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Volume 133 (2003)
- Issue 6 Pages 699-
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- Issue 4 Pages 397-
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- Issue 2 Pages 155-
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Volume 133, Issue 4
Displaying 1-20 of 20 articles from this issue
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Osamu Kuge, Masahiro Nishijima2003 Volume 133 Issue 4 Pages 397-403
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn mammalian cells, phosphatidylserine (PtdSer) is synthesized through the action of the endoplasmic reticulum enzymes, PtdSer synthase 1 and 2, and the decarboxylation of PtdSer accounts for the majority of phosphatidylethanolamine (PtdEtn) synthesis. PtdSer decarboxylation for PtdEtn formation occurs in the mitochondria. In addition, the transport of PtdSer from the endoplasmic reticulum to the mitochondria is probably a rate limiting step for PtdEtn synthesis through the decarboxylation pathway. Therefore, the regulation of PtdSer synthesis and its intracellular transport appear to be essential events for the maintenance of normal cellular PtdSer and PtdEtn levels. Here we describe the current understanding of the regulation of PtdSer biosynthesis and the transport of PtdSer from the ER to the mitochondria in mammalian cells.View full abstractDownload PDF (887K) -
Kentaro Mori, Tadashi Furusawa, Tasashi Okubo, Toshiaki Inoue, Shuntar ...2003 Volume 133 Issue 4 Pages 405-413
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe previously cloned a gene for a novel myosin (called MysPDZ) containing a PDZ-domain from bone marrow stromal cells. This new myosin is found in humans and classified as one of the class XVIII myosins (Myo18A). Here, we report the hematopoietic cell-specific splicing isoform (MysPDZβ) in addition to the previously reported isoform (MysPDZα). Combined with mouse genome sequence data, the overall genome structure and generation of the two spliced isoforms are deduced. The MysPDZβ protein lacks a PDZ-domain in the N-terminal region. Studies of the subcellular localization of the two spliced isoforms indicated that MysPDZα containing the PDZ domain co-localizes with the ER-Golgi complex, while MysPDZβ, which lacks the PDZ domain, localizes diffusely in the cytoplasm. These results suggest that the isoforms differ in their subcellular localization and may have different functions in membrane ruffling and membrane traffic pathways. The PDZ-containing spliced isoform (MysPDZα) is not expressed in bone marrow hematopoietic cells, whereas MysPDZβ lacking the PDZ is specifically expressed in most hematopoietic cells. It is noted that neither isoform is expressed in red blood cells. Interestingly, MysPDZα was detected in mature but not in immature macrophages, and its level increased after the induction of differentiation of M1 cells, suggesting a functional role of PDZ-containing myosin in macrophages.View full abstractDownload PDF (5797K) -
Yoshimitsu Takahashi, Akio Toh-e, Yoshiko Kikuchi2003 Volume 133 Issue 4 Pages 415-422
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSUMO/Smt3, a ubiquitin-like modifier, is known to conjugate other proteins and modulate their functions in various processes. Recently, Ull1/Sizl was discovered as a novel PIAS-type E3 required for septin sumoylation in yeast. We demonstrate here that the second PIAS-type Nfi1/Siz2 is also a SUMO ligase. It interacted with Smt3, SUMO/Smt3 conjugating enzyme Ubc9 and a septin component Cdc3 in the twohybrid system. The region containing the RING-like domain of Nfi1/Siz2 bound directly to Ubc9 and Cdc3, but not to Smt3. Nfi1/Siz2 stimulated Smt3 conjugation to Cdc3 in vitro. In this in vitro system, Smt3 formed polymeric chains in the presence of higher concentrations of E1 and E2 enzymes. When the lysine15 residue of Smt3 was substituted with arginine, Smt3 chain-polymerization was abolished. Using this polysumoylation-deficient mutant Smt3, we found that Cdc3 and Nfi1/Siz2 were modified with Smt3 at multiple sites. Finally we found that the C-terminal truncated form of Ull1/Siz1 was mis-localized in vivo, but retained its SUMO ligase activity in vitro. We discuss the regulation of these SUMO ligases in vivo and in vitro.View full abstractDownload PDF (6218K) -
Jingjing Jiang, Eiji Yamato, Jun-ichi Miyazaki2003 Volume 133 Issue 4 Pages 423-427
