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Shigetsugu Hatakeyama, Keiichi I. Nakayama
2003 Volume 134 Issue 1 Pages
1-8
Published: 2003
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Quality control of intracellular proteins is essential for cellular homeostasis. Molecular chaperones recognize and contribute to the refolding of misfolded or unfolded proteins, whereas the ubiquitin-proteasome system mediates the degradation of such abnormal proteins. Ubiquitin-protein ligases (E3s) determine the substrate specificity for ubiquitylation and have been classified into HECT and RING-finger families. More recently, however, U-box proteins, which contain a domain (the U box) of about 70 amino acids that is conserved from yeast to humans, have been identified as a new type of E3. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. Yeast Ufd2 is functionally implicated in cell survival under stressful conditions. This review addresses recent progress in characterization of the role of E3 enzymes, especially that of U-box proteins, in quality control of intracellular proteins.
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Jung Hwa Kim, Kyung Chan Park, Sung Soo Chung, Oksun Bang, Chin Ha Chu ...
2003 Volume 134 Issue 1 Pages
9-18
Published: 2003
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Modification of proteins by the covalent attachment of ubiquitin is a key regulatory mechanism of many cellular processes including protein degradation by the 26 S proteasome. Deubiquitination, reversal of this modification, must also regulate the fate and function of ubiquitin-conjugated proteins. Deubiquitinating enzymes catalyze the removal of ubiquitin from ubiquitin-conjugated substrate proteins as well as from its precursor proteins. Deubiquitinating enzymes occupy the largest family of enzymes in the ubiquitin system, implying their diverse function in regulation of the ubiquitin-mediated pathways. Here we explore the diversity of deubiquitinating enzymes and their emerging roles as cellular regulators.
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Daiju Okuda, Katsunori Horii, Hiroshi Mizuno, Takashi Morita
2003 Volume 134 Issue 1 Pages
19-23
Published: 2003
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EMS 16 is a member of the snake venom-derived C-type lectin family of proteins (CLPs) found in the venom of
Echis multisquamatus. It binds to glycoprotein Ia/IIa (integrin α2β1), a major collagen receptor of platelets, acting as a potent antagonist of platelet aggregation and cell migration. Amino acid sequencing and cDNA cloning of EMS 16 have revealed that it is composed of an A chain of 134 amino acid residues and a B chain of 128 residues. Crystals of EMS 16 belong to space group
P2
12
12
1, with unitcell parameters
a=46.57,
b=59.93, and
c=115.74 Å, and diffract to a resolution of 1.9 Å. Phase determination is underway by means of molecular replacement with the structure of blood coagulation factor IX-binding protein (IX-bp) from habu snake venom (PDB code 1bj3) as the search model.
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Yasuhiro Kashima, Kazuhiko Ishikawa
2003 Volume 134 Issue 1 Pages
25-29
Published: 2003
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Pyrococcus horikoshii is an obligate anaerobic hyperthermophilic archaeon. In
P. horikoshii cells, a hydroperoxide reductase homologue ORF (PH1217) was found to be induced by oxygen. The recombinant protein, which was expressed in
E. coli under aerobic conditions, exhibited no activity. However, the recombinant protein prepared under semi-anaerobic conditions exhibited alkyl hydroperoxide reductase activity. Furthermore, it was clarified that it was coupled with the thioredoxin-like system in
P. horikoshii. Western blot analysis revealed that the protein was induced by oxygen and hydrogen peroxide. This protein seems to be sensitive to oxygen but forms a thioredoxin-dependent system to eliminate reactive oxygen species in
