotide markedly inhibited the migration of BAE cells. Furthermore, the abilities to proliferate and migrate of BAE cells, which were stably transfected with expression vectors that generate the antisense RNA of CTGF cDNA, were markedly lower than those of the control. These findings suggest that endogenous CTGF expression is involved in the proliferation and migration of BAE cells.View full abstractDownload PDF (7760K) -
Hiroto Kondo, Yuji Shibano, Teruo Amachi, Nora Cronin, Kohei Oda, Ben ...1998 Volume 124 Issue 1 Pages 141-147
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2) Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km=0.012mM, kcat=14.4 s-1, kcat/Km=1, 200M-1•s-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A.View full abstractDownload PDF (721K) -
Hideki Nakajima, Yoshiaki Miura, Tatsuya Yamagata1998 Volume 124 Issue 1 Pages 148-156
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe incubation of amphipathic lactosides with cultured cells was found to prime the glycosylation of lactosides whose oligosaccharide structures were exactly the same as those of glycosphingolipids produced by cells: B16 melanoma cells produced α2-3 sialylated lactosides; and PC12 cells, Galα1-4- and Galα1-3Galα1-4lactosides. Analysis of the cell-associated glycosylated products indicated that C16 series lactoside primers function 5-6 times more efficiently as acceptors than C12 series primers. The glycosylated lactosides were also secreted into the culture medium. Lactoside primers with longer hydrophobic chains hampered the release of glycosylated products from cells. The presence of an N-acyl chain in the lipophilic moiety of primers suppressed the secretion of glycosylated products. Owing to its overall availability, lactosides with the C12 alkyl chain were glycosylated 2-3 times more than C16 series lactosides and 1.4 times more than lactosides with the C12 acyl chain. C8-lactosides did not function as primers under the conditions of this study, but they were found to be the best acceptors for sialic acid transfer with the soluble enzyme fraction. The incubation of cells with 10 μM N-hexadecanoylaminoethyl-β-O-lactoside caused a 30% decrease in endogenous GM3 of B16 cells and a 34% decrease in Gb3Cer synthesis of PC12 cells. The results of the present study demonstrate that lactoside primers serve as an efficient means to inhibit endogenous glycosphingolipids in studies to clarify glycosphin-golipid functions.View full abstractDownload PDF (3940K) -
Takashi Nonaka, Yohichi Hashimoto, Koji Takio1998 Volume 124 Issue 1 Pages 157-162
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo zinc-metalloendopeptidases, GFMEP (accession number P81054) and POMEP (accession number P81055), from the fruiting bodies of two edible mushrooms, Grifola frondosa and Pleurotus ostreatus, respectively, specifically hydrolyze peptidyl-lysine bonds (-X-Lys-) in polypeptides. To understand detailed substrate specificities and kinetic characters of these enzymes, we have synthesized various intramolecularly quenched fluorescent peptide substrates and determined their kinetic constants with these substrates. Each synthesized fluorogenic peptide has a fluorescent residue, tryptophan, at its carboxyl terminus and a quenching group, dinitrophenyl (Dnp), at its amino terminus. Quenching of the Trp fluorescence in an intact substrate is relieved on hydrolysis of the -X-Lys- bond, giving rise to a continuous increase in fluorescence. The octapeptide substrate, Dnp-Ser-Thr-Ala-Thr-Lys-Leu-Ser-Trp, was an efficient substrate for both enzymes, the kcat/Km values being 9.8×106 and 7.0 ×105M-1•s-1for GF- and POMEP, respectively. Peptides with aspartic acid adjacent to the Lys residue were found to be poor substrates for both enzymes. Neither the shortest peptide, Dnp-Thr-Lys-Trp, nor peptides with substitution of L-Arg, L-ornithine, or D-Lys for Lys were hydrolyzed by either enzyme. These results confirmed the strict specificities of GF- and POMEP toward the peptide bond, -X-Lys-. Substitution of Co2+ for Zn2+ enhanced the activity, while the Km values were comparable. All peptides not hydrolyzed by either enzyme had inhibitory effects on GFMEP activity. The active site structure is discussed in relation to these observations.View full abstractDownload PDF (1719K) -
Kaoru Konishi, Tsuyoshi Katoh, Fumi Morita, Michio Yazawa1998 Volume 124 Issue 1 Pages 163-170