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe previously demonstrated that cytokine expression following intramuscular gene transfer of a naked plasmid is increased 2 logs by in vivo electroporation, but the relatively low expression levels of the encoded protein is still a limitation for successful gene therapy and gene function studies. We recently reported that the serum viral IL-10 levels achieved by electroporation-mediated intramuscular delivery of pCAGGS-vIL10, a viral IL-10-expressing plasmid, can be further enhanced by modifying the plasmid into an immunoglobulin fusion protein expression plasmid, pCAGGS-vIL10/Fc. Here we examined the applicability of this approach to the expression of an endogenous cytokine, IL-10, in two different inbred mouse strains. We obtained sustained high serum levels of IL-10 in C3H/HeJ mice (C3H), but the level and duration of the gene expression was mouse-strain dependent. Although the serum IL-10 level was also increased by using the IL-10/Fc gene plasmid in C57BL/6 mice (B6), IL-10/Fc and a luciferase reporter showed significantly lower levels in B6 than in C3H mice, and the persistence of pCAGGS-IL10/Fc expression ranged from several days in B6 mice to more than one month in C3H mice. These results suggest that the electroporationmediated intramuscular delivery of the immunoglobulin fusion protein expression plasmid is simple and very efficient, but mouse strain differences in transgene expression should be taken into consideration in its use.View full abstractDownload PDF (1627K) -
Takashi Sakai, Li Liu, Yuji Shishido, Kiyoshi Fukui2003 Volume 133 Issue 4 Pages 429-436
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSEC cells are characterized by their potent capacity to differentiate into several cell types, such as mesoderm-like cells, endoderm-like cells, or ectoderm-like cells. By subtracting the mRNAs expressed by one EC cell clone, F9 cells, with the mRNAs expressed by another EC cell clone, P19 cells, we identified six novel genes that are expressed selectively by F9 cells. One of these genes (Nucling) encodes a polypeptide of 1411 amino acids containing an ankyrin repeat, aspartyl protease motif, a leucine zipper motif, and two t-SNARE coiled-coil domains. Northern blot analyses revealed the Nucling mRNA to be detected predominantly in heart, liver, kidney and testis, but not in brain or spleen. Immunostaining analyses revealed a unique feature of Nucling that the transiently expressed protein forms aggregates exclusively around nuclear membranes. Moreover, the expression level of the Nucling gene transcript increases progressively during the early developmental stages in mice, and specifically at cardiomuscular differentiation in vitro and in vivo. These results suggest that Nucling may play some role in the gene regulation of cell differentiation during embryonal development.View full abstractDownload PDF (3976K) -
Toshihiro Tsudzuki2003 Volume 133 Issue 4 Pages 437-443
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSI attempted to isolate synaptic vesicles by gel filtration. The rat brain synaptic vesi-cles in a synaptosomal lysate were collected by ammonium sulfate salting-out and fractionated on a Sephacryl S-500 with a mean exclusion size of 200nm. Peak I at the void volume contained large vesicular membranes and coated vesicles besides synaptic vesicles; Peak II consisted almost entirely of small agranular synaptic vesicles of 40-50nm diameter; and Peak III comprised soluble proteins. Western blotting revealed that components of 72 kDa in peaks I and II reacted with an anti-H+-ATPase A-subunit antibody [Moriyama et al. (1995) FEBS Lett. 367, 233-236]. When examined for Mg2+-ATPase activity, peak I showed specific activity of 4.52 (μmol ATP hydrolyzed/mg protein/30 min), while that of peak II was as low as 0.22. As estimated from the inhibition by bafilomycin A1 [Bowman et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7972-7976], the percentage of H+-ATPase as to total Mg2+-ATPase, 18-22%, was unchanged, indicating no accumulation of the H+-ATPase in peak II even on the chromatography. In brief, the small agranular synaptic vesicles in peak II showed little or no Mg2+-ATPase activity, although they reacted with the H+-ATPase antibody. The reason for this is obscure. Mg2+-ATPase might not be a constituent of small agranular synaptic vesicles of rat brain.View full abstractDownload PDF (3429K) -
Tomoaki Ando, Terumichi Tanaka, Yo Kikuchi2003 Volume 133 Issue 4 Pages 445-451