P. horikoshii.
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Stuart N. Ryan, Michael T. McManus, William A. Laing
2003 Volume 134 Issue 1 Pages
31-42
Published: 2003
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Trypsin and papain proteinase inhibitors have been identified and purified from aqueous extracts of apple seeds (
Malus domestica). Superdex G 75 gel filtration chromatography identified a higher molecular weight (HMW) papain inhibitory fraction (22-26 kDa) and a lower molecular weight papain and trypsin inhibitory fraction (6-12 kDa). The lower molecular weight fraction was separated into a trypsin inhibitor (designated Trpl) and early (designated Papl) and late (designated Pap 2) eluting papain inhibitors after anion exchange (Hitrap SP) chromatography. For Pap 2, two inhibitory peaks (designated Pap 2-1 and Pap 2-2) were identified after further anion exchange (Resource S) chromatography. Each of these lower molecular weight inhibitors was purified by reverse phase HPLC to homogeneity as determined by SDSPAGE and by mass spectrometry. The HMW papain inhibitory fraction was purified further by anion-exchange (Hitrap Q followed by Resource Q) column chromatography where a minor inhibitor (HMWPap 1) and major inhibitor (HMWPap 2) fraction were identified. The relative abundance in seeds of apple and the spectrum of proteinase inhibition has been determined for all of these inhibitors. Reverse-phase HPLC separated HMWPap 2 into a minor (HMWPap 2-1) and a major (HMWPap 2-2) inhibitory fraction, and SDS-PAGE and mass spectrometry confirmed that HMWPap 2-2 was purified to homogeneity. Amino acid composition data were obtained from Trpl, Papl, Pap 2-2, and HMWPap 2-2, and N-terminal sequence data from Trpl, Pap 2-1, Pap 2-2, and HMWPap 2-2, with two of these sequences (Pap 2-2 and HMWPap 2-2) perfectly matching predicted protein sequences based on EST sequences from an apple database. The relationship of these inhibitors with those of other species is discussed.
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Mohamad Radwan Almofti, Tomokazu Ichikawa, Kikuji Yamashita, Hiroshi T ...
2003 Volume 134 Issue 1 Pages
43-49
Published: 2003
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Various reagents are known to open the mitochondrial permeability pore (PTP) and induce a permeability transition (PT), releasing apoptogenic proteins from the intermembrane space and triggering apoptosis. In this study, we examined the effect of Ag
+, a known cytotoxic sulfhydryl-reactive heavy metal, on isolated rat liver mitochondria. The following results were obtained: (1) Upon addition, Ag
+ instantly induced mitochondrial swelling and acceleration of respiration. (2) Cyclosporine A, a specific inhibitor of classical PT, was ineffective against the effect of Ag
+, indicating that silver ions induced non-classic PT. (3) Sulfhydryl reagents such as reduced glutathione completely inhibited the effects of Ag
+ on the mitochondria. (4) Experimental results using polyethylene glycol indicated that Ag
+ induced opening of a pore in the inner mitochondrial membrane, which could be PTP of another open state or a distinct pore. (5) Electron microscopic analysis of mitochondria treated with Ag
+ showed a novel mitochondrial configuration that was apparently different from that of normal mitochondria or Ca
2+-treated mitochondria. (6) Ag
+ also induced the release of apoptogenic cytochrome c in a CsA-insensitive but GSH-sensitive manner. These results suggest that Ag
+ promotes a nonclassical permeability increase in the mitochondrial inner membrane that is clearly distinguishable from the classical PT and releases apoptogenic cytochrome
c in a classical PT independent manner.
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Chiaki Takahashi, Shin-ichi Nakakita, Sumihiro Hase
2003 Volume 134 Issue 1 Pages
51-55
Published: 2003
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Pyridylamino sugar chains were converted to the corresponding reducing sugar chains by first converting them to 1-amino-1-deoxy derivatives using the method previously reported [S. Hase,
J. Biochem. 112, 266-268 (1992)] and then converting the products to the corresponding reducing sugar chains using the Sommlet reaction. The reaction conditions were optimized so as to obtain the maximal product yield using 1-amino-1-deoxylactose and 1-amino-1-deoxy-
N-acetylglucosamine. When the established procedure was successively applied to pyridylamino high-mannose and complex-type sugar chains, the corresponding reducing sugar chains were obtained in yields of 30%.
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Ayumu Yoshikawa, Hiroaki Mitsuhashi, Noboru Sasagawa, Toshifumi Tsukah ...
