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Yasuo Mukohata, Kunio Ihara, Takeshi Tamura, Yasuo Sugiyama
1999 Volume 125 Issue 4 Pages
649-657
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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Following the discovery of the bacteriorhodopsin proton pump in
Halobacterium halobium (
salinarum), not only the halorhodopsin halide pump and two photosensor rhodopsins (sensory rhodopsin and phoborhodopsin) in the same species, but also homologs of these four rhodopsins in strains of other genera of
Halobacteriaceae have been reported. Twenty-eight full (and partial) sequences of the genomic DNA of these rhodopsins have been analyzed. The deduced amino acid sequences have led to new strategies and tactics for understanding bacterial rhodopsins on a comparative basis, as summarized briefly in this article. The data discussed include (i) alignment of the sequences to qualify/characterize the conserved residues; (ii) assignment of residues that cause differences in function(s)/properties; and (iii) phylogeny of the halobacterial rhodopsins to suggest their evolutionary paths. The four kinds of rhodopsin in each strain are assumed, on the basis of their genera-specific distributions, to have arisen by at least two gene-duplication processes during evolution prior to generic speciation. The first duplication of the rhodopsin ancestor gene yielded two genes, each of which was duplicated again to give four genes in the ancestor halobacterium. The bacterium carrying four rhodopsin genes, after accumulating mutations, became ready for generic speciation and the delivery of four rhodopsins to each species. The original rhodopsin ancestor is speculated to be closest to the proton pump (bacteri orhodopsin).
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Naoko Nagata, Kazutaka Momose, Yukisato Ishida
1999 Volume 125 Issue 4 Pages
658-661
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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4, 5-Diaminofluorescein (DAF-2) is a newly developed indicator of nitric oxide (NO). Two amino groups of DAF-2 are oxidized by NO. We investigated the effects of reducers on the NO-induced oxidation of DAF-2. NOC-5 (0.1-10 μM), a NO-donor, concentration-dependently elicited fluorescence with 10 μM DAF-2. The rate of the fluorescence reaction was dependent on the width of the excitation band path. The presence of catecholamines (1 μM), but not tyrosine or phenylephrine, attenuated the fluorescence induced by NOC-5. Ascorbate and other reducers like dithiothreitol, 2-mercaptoethanol, or glutathione (all 1mM) abolished the fluorescence. These results suggest that reducers attenuate the NO-induced fluorescence of DAF-2 mainly through an anti-oxidative action.
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Atsushi Shimizu, Takashi Sasaki, Jung Hee Kwon, Akito Odaka, Takanori ...
1999 Volume 125 Issue 4 Pages
662-668
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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In our previous paper, we reported a mutant of recombinant
Myrothecium verruca riabilirubin oxidase, in which the Met 467 residue was replaced by Gly [Shimizu, A
et al. (1999)
Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met 467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met 467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.
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Naohito Aoki, Miyuki Kawamura, Yumiko Yamaguchi-Aoki, Sachiyo Ohira, T ...
1999 Volume 125 Issue 4 Pages
669-675
Published: April 01, 1999
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Detailed analysis of protein tyrosine phosphatase (PTP) expression in mouse mammary gland and mammary epithelial cells using a set of degenerate primers corresponding to the PTP core domain sequence revealed the presence of 16 different receptor-type and intracellular PTPs. Northern blot and RT-PCR analyses revealed that some PTPs were up-regulated during gestation, suggesting that these enzymes are involved in development of mammary gland. However, expression of most PTPs dramatically decreased during lactation, whereas the β-casein gene expression was increased and remained at a high level. At the involution stage after weaning, most PTPs were up-regulated and their expression returned almost to the virgin level. Such up-regulation was also induced by forced weaning in lactating mother mice. These results suggest the possible contribution of PTPs to the development, involution, and remodeling of mammary gland and their possible inhibitory action on maintaining high expression of milk genes during lactation.