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSingle-headed myosin was prepared by digestion of porcine aorta smooth muscle myosin with Staphylococcus aureus V8 protease in the presence of actin. The single-headed myosin preparation contained intact light chains, a rod fragment of a heavy chain, and a heavy chain of which only a minor fraction contained a nick in the head segment. Below 0.2M NaCl, the single-headed myosin showed a decrease in Ca2+-ATPase activity and an increase in the elution time on gel filtration HPLC in a phosphorylation-dependent manner, indicating a phosphorylation-dependent conformational transition between the extended and folded forms. These conformations were confirmed by electron microscopic observation of rotary-shadowed samples of single-headed myosin. However, the conformational transition of single-headed myosin occurred in a narrower range with lower salt concentrations than that of double-headed myosin. The filament assembly of single-headed myosin was thus facilitated and phosphorylation-independent. The single-headed myosin also showed high actin-activated ATPase activity independent of phosphorylation. These results indicate that the two-headed structure of smooth muscle myosin is not essential for the conformational transition, but is required for the phosphorylation-dependent regulation of enzymatic activity and filament assembly.View full abstractDownload PDF (6339K) -
Nobuhiro Ishida, Miyuki Ito, Shigemi Yoshioka, Ge-Hong Sun-Wada, Masao ...1998 Volume 124 Issue 1 Pages 171-178
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe recently described the cloning of putative human CMP-sialic acid transporter (hCST) cDNA [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. The hCST cDNA coded for a hydrophobic protein with an amino acid sequence showing a high degree of similarity (92% identity) to that of murine CMP-sialic acid transporter. In this report, we demonstrate that hCST corrects the CMP-sialic acid transporter-deficient phenotype of CHO-derived Lec2 cells, as judged from the recovery of WGA-sensitivity by transformants, and the recovery of CMP-sialic acid transporting ability by microsomal vesicles prepared from them. A peptide antibody against the C-terminus of the hCST protein detected the cDNA products expressed in the microsomes of the transformants. The subcellular localization of the hCST protein in the Golgi membrane was demonstrated by immunofluorescence microscopy, using the hCST-specific antibody. These result clearly indicate that hCST cDNA encodes the human CMP-sialic acid transporter protein. Plasma membrane-selective permeabilization combined with immunofluorescence microscopy provided strong evidence that the C-terminus of the human CMP-Sia transporter is exposed to the cytosol on the outer surface of the Golgi membrane.View full abstractDownload PDF (3309K) -
Jamil Ahsan Kazi, Osamu Nakamura, Tomokazu Ohnishi, Naokatu Arakaki, T ...1998 Volume 124 Issue 1 Pages 179-186
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat fetuin, a counterpart of human α2-HS glycoprotein and bovine fetuin, shows strong intermolecular binding and association with other serum proteins. Therefore, to measure its concentration in rat serum, we pretreated serum samples with 1% SDS plus 5% (ca. 0.7M) 2-mercaptoethanol at 100°C for 3min, and then subjected them to SDS-PAGE under reducing conditions followed by Western blotting. We found that the fetuin concentrations in normal rat serum determined by Western blotting were 2.5-4.5mg/ml. These concentrations were three orders of magnitude higher than the previously reported concentrations. We also tried to measure the fetuin concentration in rat serum by means of an enzymelinked immunosorbent assay after treatment of the samples with 0.1% sodium dodecyl sulfate (SDS) plus 10mM 2-mercaptoethylamine at 100°C for 3min, but it gave a value of about 1/4 of that on Western blotting. Rat fetuin is expressed mainly in the liver, with a peak 2-4 weeks after birth, as determined by Northern blot analysis. The fetuin mRNA level in the liver changes almost in parallel with its serum concentration. The tibia also expresses fetuin, but much less than the liver.View full abstractDownload PDF (4583K) -
Tomoyuki Mine, Yuji Morita, Atsuko Kataoka, Tohru Mizushima, Tomofusa ...1998 Volume 124 Issue 1 Pages 187-193