2003 Volume 134 Issue 1 Pages
57-61
Published: 2003
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It is known that denervation of rat skeletal muscle causes atrophy and this is often adopted as a model for human muscle atrophy. To understand the molecular changes that occur, it is important to identify the profiles of differential gene expression. In the present study, we investigated differentially expressed genes in denervated muscle using DNA microarrays with printed genes preferentially expressed in skeletal muscle. We found that several genes are differentially expressed. Of these genes, ARPP-16/19 (cAMP-regulated phosphoprotein 16/19) is selectively enhanced after denervation. The expression of ARPP-16/19 in denervated muscles starts to increase from two days after denervation surgery. On the other hand, the expression of ARPP-16/19 does not change in hind-limb suspended muscles, such as EDL and soleus muscles. These results suggest that the increase in ARPP-16/19 mRNA expression is regulated by unknown factor (s) secreted from nerves, and not by electrical muscle activity.
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Yoko Ishida, Keiichi Komaru, Masahiro Ito, Yoshihiro Amaya, Shoji Kohn ...
2003 Volume 134 Issue 1 Pages
63-70
Published: 2003
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A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A.
et al. (1999)
Hum. Mut. 13, 171-172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-β-
N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu
218→Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca
2+ coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule.
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Junko Fukumura, Eishi Noguchi, Takeshi Sekiguchi, Takeharu Nishimoto
2003 Volume 134 Issue 1 Pages
71-82
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ts ET 24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK 21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of is ET 24 cells was isolated and screened for an open reading frame in the deposited human genomic library.
X chromosomal
DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and la of the hamster DBX of is ET 24 cells. At the nonpermissive temperature of 39.5°C, is ET 24 cells were arrested in the G 1-phase and survived for more than 3 days. In is ET 24 cells, total protein synthesis was not reduced at 39.5°C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5°C for 17 h. The amount of
cyclin A mRNA decreased in is ET 24 cells within 4 h after the temperature shift to 39.5°C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.
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Yasuo Kikuchi, Shizue Ohsawa, Junsei Mimura, Masatsugu Ema, Chikahisa ...
2003 Volume 134 Issue 1 Pages
83-90
Published: 2003
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AhR (aryl hydrocarbon receptor), AhRR (AhR repressor), and Arnt (AhR nuclear translocator) are members of the bHLH (basic-helix-loop-helix) -PAS (Per-AhR/Arnt-Sim homology sequence) transcription factor superfamily. They associate with each other to form heterodimers, AhR/Arnt or AhRR/Arnt, and bind the XRE (xenobiotic responsive element) sequences in the promoter regions of the target genes to regulate their expression. Their basic regions and HLH motifs mediate DNA binding activity and protein dimerization, respectively. The PAS domain includes two incomplete repeats, PAS-A and PAS-B, and is considered to determine the specificity on protein dimerization. However, the three-dimensional structures of PAS folds reported so far are all monomeric, therefore, little is known about the structural basis of protein interaction through PAS domains. In the present study, we prepared heterodimeric bHLH-PAS domains derived from AhR and Arnt, and AhRR and Arnt by co-expressing each pair in
E. coli, and showed that the heterodimers formed exhibited full DNA-binding activity, which was not apparently affected by deletion of the highly basic amino acid cluster most N-terminal as to the HLH region of AhR or AhRR. Methylation of the two CpG sites in the XRE core sequence reduced the binding affinity to heterodimeric proteins, with 5-methylcytosine in the AhR recognition site exhibiting a greater inhibitory effect than that in the Arnt recognition site.
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Tian-Ming Yao, Koji Tomoo, Toshimasa Ishida, Hiroshi Hasegawa, Masahir ...