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Shunji Hattori, Eijiro Adachi, Tetsuya Ebihara, Tomoko Shirai, Iori So ...
1999 Volume 125 Issue 4 Pages
676-684
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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Alkaline treatment is a good method for extracting collagen with high recovery even from an aged animal specimen. However, the properties of collagen treated under alkaline conditions have not been well established yet. By the treatment with a solution of 3% sodium hydroxide and 1.9% monomethylamine, the isoelectric point of type I collagen was lowered from 9.3 to 4.8 because of the conversions of Asn and Gln to Asp and Glu. With the acidification of the pI, the denaturation temperature of the collagen was decreased from 42 to 35°C after 20 d treatment, but the collagen-specific triple helical conformation was maintained. Human keratinocytes and fibroblasts adhered to the alkali-treated collagen via the collagen receptor integrin α2β1. This indicates that the alkali-treated collagen maintained its property as a biological adherent molecule. Unlike acid-soluble collagen, alkali-treated collagen lost the ability to form fibrils at neutral pH under physiological conditions. This ability was lost even after 4h of alkaline treatment, when the denaturation temperature of the collagen did not change. On the other hand, the alkali-treated collagen formed a fibrous precipitate with a uniform diameter of 50-70 nm under acidic conditions at 30°C.
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Takuya Watanabe, Tomonori Fujiwara, Shinji Komazaki, Kazuhiko Yamaguch ...
1999 Volume 125 Issue 4 Pages
685-689
Published: April 01, 1999
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The membrane protein syntaxin (originally named HPC-1) is involved in vesicle trafficking and required for neurotransmitter release at nerve terminals. The presence of syntaxin on target membranes is hypothesized to confer specificity to targeting and fusion
via interactions with complementary vesicle-associated proteins. To elucidate the function of syntaxin 1 A in exocytosis, HPC-1/syntaxin 1A-reduced PC12h cells (PC12h/
Δsyx) that were stably transfected with a plasmid for antisense syntaxin 1 A expression were constructed. Depolarizing stimulation of PC12h/Jsyx enhanced dopamine release, compared with PC12h. There was a strong inverse correlation between syntaxin 1 A protein expression and enhancement of dopamine release. Reduction of syntaxin 1 A had no effect on increase of the cytoplasmic free Ca
2+ concentration by depolarized stimulation. Moreover, PC12h/Jsyx clones similarly enhanced of exocytosis by native secretagogues. These results indicate that syntaxin 1 A has more than one function in exocytosis.
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Hiroki Tsuruta, Yasuo Aizono
1999 Volume 125 Issue 4 Pages
690-695
Published: April 01, 1999
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Phosphatase I purified from a psychrophile (
Shewanella sp.) [Tsuruta
et al. (1998)
J. Biochem. 123, 219-225] dephosphorylated
O-phospho-L-tyrosine and phospho-tyrosyl residues in phosphorylated poly(Glu
4, Tyr
1) random polymer (polyEY) and phosphorylated myelin basic protein (MBP) but not phosphoseryl and/or phosphothreonyl residues in phosphorylated histone H 1, casein and phosphorylase
a, indicating that the enzyme showed protein-tyrosine-phosphatase (PTPase, EC 3. 1. 3. 48)-like activity
in vitro. The enzyme was remarkably inhibited by diethylpyrocarbonate (DEPC), monoiodoacetic acid (MIAA), and monoiodoacetamide (MIAM). Binding of 1 mol of DEPC to 1 mol of the enzyme caused complete inhibition of the enzyme; and 0.88 mol of 1-carboxymethylated histidine per mole of the enzyme was found when 90% of enzyme activity was lost by modification with
14CMIAA. These results indicated that this psychrophilic enzyme was a PTPase-like enzyme with histidine as its catalytic residue.
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Masaki Nojiri, Masafumi Yohda, Masafumi Odaka, Yusuke Matsushita, Masa ...