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe detected chloramphenicol/H+ antiport activity in membrane vesicles of Escherichia coli and cloned a gene for the antiporter from chromosomal DNA of E. coli. Introduction of the gene into E. coli cells conferred resistance to chloramphenicol and ethidium. A slight increase in resistance to acridine orange was also observed. Elevated chloramphenicol efflux and ethidium efflux were observed in cells harboring a plasmid carrying the gene. Addition of chloramphenicol to the assay mixture reduced the efflux of ethidium. Elevated chloramphenicol/H+ antiport activity was observed in membrane vesicles prepared from cells harboring the plasmid. The pH optimum for the activity was 6.5. We sequenced the gene and deduced the amino acid sequence of its product. A sequence homology search revealed that it was same as that of Cmr (or MdfA). Thus, it became clear that Cmr (MdfA) is the chloramphenicol(and ethidium)/H+ antiporter.View full abstractDownload PDF (760K) -
Keiko Nohara, Manabu Kunimoto, Hidekazu Fujimaki1998 Volume 124 Issue 1 Pages 194-199
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGangliosides have long been implicated in T cell activation. GD1c with two N-glycolylneuraminic acids [GD1c (NeuGc, NeuGc)] is the predominant ganglioside in rat T cells. In the present study, the anti-GD1c (NeuGc, NeuGc) mAb, AC1, which binds to the NeuGcα2-8Neu-Gcα2- sequence, was found to enhance Con A-activated cellular proliferation at a concentration at which AC1 alone did not activate the cells. The potentiation by AC1 was observed more consistently and effectively in the cellular activation elicited by cross-linking of anti-CD3 and anti-CD4, rather than in the cell growth induced by immobilized anti-CD3 alone. Moreover, the combination of immobilized anti-CD4 and soluble AC1 had a remarkable mitogenic effect. In addition, we have demonstrated the existence of a 100 kDa protein in rat T cell lysates which reacts with AC1 on Western blots, and this interaction is abolished by sialidase-treatment of the membrane. Pronase treatment of the T cells, which rendered the 100 kDa protein undetectable on Western-blotting, reduced the number of AC1-positive cells by 40-50% on flow cytometry. On the other hand, all cells became AC1-negative after sialidase treatment. These findings indicated that AC1 reacts with both GD1c (NeuGc, NeuGc) and the 100 kDa glycoprotein on rat T cells. Taken together, these results predict the presence of a novel regulatory mechanism of T cell activation involving CD4 and the NeuGcα2-8NeuGcα2- sequence.View full abstractDownload PDF (1916K) -
Yasuzo Nishina, Kyosuke Sato, Retsu Miura, Kunio Matsui, Kiyoshi Shiga1998 Volume 124 Issue 1 Pages 200-208
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS[4-Carbonyl-18O]-enriched lumiflavin, riboflavin, and FMN were prepared by incubating each corresponding non-labeled flavin in 1M Na18OH (H218O) at 25°C. [4-Carbonyl-18O] FAD was prepared from the corresponding riboflavin by using FAD synthetase. Isotope effects by [4-carbonyl-18O]-labeling confirmed that the 1, 709-cm-1 band in the IR spectrum of lumiflavin and the 1, 711-cm-1 band in the Raman spectrum of FAD are mainly derived from C (4)=O stretching vibrational mode. The 1, 605-cm-1 Raman band of the anionic reduced flavin in the purple intermediate of D-amino acid oxidase (DAO) with D-proline or D-alanine does not shift in DAO reconstituted with [4-carbonyl-18O]FAD, although it shifts with [4, lOα-13C2]- or [4α-13C] FAD. Thus the band is mainly due to the C(4α)=C(10α) stretching vibrational mode and includes no contribution from C (4)=O stretching vibration. The band frequencies cover a fairly wide range (1, 602-1, 620cm-1) depending on the enzymes. The frequencies of the reduced flavin in the purple intermediates of the dehydrogenases (medium-chain acyl-CoA, short-chain acyl-CoA, and isovaleryl-CoA dehydrogenases) are higher than those of the oxidases (DAO and L-phenylalanine oxidase). This indicates that the C (4α)=C (10α) bond order of reduced flavin in the dehydrogenases with the low reactivity for molecular oxygen is stronger than that in the oxidases with high reactivity. Therefore, the band frequency of C (4α)=C (10α) stretching may serve as an indicator of the reactivity of flavoprotein with molecular oxygen. Furthermore, strong hydrogen bonding of flavin at the N(1) moiety with the hydroxyl group of Thr136 in MCAD is probably responsible for the strong bond of the C (4α)=C (10α) of reduced flavin in the dehydrogenase.View full abstractDownload PDF (5624K) -
Yo Sato, Hea Nam Hong, Nobuaki Yanai, Masuo Obinata1998 Volume 124 Issue 1 Pages 209-216