2003 Volume 134 Issue 1 Pages
91-99
Published: 2003
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The microtubule-associated protein tau is a highly soluble protein that shows hardly any tendency to assemble under physiological conditions. In the brains of Alzheimer's disease (AD) patients, however, tau dissociates from the axonal microtubule and abnormally aggregates to form paired helical filaments (PHFs). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. In recent years, several factors regulating tau assembly have come to light, yet some important questions remain to be answered. In this work, the His-tagged gene constructs of the fourrepeat microtubule binding domain (4 RMBD) in tau protein and its three mutants, 4 RMBD S305N, N279K, and P301L, were expressed in E. coli and purified. Gel filtration chromatography and dynamic light scattering measurement yielded a Stokes radius of 3.1 nm, indicating that the His-tagged 4 RMBD normally exists in buffer solution in a dimer state, which is formed by non-covalent intermolecular interactions. This non-covalent dimer can further polymerize to form filaments in the presence of polyanions such as heparin. The kinetics of the
in vitro aggregation was monitored by thioflavine S dye fluorescence and CD measurements. The aggregation of 4 RMBD was suggested to be a nucleation-dependent process, where the non-covalent dimer acts as an effective structural unit. The aggregation rate was strongly affected by the point mutation. Among the 4 RMBD mutants, the rate of S305N was exceptionally fast, whereas N279K was the slowest, even slower than the wild-type. The aggregations were optimal in a weakly reducing environment for all the mutants and the wild type. However, the aggregations were affected differently by buffer pH, depending on the 4 RMBD mutation.
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Gyu Hyun Nam, Do-Hyung Kim, Nam-Chul Ha, Do Soo Jang, Young Sung Yun, ...
2003 Volume 134 Issue 1 Pages
101-110
Published: 2003
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Ketosteroid isomerase (KSI) from
Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Δ
5-3-ketosteroids at a rate of the diffusion-controlled limit. The dimeric interactions mediated by Arg 72, Glu 118, and Asn 120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme. The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A. Equilibrium unfolding analysis revealed that the ΔG
UH2O values for the R72A, E118A, N120A, and E118A/N120A mutants were decreased by about 3.8, 3.9, 7.8, and 9.5 kcallmol, respectively, relative to that of the wild-type enzyme. The interface mutations not only decreased the
kcat/
KM value by about 8- to 96-fold, but also increased the
KD value for
d-equilenin, a reaction intermediate analogue, by about 7- to 17.5-fold. The crystal structure of R72A determined at 2.5 Å resolution and the fluorescence spectra of all the mutants indicated that the interface mutations altered the active-site geometry and resulted in the decreases of the conformational stability as well as the catalytic activity of KSI. Taken together, our results strongly suggest that the conserved interface residues contribute to stabilization and structural integrity of the active site in the dimeric KSI.
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Tetsuya Okuda, Toshihiko Sawada, Hirofumi Nakano, Kazumi Matsubara, Yo ...
2003 Volume 134 Issue 1 Pages
111-120
Published: 2003
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N-Acetylgalactosamine 4-sulfotransferase (GalNAc4ST) transfers sulfate to position 4 of nonreducing terminal GalNAc residues. We previously cloned human GalNAc4ST-1 cDNA. In this paper, we report the cloning, characterization and chromosomal mapping of mouse GalNAc4ST-1 and GalNAc4ST-2. Mouse GalNAc4ST-1 and GalNAc4ST-2 contain single open reading frames that predict type II transmembrane proteins composed of 417 and 413 amino acid residues, respectively. The amino acid sequence identity between the two isoforms is 49%. When the cDNA was transfected to COS-7 cells, sulfotransferase activities toward carbonic anhydrase VI and GalNAcβ1-4GlcNAcβ1-octyl were overexpressed, but the sulfotransferase activity toward chondroitin showed no increase over the control level. Northern blot analysis showed that the 2.4 kb messages of GalNAc4ST-1 and GalNAc4ST-2 were strongly expressed in the kidney, where both of the human isoforms were hardly expressed. Reverse transcription-PCR analysis showed that, unlike human GalNAc4ST-1, the expression of mouse GalNAc4ST-1 in the pituitary gland was only marginal, while that of GalNAc4ST-2 in the pituitary gland was as high as that in the kidney. These results suggest that the functions of the two GalNAc4ST isoforms may differ between human and mouse. By fluorescence
in situ hybridization, the GalNAc4ST-1 and GalNAc4ST-2 genes were localized to mouse chromosome 7B3 distal-B5 proximal and chromosome 18A2 distal-B1 proximal, respectively.