1999 Volume 125 Issue 4 Pages
696-704
Published: April 01, 1999
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The nitrile hydratase (NHase) from
Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrite hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase α subunit, NHase β subunit and NHase activator. We overproduced the NHase in
Escherichia coli using a T 7 expression system. The NHase was functionally expressed in
E. coli only when the NHase activator encoded downstream of the β subunit gene was co-expressed and the transformant was grown at 30°C or less. A ligand cysteine, αCys 112, of the recombinant NHase was also posttranslationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of αCys 114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.
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Yuichiro Higashimoto, Hiroaki Kodama, Masood Jelokhani-Niaraki, Fumio ...
1999 Volume 125 Issue 4 Pages
705-712
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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Peptaibols comprise a family of peptide antibiotics with high contents of 2-aminoiso-butyric acid (Aib) residues and C-terminal amino alcohols. These peptides form α-helical structures leading to voltage-gated ion channels in lipid membranes. In the present study, amphiphilic helical Aib-containing peptides of various chain-lengths, Ac-(Aib-Lys-Aib-Ala)
n-NH
2 (
n=1-5), were designed to investigate the mechanisms of the aggregation and transmembrane orientation of helical motifs in lipid bilayer membranes. Peptide synthesis was performed by the conventional stepwise Fmoc solid-phase method. The crude peptides were obtained in high yields (66-85%) with high purities (69-95%). Conformational analysis of the synthetic peptides was performed by CD spectroscopy. It was found that these peptides take on highly helical structures, and the helicity of the peptides increases with an increase in chain-length. The longest peptide, Ac-(Aib-Lys-Aib-Ala)
5-NH
2, self-aggregates and adopts a barrel-stave conformation in liposomes. Ac-(Aib-Lys-Aib-Ala)
5-NH
2 exhibited potent antimicrobial activity against Gram-positive bacteria. Patch-clamp measurements revealed that this peptide can form well-defined ion channels with a long lifetime at relatively low transbilayer potentials and peptide concentrations. For this peptide, the single-channel conductance of the most frequent event is 227 pS, which could be related to a single-state tetrameric pore.
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Tatsuya Oda, Naoko Shinmura, Yuka Nishioka, Nobukazu Komatsu, Tomomits ...
1999 Volume 125 Issue 4 Pages
713-720
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphtha-lenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca
2+-dependent and strongly inhibited by 0.1M lactose, indicating that Ca
2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-Ill, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.
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Namchai Chewawiwat, Masato Yano, Kazutoyo Terada, Nicholas J. Hoogenra ...
1999 Volume 125 Issue 4 Pages
721-727
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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Tom 34 is a newly-found component of the mitochondrial protein import machinery in mammalian cells with no apparent counterpart in fungi. RNA blot and immunoblot analyses showed that the expression of Tom 34 varies among tissues and differs from that of the core translocase component Tom 20. In contrast to a previous report [Nuttal, S. D.
et al. (1997)
DNA Cell Biol. 16, 1067-1074], the present study using a newly-prepared anti-Tom 34 antibody with a high titer showed that Tom 34 is present largely in the cytosolic fraction and partly in the mitochondrial and membrane fractions after fractionation of tissues and cells, and that the membrane-associated form is largely extractable with 0.1M sodium carbonate. The
in vitro import of preproteins into isolated rat mitochondria was strongly inhibited by
ΔTom 34 which lacks the NH
2-terminal hydrophobic region of human Tom 34 (hTom 34). Import was also strongly inhibited by anti-hTom 34. In pulsechase experiments using COS-7 cells, pre-ornithine transcarbamylase (pOTC) was rapidly processed to the mature form. Coexpression of hTom 34 resulted in a stimulation of pOTC processing, whereas the coexpression of hTom 34 antisense RNA caused inhibition. The results confirm that Tom 34 plays a role in mitochondria) protein import in mammals, and suggest it to be an ancillary component of the translocation machinery in mammalian cells.
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Shou Takashima, Akira Nakamura, Makoto Hidaka, Haruhiko Masaki, Takesh ...