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSErythropoiesis is regulated by the hematopoietic microenvironment of the spleen and fetal liver in mice. We showed that established stromal cells of these organs selectively support erythropoiesis in vitro. To identify the cell surface molecule (s) on the stromal cells involved in erythropoiesis, we raised monoclonal antibodies against the stromal cells. Using one of these antibodies (11D), we cloned a new gene named smap-1 (stroma membrane-associated protein-1). The SMAP-1 protein deduced from the nucleotide sequence of the cDNA was a newly identified membrane protein with direct repeats of the KKD/E units found in MAP1A and MAP1B, which is involved in the association with microtubules. By transfection of the anti-sense smap-1 cDNA into the stromal cells, we showed that SMAP-1 may have a stimulatory effect on stroma-supported erythropoiesis. Its expression was detected in the yolk sac, fetal liver, spleen, and bone marrow, and was correlated with their erythropoietic activity.View full abstractDownload PDF (5909K) -
Masatoshi Nomura, Ken Kawabe, Sumie Matsushita, Sanae Oka, Osamu Hatan ...1998 Volume 124 Issue 1 Pages 217-224
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAd4BP/SF-1 is a transcription factor essential for the development of the adrenal gland and the gonads as well as for the maintenance of their functions through regulating tissue-specific gene transcription. In the whole body, hypothalamo-pituitary-gonadal and -adrenal axes are known to play prominent roles in mediating the function of the gonads and adrenal. In this study, the effects of the tropic peptide hormones secreted by the pituitary on the regulation of the rat Ftz-F1 (rFtz-F1) gene encoding Ad4BP/SF-1 were investigated. Immunochemical studies revealed that Ad4BP/SF-1 was expressed even in the adrenal cortex of hypophysectomized rats. Such persistent expression of Ad4BP/SF-1 was also observed in the testes and ovaries of the hypophysectomized animals. In contrast to Ad4BP/SF-1, the expressions of steroidogenic P450s were reduced significantly. The transcriptional activities of the endogenous and transfected rFtz-F1 genes were examined with Y-1 and I-10 cells derived from mouse adrenocortical and testicular Leydig cell tumors, respectively. Neither gene appeared to be activated significantly by cAMP, whereas both endogenous and exogenous CYP11A genes encoding P450 (SCC) were activated. Taken together, these observations indicate that the expression of the rFtz-F1 gene is mainly regulated by a mechanism independent of the neuroendocrine axes.View full abstractDownload PDF (4907K) -
Jau-Song Yu1998 Volume 124 Issue 1 Pages 225-230
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFreshly isolated protein phosphatase 2A (PP2A) was highly active as to the dephosphorylation of protein substrates, but lost most of its spontaneous activity on prolonged storage, and was converted to a latent form requiring Mn2+ or Co2+ ions for activity. In this report, we show that the latent form of PP2A can be activated by the Fe2+/ascorbate system. Activation of the phosphatase required both Fe2+ ions and ascorbate, and the level of activation was dependent on the concentrations of both Fe2+ ions and ascorbate. Both the holoenzyme and catalytic subunit of phosphatase 2A could be activated by the Fe2+/ascorbate system, indicating that direct modulation of the catalytic subunit of the phosphatase by the Fe2+/ascorbate system may cause this activation. Several common divalent metal ions, including Ca2+, Mg2+, Cu2+, Zn2+, and Ni2+ ions, cannot cooperate with ascorbate to activate the phosphatase. Dithiothreitol, a SH-containing reducing agent, could replace ascorbate in the Fe2+/ascorbate system to activate the phosphatase, whereas H2O2 a strong oxidizer, significantly diminished the phosphatase activation by the Fe2+/ascorbate system. The results indicate that iron ions stabilized in the +2 state by reducing agents can activate the phosphatase. Overall, the present study provides initial biochemical evidence suggesting that Fe2+ could be a biologically important metal ion cofactor responsible for PP2A activation.View full abstractDownload PDF (1939K) -
Kyoko Hiramatsu, Hiroko Miura, Sachiko Kamei, Kentaro Iwasaki, Masao K ...1998 Volume 124 Issue 1 Pages 231-236