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Maki Onda, Yukari Inoue, Manami Kawabata, Tomoyoshi Mita
2003 Volume 134 Issue 1 Pages
121-128
Published: 2003
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To investigate the susceptibilities of fungal and mammalian cells to amphotericin B (AmB), AmB-loaded lysophosphatidylcholine (LPC) micelles as drug delivery vehicles were incubated at 37°C with phosphatidylcholine vesicles containing different sterols as model systems for fungal and mammalian cells. The binding and kinetics of AmB to sterols in the membranes were judged by UV visible spectroscopy. In the 91% monomeric form, AmB interacted rapidly with ergosterol and slowly with 7-dehydrocholesterol (7-DHC), while it did not interact with cholesterol. In the 50% monomeric form, AmB formed complexes more rapidly with ergosterol or 7-DHC than in the monomeric form, whereas it did not still interact with cholesterol. The interaction was also characterized by resonance energy transfer between the fluorescent probe trimethylammonium diphenylhexatriene (TMA-DPH) and AmB. In the 91% monomeric form, AmB caused initial fluorescence quenching in bilayer membranes containing any sterol as well as sterol-free bilayer membranes due to the release of AmB and its incorporation within the membranes. However, a second phase of increasing fluorescence was found in the case of ergosterol alone. On the other hand, in the 47% monomeric form, AmB gave a biphasic intensity profile in membranes containing any sterol as well as sterol-free membranes. However, the extent of the second phase of increasing fluorescence intensity was markedly dependent upon sterol composition. Studies using sterol-containing vesicles provide important insights into the role of the aggregation state of AmB in its effects on cells.
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Daisuke Kameoka, Tadashi Ueda, Taiji Imoto
2003 Volume 134 Issue 1 Pages
129-135
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A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-α-Asp, and MALDUTOF-mass spectrometry. To achieve better resolution, we employed digests of
15N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.
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Jeong Hee Ryu, Youngjoo Lee, Seung Kee Han, Hak Yong Kim
2003 Volume 134 Issue 1 Pages
137-142
Published: 2003
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Polychlorinated biphenyls (PCBs) are well-known recalcitrant environmental pollutants. Although the metabolism of the PCBs has been intensively studied, very little is known about their mechanism of toxicity in living organisms or how they are degraded. We have examined the effects of PCBs on two different yeast strains to determine their mechanism of action. One yeast strain (K 601, wild type) is resistant to the growth-inhibitory effect of PCBs, whereas the other strain (AA 542,
PMR 1 mutant) is susceptible. PCBs increased the level of intracellular hydrogen peroxide in AA 542 cells but not in K 601 cells. In the presence of α-tocopherol or ursolic acid the growth of AA 542 cells was not inhibited by treatment with PCBs. These results suggest that PCBs block cell growth through production of hydrogen peroxide in the
PMR 1 mutant strain, AA 542. We compared superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase activities in both strains. The catalase activity in K 601 cells was 10 times higher than that in AA 542 cells. In contrast, there was no difference in activities of SOD and GPx between the two strains. Collectively, these observations indicate that oxidative stress causes the inhibition of cell growth observed in catalase-deficient yeast cells exposed to PCBs.
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Hideo Saito, Kazuhisa Takeda, Ken-ichi Yasumoto, Haruo Ohtani, Ken-ich ...
2003 Volume 134 Issue 1 Pages
143-150
Published: 2003
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The gene coding for microphthalmia-associated transcription factor (Mitt) contains many promoters that could generate multiple Mitf isoforms with distinct amino-termini, such as ubiquitously expressed Mitf-A and Mitf-H. To gain further insight into Mitf isoform multiplicity and the regulation of the promoter usage of the
Mitf gene, we have analyzed the function of the amino-terminal domains of Mitf isoforms and the expression of Mitf mRNA in mouse postnatal testis, which is characterized by spermatogenesis and by a cool temperature because of its unique location. Here we show that the amino-terminal domain of Mitf-A possesses a transactivation activity, as judged by yeast expression analysis. We also show the expression of Mitf-A and Mitf-D mRNAs in testis by PCR-based methods. Moreover,
in situ hybridization analysis revealed that an Mitf mRNA, probably representing Mitf-A and/or Mitf-D, is expressed in germ cells, including spermatogonia, spermatocytes that undergo meiosis, and round spermatids with the haploid genome, but is undetectable in elongated spermatids with remodeled and condensed chromatin. Notably, Mitf mRNA is undetectable in somatic Leydig cells and peritubular cells. Therefore, multiple promoters may direct differential expression of the
Mitf gene in the testis and contribute to functional diversity of Mitf isoforms.