1999 Volume 125 Issue 4 Pages
728-736
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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A novel fungal β-glucosidase gene (
bgl 4) and its homologue (
bgl 2) were cloned from the cellulolytic fungi
Humicola grisea and
Trichoderma reesei, respectively. The deduced amino acid sequences of
H. grisea BGL 4 and
T. reesei BGL 2 comprise 476 and 466 amino acids, respectively, and share 73.1% identity. These β-glucosidases show significant homology to plant β-glucosidases belonging to the β-glucosidase A (BGA) family. Both genes were expressed in
Aspergillus oryzae, and the recombinant β-glucosidases were purified. Recombinant
H. grisea BGL 4 is a thermostable enzyme compared with recombinant
T. reesei BGL 2. In addition to β-glucosidase activity, recombinant
H. grisea BGL 4 showed a significant level of β-galactosidase activity, while recombinant T. reesei BGL 2 showed weak β-galactosidase activity. Cellulose saccharification by
Trichoderma cellulases was improved by the addition of recombinant
H. grisea BGL 4.
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Elizabeth A. Sweeney, Yasuyuki Igarashi
1999 Volume 125 Issue 4 Pages
737-745
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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Sphingolipids, ceramide in particular, have come to be regarded as having roles in cellular signaling, most recently being associated with stress and the cellular responses to stress. In the present study we first examined the mechanisms involved in the changes in cellular ceramide levels in normal human mesangial cells (NHMC) in the growth, quiescent, and senescent phases as well as those resulting from environmental stimuli. We found that in NHMC total ceramide levels increase in response to cellular stresses as a result of a combination of enzyme activities. Furthermore, different stresses cause different alterations in various enzyme activities, with age and growth influencing acidic enzymes, but cell density affecting neutral, resulting in final ceramide level increases which most likely are associated with distinct pools of ceramide. Secondly, we examined the influence of changes in ceramide levels on apoptosis induced by sphingosine and its methylated derivative
N, N-dimethylsphingosine. We found that increases in cellular ceramide levels prohibited the apoptosis and caused a quiescent state in the cells. The data presented here provide additional insight into the roles of ceramide and related enzymes in cellular responses to stress and suggest a possible relevance to
in vivo disease states.
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Motohiro Tani, Nozomu Okino, Susumu Mitsutake, Makoto Ito
1999 Volume 125 Issue 4 Pages
746-749
Published: April 01, 1999
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A fluorescent analogue of ceramide, C12-NBD-ceramide, was found to be hydrolyzed much faster than
14C-labeled ceramide by alkaline ceramidase from
Pseudomonas aeruginosa and neutral ceramidase from mouse liver, while this substrate was relatively resistant to acid ceramidase from plasma of the horseshoe crab. The radioactive substrate was used more preferentially by the acid ceramidase. It should be noted that C6-NBD-ceramide, which is usually used for ceramidase assays, was hardly hydrolyzed by any of the enzymes examined, compared to C12-NBD-ceramide. For the alkaline and neutral enzymes, the
Vmax and
k (
Vmaλ/
Km) with C12-NBD-ceramide were much higher than those with
14C-ceramide. In contrast, for the acid enzyme these parameters with C12-NBD-ceramide were less than half those with the radioisotope-labeled substrate. It is noteworthy that the labeling of ceramide with NBD did not itself reduce the
Km of the alkaline enzyme, but did that of the neutral enzyme. It was also found that C12-NBD-ceramide was preferentially hydrolyzed by the alkaline and neutral enzymes, but not the acid one, in several mammalian cell lines. This study clearly shows that the attachment of NBD, but not dansyl, increases the susceptibility of ceramide to alkaline and neutral enzyme, and decreases that to acid enzymes. Thus the use of this substrate provides a specific and sensitive assay for alkaline and neutral ceramidases.
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Shan Ding, Sachiko Kuroki, Asako Kameyama, Akihiko Yoshimura, Masaki K ...