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe bacterial RNase P ribozyme can accept a hairpin RNA with CCA-3' tag sequence as well as a cloverleaf pre-tRNA as substrate in vitro, but the details are not known. By switching tRNA structure using an antisense guide DNA technique, we examined the Escherichia coli RNase P ribozyme specificity for substrate RNA of a given shape. Analysis of the RNase P reaction with various concentrations of magnesium ion revealed that the ribozyme cleaved only the cloverleaf RNA at below 10mM magnesium ion. At 10mM magnesium ion or more, the ribozyme also cleaved a hairpin RNA with a CCA-3' tag sequence. At above 20mM magnesium ion, cleavage site wobbling by the enzyme in tRNA-derived hairpin occurred, and the substrate specificity of the enzyme became broader. Additional studies using another hairpin substrate demonstrated the same tendency. Our data strongly suggest that raising the concentration of metal ion induces a conformational change in the RNA enzyme.View full abstractDownload PDF (2534K) -
Yukinobu Goso, Tomoaki Ikezawa, Makoto Kurihara, Masaki Endo, Kyoko Ho ...2003 Volume 133 Issue 4 Pages 453-460
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA novel anti-mucin monoclonal antibody (mAb), designated RGM23, was developed against mucin purified from rat gastric mucosa. RGM23 reacted with the mucin attached to the ELISA well. The reactivity was lost by trypsin treatment, but not by periodate oxidation, indicating that RGM23 recognizes the peptide moiety of the mucin molecule. Histochemical study showed that RGM23 stained the corpus and antral surface mucosa of rat stomach, but not their glandular mucosa, nor duodenal, small intestinal or large intestinal mucosa. The area stained with RGM23 was coinci-dent with that stained with 45M1, a mAb reacting with MUC5AC mucin. Examination of the mucin subunits extracted from rat stomach by Sepharose CL-4B and Q-Sepha-rose chromatography and CsTFA equilibrium centrifugation showed that RGM23 reacted with the surface mucous cell-type mucins that were stained with periodate-Schiff (PAS) and reacted with mAb RGM21. The gastric gland-type mucin, which reacted with mAb HIK1083, did not react with RGM23. On Q-Sepharose chromatogra-phy, a part of the RGM21-reactive mucins was only faintly stained with PAS and did not react with RGM23. The results together indicated that RGM23 probably reacted with the rat MUC5AC (rMuc5AC) mucin present in the surface mucosa of the stomach, and that the surface mucosal cells in rat stomach may contain mucin bearing non-rMuc5AC core protein in addition to rMuc5AC mucins.View full abstractDownload PDF (2672K) -
Naoki Takaya, Seigo Kuwazaki, Yoshiaki Adachi, Sawako Suzuki, Tomoko K ...2003 Volume 133 Issue 4 Pages 461-465
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSInduction of the mitochondrial nitrate-respiration (denitrification) system of the fungus Fusarium oxysporum requires the supply of low levels of oxygen (O2). Here we show that 02 and nitrate (NO3-) respiration function simultaneously in the mitochondria of fungal cells incubated under hypoxic, denitrifying conditions in which both O2 and NO3- act as the terminal electron acceptors. The NO3- and nitrite (NO2-) reduct-ases involved in fungal denitrification share the mitochondrial respiratory chain with cytochrome oxidase. F. oxysporum cytochrome c549 can serve as an electron donor for both NO2- reductase and cytochrome oxidase. We are the first to demon-strate hybrid respiration in respiring eukaryotic mitochondria.View full abstractDownload PDF (584K) -
Takuji Oyama, Hideo Miyake, Masami Kusunoki, Yasunori Nitta2003 Volume 133 Issue 4 Pages 467-474
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe crystal structures of β-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large con-formational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 Å away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, α-EPG and α-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the β-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.View full abstractDownload PDF (5330K) -
Tatsuya Fukushima, Shu Ishikawa, Hiroki Yamamoto, Naotake Ogasawara, J ...2003 Volume 133 Issue 4 Pages 475-483
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA Bacillus subtilis veg mutant exhibited a small reduction of absorbance, a large reduction of hexosamine release, and slow dipicolinic acid release from spores during germination with L-alanine as a germinant. But veg spores exhibited normal resistance to chloroform, 2-propanol, lysozyme, and heat. SDS-polyacrylamide gel electrophoresis of spore coat proteins revealed no difference in coat proteins between the wild type and the veg mutant. Northern and veg-lacZ fusion analyses indicated that the veg gene is transcribed in both the vegetative growth and sporulation phases, and primer extension analysis indicated an identical transcriptional start point in both phases. The upstream sequence suggests that veg is transcribed by EσA RNA polymerase. Veg-GFP fusion protein was detected for vegetative cells and spores, but the fluorescence of mother cells disappeared completely in the late sporulation phase. Decoated spores containing Veg-GFP exhibited a strong green fluorescence in the core, but much weaker fluorescence in the cortex.View full abstractDownload PDF (5332K) -