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Maki Sato, Takashi Kikukawa, Tsunehisa Araiso, Hirotaka Okita, Kazumi ...
2003 Volume 134 Issue 1 Pages
151-158
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Pharaonis halorhodopsin (
phR.) is an inward light-driven chloride ion pump in
Natronobacterium pharaonis. In order to clarify the roles of the Ser 130
phR and Thr 126
phR residues, which correspond to Ser115
shR and Thr111
shR of
salinarum hR (
shR), with regard to their Cl-binding affinity and the photocycle, the wild-type
phR, and S 130 and T 126 mutants were expressed in
Escherichia coli cells. The photocycles of the wild-type
phR, and S 130 and T 126 mutants were investigated in the presence of 1M NaCl. Based on results of kinetic analysis involving singular value decomposition and global fitting, typical photointermediates K, L and O were identified, and the kinetic constants of decay or formation varied depending on the mutant. The photocycle scheme was linear for the wild-type
phR, and S130C, S130T and T126V mutants. On the other hand, the S130A mutant showed a branched pathway between the L-hR and L-O steps. The present study revealed the following two facts with respect to the Ser130
phR residue: 1) The OH group of this residue is important for Cl
- ion binding next to the Schiff base nitrogen, and 2) replacement of an Ala residue, which is unable to form a hydrogen bond, results in a branched photocycle. The implication of this branching was discussed.
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Nami Hirota-Nakaoka, Kazuhiro Hasegawa, Hironobu Naiki, Yuji Goto
2003 Volume 134 Issue 1 Pages
159-164
Published: 2003
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Increasing numbers of proteins have been found to aggregate into insoluble fibers, collectively referred to as amyloid fibrils. To address the conformational stability of amyloid fibrils, we studied the effects of dimethylsulfoxide (DMSO), 2, 2, 2-trifluoroethanol (TFE), and 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP) on β
2-microglobulin amyloid fibrils by circular dichroism, thioflavin T fluorescence, light scattering, and electron microscopy. When measured by circular dichroism and thioflavin T fluorescence, HFIP, and TFE dissolved the fibrils, producing predominantly helical conformations. However, these alcohols did not dissolve the amyloid fibrils completely as monitored by light scattering and electron microscopy. On the other hand, DMSO completely dissolved the amyloid fibrils although a high concentration [
i.e., 80% (v/v)] was required. These results are consistent with the important role of hydrogen bonds in stabilizing amyloid fibrils.
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You Hachikubo, Kohji Ito, Keiichi Yamamoto
2003 Volume 134 Issue 1 Pages
165-171
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Myosin is a molecular motor and a member of a protein family comprising at least 18 classes. There is an about 1, 000-fold difference in the
in vitro sliding velocity between the fastest myosin and the slowest one. Previous studies revealed that the hydrophobic triplet in the motor domain (Val 534, Phe 535, and Pro 536 in
Dictyostelium myosin) is important for the strong binding of myosin to actin. We studied the role of the triplet in the sliding motion of myosin by means of site directed mutagenesis because the sliding velocity is determined by the time that myosin interacts with actin strongly. We produced mutant
Dictyostelium myosins and subfragment-1s that have the triplet sequences of various classes of myosin with different sliding velocities. The
Vmax and
Kactin values of the actin-activated ATPase for all these mutant subfragment-1s were lower than those of the wild-type
Dictyostelium myosin. The mutant myosins exhibited much lower sliding velocities than the wild type. The time that the mutant subfragment-1s are in the strongly bound state did not correlate well with the sliding velocity. Our results suggested that (i) the hydrophobic triplet alone does not determine the sliding velocity of myosin, (ii) the size of the amino acid side chain in the triplet is crucial for the ATPase activity and the motility of myosin, and (iii) the hydrophobic triplet is important not only for strong binding to actin but also for the structural change of the myosin motor domain during the power stroke.
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2003 Volume 134 Issue 1 Pages
173
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