1999 Volume 125 Issue 4 Pages
750-759
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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Complimentary DNA clones encoding the α
1c and β
2a subunits of guinea-pig cardiac L-type Ca
2+ channels were isolated using the PCR method. The open reading frame encoded 2, 169 amino acids for the a, c and 597 amino acids for the α
2a subunit. The proteins showed 94.2 and 94.8%, respectively, identity to the respective subunit of the rabbit protein. The message size of the guinea pig α
1c and β
2a subunits was 8.0 and 3.5/4.0 kb, respectively. RT-PCR analysis revealed that the α
1c subunit is expressed exclusively in the heart, while the β
2a subunit is expressed in the heart, cerebellum, whole brain, and stomach. The α
1c and β
2a subunits are transiently expressed in BHK (baby hamster kidney) cells, and the channel currents were studied using the whole-cell patch clamp technique in medium containing 30mM Ba
2+. In cells expressing α
1c alone, the Ba
2+ current was activated at -30 mV and more positive potentials and peaked at about 10 mV. The co-expression of β
2a with α
1c did not affect the voltage-dependence of the current, but increased the peak current and accelerated current decay. In cells transfected with guinea pig α
1c and rabbit β
1+α
2/δ, a Ba
2+ current comparable to those in native myocytes was observed. The Ba
2+current can be blocked completely by nifedipine and is enhanced 3-fold by Bay K 8644. On the other hand, neither forskolin nor okadaic acid affects the Ba
2+ current, suggesting that cAMP-mediated modulation is not easily reproduced in transfected cells, unlike that seen in native cardiac myocytes.
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Akira Yamamura, Toshifumi Sakaguchi, Yuji Murakami, Kenji Yokoyama, Ei ...
1999 Volume 125 Issue 4 Pages
760-769
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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L-Glutamate dehydrogenase (GLDH) independent of NAD (P) and oxygen was first obtained from the psychrotrophic bacterium
Aeromonas sp. L 101, originally isolated from the organs of salmon (
Oncorhynchus keta). GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200 pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5. 7. Maximum activity was obtained at 55°C and pH 8. 5. The activity of GLDH at 4 and 20°C was 38 and 50%, respectively, of that at 50°C. GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo[α]phenoxazin-7-ium chloride (meldola's blue), 3, 3'-[3, 3'-dimethoxy-(1, 1'-biphenyl)-4, 4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2 H tetrazolium chloride] (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2 H tetrazolium (INT). The presence of NAD (P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.
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Kazumi Ishidoh, Mitsue Takeda-Ezaki, Sumio Watanabe, Nobuhiro Sato, Mi ...
1999 Volume 125 Issue 4 Pages
770-779
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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Lysosomal proteinases are translated as preproforms, transported through the Golgi apparatus as proforms, and localized in lysosomes as mature forms. In this study, we analyzed which subclass of proteinases participates in the processing of lysosomal proteinases using Bafilomycin A 1, a vacuolar ATPase inhibitor. Bafilomycin A 1 raises lysosomal pH resulting in the degradation of lysosomal proteinases such as cathepsins B, D, and L. Twenty-four hours after the withdrawal of Bafilomycin A 1, NIH3T3 cells possess these proteinases in amounts and activities similar to those in cells cultured in DMEM and 5% BCS. In the presence of various proteinase inhibitors, procathepsin processing is disturbed by E-64-d, resulting in abnormal processing of cathepsins D and L, but not by APMSF, Pepstatin A, or CA-074. In the presence of
Helicobacter pylori Vac A toxin, which prevents vesicular transport from late endosomes to lysosomes, the processing of procathepsins B and D occurs, while that of procathepsin L does not. Thus, procathepsins B and D are converted to their mature forms in late endosomes, while procathepsin L is processed to the mature form after its arrival in lysosomes by some cysteine proteinase other than cathepsin B.
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Kazuo Kasai, Hye-Won Shin, Chisa Shinotsuka, Kazuo Murakami, Kazuhisa ...