Bei-Wen Ying, Tsutomu Suzuki, Yoshihiro Shimizu, Takuya Ueda2003 Volume 133 Issue 4 Pages 485-491
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHere we describe the application of an in vitro translation system for genetic screening, to identify RNA-binding proteins that bind to their own mRNAs. It is a relatively novel system designed using an advanced cell-free translation system reconstructed with purified translational components. Due to the absence of nucleases and proteases, the complex of mRNA and nascent polypeptide synthesized in this system is expected to exhibit high stability ensuring the following efficient selection toward the protein. Escherichia coli ribosomal protein S15, which is known to bind to its own mRNA, was employed as a model molecule to evaluate the system. Wild-type S15 mRNA specifically isolated from a mutant mRNA lacking the secondary structure responsible for binding the S15 protein accumulated markedly after several rounds of selection-amplification. The success of this selection demonstrates the potentiality of the systematic screening of self-mRNA targeting proteins through direct and functional selection. This strategy as a method to identify peptides or proteins that bind to their own mRNAs, is of general interest and has different potential applications, such as, the identification of new regulatory proteins or peptide motifs for RNA recognition, the study of self-mRNA-protein interactions, etc.View full abstractDownload PDF (2521K) -
Kayoko Saso, Takahiro Ito, Shunji Natori, Kazuhisa Sekimizu2003 Volume 133 Issue 4 Pages 493-500
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTranscription elongation factor S-II was originally purified as a specific stimulator of transcription by RNA polymerase II. Recent studies suggest that S-II participates in gene-specific transcriptional activation in vivo, despite the fact that it directly binds RNA polymerase II and does not recognize specific DNA sequences. In this study, under the hypothesis that S-II requires co-factors to regulate the expression of specific-genes in vivo, we searched for factors that directly interact with S-II using a yeast two-hybrid system, and isolated a novel nuclear protein, FESTA. FESTA is expressed specifically in kidney and spleen, supporting our notion that S-II participates in gene-specific regulation. Two mRNA isoforms of FESTA encoding proteins with different sizes were identified and named FESTA-S and FESTA-L. FESTA contains a serine-rich region and a C-terminal tail that are highly similar to those of the ELL-associated factor EAF1. Reporter gene assays indicated that both GAL4-FESTA-S and GAL4-FESTA-L fusion proteins have trans-activating ability. Furthermore, deletion of the C-terminal tail of FESTA dramatically reduced its trans-activating ability and abolished its interaction with S-II. This study is the first report of a transcriptional activator that directly interacts with S-II and contains a transcriptional activation domain that cooperates with S-II via direct interaction.View full abstractDownload PDF (2985K) -
Kaori Nagao, Shigeo Takenaka, Ryoichi Yamaji, Hiroshi Inui, Yoshihisa ...2003 Volume 133 Issue 4 Pages 501-505
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn cultured rat vascular smooth muscle cells (VSMC), inducible nitric oxide synthase (iNOS) expression evoked by interleukin-1β(IL-1β) or tumor necrosis factor-α was greatly enhanced in hypoxia (2% O2), compared to in normoxia. In contrast, iNOS induction by interferon-γ, lipopolysaccharide or their combination was barely influenced by hypoxia. These results indicate that iNOS induction is regulated by hypoxia in different manners, depending on the stimuli in VSMC. Nitric oxide (NO) production in response to stimulation with interferon-γ plus lipopolysaccharide was significantly decreased in hypoxia, due to a decrease in the concentration of O2 as a substrate. In contrast, the level of NO production in hypoxia was almost the same as that in normoxia when the cells were stimulated by IL-1β. In addition, cGMP increased in response to IL-1β in hypoxia to a level comparable to that in normoxia. Thus, it seems that the IL-1β-induced expression of iNOS is up-regulated in hypoxia to compensate for a decrease in the enzyme activity due to the lower availability of O2 as a substrate, and consequently a sufficient amount of NO is produced to elevate cGMP to an adequate level. In addition, the IL-1β-induced synthesis of tetrahydrobiopterin, a cofactor for iNOS, was also greatly stimulated by hypoxia in VSMC.View full abstractDownload PDF (665K) -