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN1, N12-Diacetylspermine (DiAcSpm)-specific antibodies were raised in rabbits, using N-acetylspermine coupled to mercaptosuccinylated BSA via N-(4-maleimidobutyryloxy)-succinimide as an antigen. Highly DiAcSpm-specific antibodies were enriched from crude sera through a series of affinity-based fractionations. A competitive ELISA system, intended for measuring DiAcSpm in solution, was constructed using this antibody preparation, with N-acetylspermine coupled to a synthetic peptide via N-(8-maleimidocapryloxy)-succinimide as a solid phase antigen. The K1 value for DiAcSpm with this competitive ELISA system was 33 nM, and the cross-reactivity with DiAcSpm, AcSpm, DiAcSpd, N1-AcSpd, and N8-AcSpd was 100, 0.29, 0.20, 0.033, and 0.055%, respectively. This procedure can be applied to the determination of DiAcSpm in human urine samples, giving highly reproducible results. The coefficients of variation obtained were 6.7 and 4.2% for within-run and between-run precision, respectively. The correlation coefficient between DiAcSpm concentrations in urine estimated by ELISA and those by HPLC analysis was calculated to be 0.99, and the regression equation was expressed as y=1.04x+0.026 μM.View full abstractDownload PDF (680K) -
Lisa A. Main, Kuniko Okumura-Noji, Taira Ohnishi, Shinji Yokoyama1998 Volume 124 Issue 1 Pages 237-243
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rate of the non-directional transfer of cholesteryl ester and triglyceride by human cholesteryl ester transfer protein (CETP) was measured between human plasma lipoproteins by monitoring fluorescence spectrum of pyrene-labeled lipid. The transfer rates between high density lipoproteins (HDLs) and between low density lipoproteins (LDLs) were both directly proportional to the substrate lipid concentration within the physiological range of the lipoprotein concentration. Higher preference of cholesteryl ester transfer to triglyceride was demonstrated with HDL than LDL. Although the highly selective binding of CETP to HDL was observed in the electrophoretic analysis, the transfer rate was only moderately higher with HDL for cholesteryl ester and not so at all for triglyceride. In addition, the rate of cholesteryl ester transfer between LDLs was uninfluenced by the presence of a small amount of HDL that is just sufficient to absorb all the CETP in the reaction mixture. The results indicated the preferential transfer of cholesteryl ester over triglyceride by CETP in the interaction with HDL in non-directional lipid transfer reaction among lipoproteins. However, the apparent binding of CETP to HDL does not seem to play an essential role in this type of lipid transfer by CETP.View full abstractDownload PDF (1646K) -
Kunio Fujiwara, Yoshie Kaminishi, Tsunehiro Kitagawa, Daisuke Tsuru, M ...1998 Volume 124 Issue 1 Pages 244-249
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have developed three mouse monoclonal antibodies (mAb) of types IgG1 and IgG2b, i.e. anti-acetylspermine (Ac-Spm)-1 and 2 (ACSPM-1 and 2), and anti-acetylspermine (Ac-Spm)-3 (ACSPM-3), respectively, against Ac-Spm conjugated to bovine serum albumin via a heterobifunctional cross-linker, N-(γ-maleimidobutyryloxy) succinimide (GMBS). Among these mAbs, ACSPM-2 was the most useful for the development of an enzyme-linked immunosorbent assay (ELISA) for acetylpolyamines (Ac-PAs) with glutaraldehyde (GA)-conjugated N1, N12-diacetylspermine (2Ac-Spm) or acetylspermine (Ac-Spm) as the solid phase antigen. However, GMBS-conjugated Ac-Spm did not behave as a solid phase antigen in the competitive ELISA. The ELISA is based on the principle of competition between an analyte and the conjugated antigen for the mAb, followed by immunoreaction with biotinylated anti-mouse immunoglobulin and horseradish peroxidase-streptavidin. The ACSPM-2 mAb reacted with 2Ac-Spm to the highest degree, followed by Ac-Spm, N1-acetylspermidine (N1-Ac-Spd), N1, N8-diacetylspermidine (2Ac-Spd), and spermine (Spm), the EC50 values being 0.06, 0.25, 7.0, 10, and 60 μM, respectively, but exhibited almost no cross-reaction with other polyamine-related compounds or amino acids. The method was used to determine the urinary Ac-PA levels in healthy subjects, the average value of 0.36 μg of 2Ac-Spm/g creatinine (n=16) being obtained. The ACSPM-2 ELISA for 2Ac-Spm, which was the PA most relevant to the analysis of human urine among the five PA analogs mentioned above, might have potential for elucidation of the correlation of urinary 2Ac-Spm levels in cancers.View full abstractDownload PDF (654K) -
1998 Volume 124 Issue 1 Pages 250
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (676K)
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