1999 Volume 125 Issue 4 Pages
780-789
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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Dynamins are a family of _??_100-kDa GTPases that are thought to play a pivotal role in the formation of endocytic coated vesicles. There are three dynamin genes in mammals: dynamin I is neuron-specific, dynamin II shows ubiquitous expression, and dynamin III is expressed in testis, brain, and lung. However, most studies on the functions of dynamins to date have been restricted to dynamin I. In the present study, we show that, like dynamin I, dynamin II is involved in receptor-mediated endocytosis. While this study was in progress, Jones
et al. [Jones, S. M., Howell, K. E., Henley, J. R., Cao, H., and McNiven, M. A. (1998)
Science 279, 573-577] reported that dynamin II is localized in the trans-Golgi network (TGN) and involved in the formation of constitutive transport vesicles and clathrin-coated vesicles from this compartment. However, immunofluorescence analyses and experiments using cells transfected with dominant-negative dynamin II failed to show any evidence for localization of dynamin II in the TGN or for its involvement in vesicle formation from this compartment. Our data thus indicate that dynamin II is involved in endocytosis but not in the formation of transport vesicles from the TGN.
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Pharhad Eli, Takeshi Ariyama, Koichi Nishigaki, Yuzuru Husimi
1999 Volume 125 Issue 4 Pages
790-794
Published: April 01, 1999
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Perpendicular temperature gradient gel electrophoresis (TGGE) profiles were analyzed for PCR products from a random pool of DNA [60 nts random region flanked by two primer (20 nts) sites]. Besides a normal transition profile of a homoduplex, unique mobility transition profiles of two kinds of heteroduplex with a big internal loop were observed, representing the successive helix-coil transitions of the DNAs. As the appearance of the heteroduplex band is an estimator of the complexity of a random pool, it will be applicable to monitor the extent of the selection process in the
in vitro selection method. When imidazole was added to the electrophoretic buffer, the transition pattern shifted to the low temperature side. At a concentration of 1M, imidazole lowered the melting temperature (
Tm) of DNA by 13±2°C for all the three chain separation transitions observed. Thus imidazole is a stronger denaturant than urea, at least at dilute concentration. Dependence of Tm on concentration of imidazole and the mobility change suggested that imidazole binds to nucleotide in the single-stranded state.
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Zaw Myint, Tetsuya Inazu, Takashi Tanaka, Kazuya Yamada, Vincent W. Ke ...
1999 Volume 125 Issue 4 Pages
795-802
Published: April 01, 1999
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A homeobox gene, Hex, is mainly expressed in haematopoietic cells and hepatocytes. It is assumed to play a role in the early stage of differentiation of these cells. To understand the mechanisms involved in the regulation of the Hex gene expression in hepatocytes, we cloned and characterized the mouse Hex gene. The gene consists of four exons and three introns, and spans about 5.7 kb. All the exon-intron boundaries are consistent with the “GT-AG” rule. A single transcription start site was identified by primer extension and S1 mapping analyses. Although the 5'-flanking region is G/C rich (69%), it contains probable “TATA and CCAAT” boxes. Potential binding sequences for transcriptional regulatory proteins including Spl and AP-2 are also present in this region. Functional analysis of the Hex promoter was performed by transfecting MH
1C
1, HeLa, COS-7, and Caco-2 cells with Hex promoter region-luciferase constructs. We found three possible positive regulatory regions, comprising of nucleotides -199 and -172, -154 and -133, and -105 and -68, respectively, required for Hex gene expression in MH
1C
1 cells by analyses of a series of 5'-deletion constructs of the fusion genes. The activities of these constructs were extremely low in HeLa, COS-7, and Caco-2 cells suggesting that they possess cell-type specificity. Further analysis revealed two GC boxes, GC box1 and GC box2, at nucleotides -197 to -188 and - 176 to - 167, respectively, necessary for Hex gene expression. Thus, multiple regulatory elements contribute to the Hex gene expression in hepatocytes.