Hiroshi Nakashima, Satoshi Fukuchi, Ken Nishikawa2003 Volume 133 Issue 4 Pages 507-513
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIt is known that in thermophiles the G+C content of ribosomal RNA linearly correlates with growth temperature, while that of genomic DNA does not. Although the G+C contents (singlet) of the genomic DNAs of thermophiles and methophiles do not differ significantly, the dinucleotide (doublet) compositions of the two bacterial groups clearly do. The average amino acid compositions of proteins of the two groups are also distinct. Based on these facts, we here analyzed the DNA and protein compositions of various bacteria in terms of the optimal growth temperature (OGT). Regres-sion analyses of the sequence data for thermophilic, mesophilic and psychrophilic bacteria revealed good linear relationships between OGT and the dinucleotide compositions of DNA, and between OGT and the amino acid compositions of proteins. Together with the above-mentioned linear relationship between ribosomal RNA and OGT, the DNA and protein compositions can be regarded as thermostability measures for RNA, DNA and proteins, covering a wide range of temperatures. Both the DNA and proteins of psychrophiles apparently exhibit characteristics diametrically opposite to those of thermophiles. The physicochemical parameters of dinucleotides suggested that supercoiling of DNA is relevant to its thermostability. Protein stability in thermophiles is realized primarily through global changes that increase charged residues (i.e., Glu, Arg, and Lys) on the molecular surface of all proteins. This kind of global change is attainable through a change in the amino acid composition coupled with alterations in the DNA base composition. The general strategies of thermophiles and psychrophiles for adaptation to higher and lower temperatures, respectively, that are suggested by the present study are discussed.View full abstractDownload PDF (851K) -
Satoshi Kagiwada, Rie Zen2003 Volume 133 Issue 4 Pages 515-522
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn the yeast Saccharomyces cerevisiae, the expression of phospholipid biosynthetic genes, including the INO1 gene (encoding inositol-l-phosphate synthase), is coordinately regulated by a cis-acting transcriptional element, UASINO (inositol-sensitive upstream activating sequence). For this paper we studied the effect of SCS2 disruption on INO1 expression. SCS2 encodes a type II membrane protein and its deletion leads to inositol auxotrophy at temperatures above 34°C. We found that the expression of the INO1 gene was reduced in the scs2Δ strain even when the cells were cultured under derepressing conditions for INO1 expression. However, the β-galactosidase gene fused with the INO1 promoter region was expressed normally in the scs2Δ strain. The phospholipid composition of scs2Δ cells was not dramatically changed compared with wild-type cells at 28°C, but the phosphatidylinositol level was reduced in scs2Δ cells cultured at 34°C. In addition, elevated phosphatidylcholine synthesis through the CDP-choline pathway was observed in the scs2Δ strain, and the disruption of genes involved in the CDP-choline pathway rescued the INO1 expression defect of the scs2Δ strain. These results indicate that Scs2p can contribute to coordinated phospholipid metabolism including INO1 expression by regulating phosphatidylcholine synthesis through the CDP-choline pathway.View full abstractDownload PDF (3024K) -
Naoshi Yamazaki, Yasuhisa Yamanaka, Yoshiko Hashimoto, Takayuki Hirama ...2003 Volume 133 Issue 4 Pages 523-532
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMuscle-type carnitine palmitoyltransferase I (M-CPTI) is the key enzyme for fatty acid β-oxidation in heart and skeletal muscles and in adipose tissue. So far, M-CPTI mRNA has been detected in white adipocytes from epididymal fat pads of rats and humans, but not in mouse adipocytes. To characterize the gene expression of M-CPTI in mice, we isolated the genomic DNA encoding mouse M-CPTI and determined its transcription initiation site. As a result, the mouse M-CPTI gene seemed to have multiple initiation sites, as in the case of the rat and human genes. Furthermore, the con-served nucleotide sequence of the response element for peroxisome proliferators was shown to exist in the upstream of the mouse gene as in that of the rat and human genes. From these observations, we suggest that the anomalous expression of M-CPTI in mouse adipocytes reported previously may be regulated by factors other than peroxisome proliferators. Previously, we reported that there were transcripts containing regions of both CK/EK-β and M-CPTI genes in humans. In this study, we found that such transcripts also exist in rodents and that the amounts of the transcripts containing regions of both of these genes did not depend on the expression level of CK/EK-β.View full abstractDownload PDF (4217K) -
Carsten Janke, Davy Martin, Marie-Josèphe Giraud-Panis, Martine ...2003 Volume 133 Issue 4 Pages 533-539
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1: a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins.View full abstractDownload PDF (5404K) -
Yoshiyuki Mizushina, Chikako Murakami, Hirosato Takikawa, Nobuyuki Kas ...2003 Volume 133 Issue 4 Pages 541-552
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHippospongic acid A (HA-A) is a novel natural triterpene metabolite that exhibits inhibitory activity against the gastrulation of starfish embryos isolated from a marine sponge, Hippospongia sp. We succeeded in chemically synthesizing the natural enantiomer and the racemate HA-A. In this study, we examined its action mode in vitro. HA-A was a rare compound that could selectively but uniformly inhibit the activities of all the vertebrate DNA polymerases tested such as α, β, δ, ε, η, κ, and λ, in the IC50 range of 5.9-17.6 μM, and interestingly also those of human DNA topoisomerases I and II (IC50 = 15-25 μM). HA-A exhibited no inhibitory effect on DNA polymerases from insects, plants and prokaryotes, or on many other DNA metabolic enzymes. HA-A was an inhibitor specific to DNA polymerases and DNA topoisomerases from vertebrates, but not selective as to a subclass species among the enzymes. Since DNA polymerase β is the smallest, we used it to analyze the biochemical relationship with HA-A. Biochemical, BIAcore and computer modeling analyses demonstrated that HA-A bound selectively to the N-terminal 8 kDa DNA template-binding domain of DNA polymerase β, and HA-A inhibited the ssDNA binding activity. HA-A could prevent the growth of NUGC-3 cancer cells at both the G1 and G2/M phases, and induce apoptosis in the cells. The LD50 value was 9.5 μM, i.e. in the same range as for the enzyme inhibition. Therefore, we concluded that one molecular basis of the gastrulation of starfish embryos is a process that requires DNA polymerases and DNA topoisomerases, and subsequently the gastrulation was inhibited by HA-A. We also discussed the in vivo role of HA-A.View full abstractDownload PDF (5445K) -
Peter Mayr, Barbara Petschacher, Bernd Nidetzky2003 Volume 133 Issue 4 Pages 553-562
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSXylose reductase has been purified to apparent homogeneity from cell extracts of the fungus Cryptococcus flavus grown on D-xylose as carbon source. The enzyme, the first of its kind from the phylum Basidiomycota, is a functional dimer composed of identical subunits of 35.3 kDa mass and requires NADP(H) for activity. Steady-state kinetic parameters for the reaction, D-xylose + NADPH + H+↔ xylitol + NADP+, have been obtained at pH 7.0 and 25°C. The catalytic efficiency for reduction of D-xylose is 150 times that for oxidation of xylitol. This and the 3-fold tighter binding of NADPH than NADP+ indicate that the enzyme is primed for unidirectional metabolic function in microbial physiology. Kinetic analysis of enzymic reduction of aldehyde substrates differing in hydrophobic and hydrogen bonding capabilities with binary enzyme-NADPH complex has been used to characterize the substrate-binding pocket of xylose reductase. Total transition state stabilization energy derived from bonding with non-reacting sugar hydroxyls is ≈15kJ/mol, with a major contribution of 5-8kJ/mol made by interactions with the C-2(R) hydroxy group. The aldehyde binding site is ≈1.2 times more hydrophobic than n-octanol and can accommodate linear alkyl chains of ≤6 carbons. Hydrophobic interactions provide a total binding energy of ≈10 kJ/mol. Specificity for the aldehyde substrate is achieved through large decreases in apparent Km (≈100-fold) and smaller but significant increases in turnover number (≈5-fold). We observed up to 250-fold preference of xylose reductase for reaction with pyridine carbaldehydes, 4-nitro-benzaldehyde, and α-oxo-aldehydes over reaction with D-xylose, perhaps reflecting a secondary role of this enzyme in detoxication metabolism of reactive endogenous aldehydes and compounds of xenobiotic origin.View full abstractDownload PDF (3582K)
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