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Miki Nakajima, Tsuyoshi Yokoi, Mayumi Mizutani, Moritoshi Kinoshita, M ...
1999 Volume 125 Issue 4 Pages
803-808
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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A genetic polymorphism was identified in the 5'-flanking region of human
CYP1A2 gene, and its effect on the transcriptional activation of the
CYP1A2 gene was investigated. Nucleotide sequence analysis revealed the existence of a point mutation from guanine (wild type) to adenine (mutated type) at position -2964 in the gene. This point mutation was detected by a polymerise chain reaction-restriction fragment length polymorphism method using
DdeI or
BslI restriction enzyme, and was proven to be genetically inherited. Allele frequency in 116 Japanese subjects showed 0.77 and 0.23 for the wild and mutated types of allele, respectively. The point mutation caused a significant decrease of CYP1A2 activity measured by the rate of caffeine 3-demethylation in Japanese smokers (
p<0.05). Gel retardation analysis showed the existence of protein bound to the polymorphic locus. These results suggest that this polymorphism is a causal factor of decreased CYP1A2 inducibility.
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Dong Yuan, Xiuguang Ma, Jun Ma
1999 Volume 125 Issue 4 Pages
809-817
Published: April 01, 1999
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Our previous studies demonstrated that the
Drosophila homeodomain protein, Bicoid (Bed), binds DNA cooperatively. In this study, we determined the patterns of adjacent DNA sites required for cooperative recognition by Bed. Our
in vitro selection and biochemical experiments demonstrated that Bcd binds preferentially to both head-to-head and tail-to-tail symmetric sites that are separated by short spacing. An increase in the spacing reduces the strict requirement of symmetric patterns of adjacent sites, permitting Bed to recognize tandem repeat sites cooperatively. Our further experiments
in vivo showed that the only pair of optimally spaced symmetric Bed sites in a
hunchback (
hb) enhancer element contributes the most to transcriptional activation by Bed, demonstrating the biological importance of the binding site patterns revealed by our
in vitro selection studies.
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Emiko Kondo, Akira Horii, Shinichi Fukushige
1999 Volume 125 Issue 4 Pages
818-825
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
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The human
PMS2 gene encodes one of the bacterial
mutL homologs that is associated with hereditary nonpolyposis colorectal cancer (HNPCC). One of the interesting features of the
hPMS2 gene is that it is part of a multiple gene family which is localized on chromosome bands 7p22, 7p12-p13, 7q11, and 7q22. Here we report four newly identified
hPMS2-like (
PAIS2L) genes. All four novel members of the
PMS2L gene family encode relatively short polypeptides composed of the amino-terminal portion of hPMS2 and are expressed ubiquitously except in the heart. To clarify whether the PMS2L polypeptides contribute to the DNA mismatch repair (MMR) pathway through an interaction with hMLH1, we have performed a yeast two-hybrid assay and an immunoprecipitation study using an hPMS2 mutant cell line, HEC-1-A. Our results clearly indicate that hMLH1 does not interact with two representative PMS2Ls, whereas the carboxyl-terminal portion of hPMS2, not the amino-terminal portion, does interact with hMLH1. Thus, PMS2Ls are not likely to participate in the MMR pathway through association with hMLH1; they must play some other roles in the living cells.
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Norifumi Watanabe, Hiroto Kawashima, Yong-Fei Li, Masayuki Miyasaka
1999 Volume 125 Issue 4 Pages
826-831
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
JOURNAL
FREE ACCESS
L-Selectin, a leukocyte adhesion molecule, mediates leukocyte rolling on the endothelium and plays a critical role in leukocyte recruitment at inflammatory sites as well as in lymphocyte homing. We have previously shown that L-selectin reactive chondroitin sulfate and heparan sulfate proteoglycans (HSPGs) are both expressed in the distal tubules of the kidney and that versican is one of the chondroitin sulfate-type ligands. In the present study, we characterized the heparan sulfate-type ligand(s) in more detail. The molecular sizes of HSPGs were approximately 600 kDa with core protein sizes of 160 and 180 kDa. Western blotting analysis showed that L-selectin reactive HSPGs were neither agrin nor perlecan, major basement membrane HSPGs in the kidney. The binding to L-selectin was mediated by the lectin domain of L-selectin in a Ca
2+-dependent manner and required heparan sulfate side chains, but not sialic acid. To our knowledge, this is the first biochemical characterization of the L-selectin reactive heparan sulfate proteoglycan(s) in the distal tubules of the kidney.
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Taku Yamada, Rika Fukuda, Michio Himeno, Kenji Sugimoto
1999 Volume 125 Issue 4 Pages
832-837
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
JOURNAL
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Human heterochromatin protein HP1
Hsα possesses two evolutionarily conserved regions in the N- and C-terminal halves, so-called chromo and chromo-shadow domains, and DNA-binding domain in the internal non-conserved region. Here, to examine its
in vivo properties, we expressed HP1
Hsα as a fusion product with green fluorescent protein in human cells. HP1
Hsα was observed to form discrete dots in interphase nuclei and to localize in the centromeric region of metaphase chromosomes by fluorescence microscopy. Interestingly, this dot-forming activity was also found in the N-terminal half retaining the chromo and DNA-binding domains and in the C-terminal chromo-shadow domain. However, the chromo domain alone stained nuclei homogeneously. To correlate this dot-forming activity with self-associating activity
in vitro, the chromo and chromo-shadow domain peptides were independently expressed in
Escherichia coli, affinity purified, and chemically cross-linked with glutaraldehyde. In a SDS-polyacrylamide gel, the former mainly produced a dimer, while the latter produced a ladder of bands up to a tetramer. When passed through a gel filtration column in a native state, these peptides were exclusively separated as a dimer and a tetramer, respectively. These results suggested that the internal DNA-binding and C-terminal chromo-shadow domains are both involved in heterochromatin formation
in vivo.
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Norihiko Kageyama, Shunji Natsuka, Sumihiro Hase
1999 Volume 125 Issue 4 Pages
838-845
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
JOURNAL
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Some α(1, 3)fucosylated oligosaccharides serve as counter receptors to lectin-like adhesion proteins or are expressed with temporal precision during embryogenesis, and α(1, 3)-fucosyltransferase is a key enzyme in the production of these oligosaccharides. Two α(1, 3)-fucosyltransferase genes, designated zFT1 and zFT2, were cloned from zebrafish. Sequence comparisons with other genes indicated that zFT1 and zFT2 share about 30% amino acid sequence identity with human α(1, 3)fucosyltransferases. Although the α(1, 3)fucosyltransferases cloned so far can be classified into three types-myeloid, Lewis, and leukocyte-by virtue of their amino acid sequences, phylogenetic analysis indicated that neither zFT1 nor zFT2 belongs to any of these categories. The expression of zFT1 or zFT2 in mammalian cells induces α(1, 3)fucosyltransferase activity to synthesize the Lewis x structure from pyridyl-aminated lacto-
N-neotetraose; however, lacto-
N-tetraose does not serve as a substrate. Reverse transcriptase-polymerase chain reaction analysis revealed that zFT1 is transcribed during a restricted period before hatching, whereas the mRNA for zFT2 was detected only after hatching.
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Tohru Saeki, Keishi Matoba, Hiroko Furukawa, Kensuke Kirifuji, Ryuhei ...
1999 Volume 125 Issue 4 Pages
846-851
Published: April 01, 1999
Released on J-STAGE: November 18, 2008
JOURNAL
FREE ACCESS
Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na
+-dependent [
3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The
Km and
Vmax, for Na
+-dependent [
3H]taurocholate transport into isolated ileocytes were calcu-lated as 27 μM and 360 pmol/mg protein/min, respectively. Uptake of [
3H]taurocholate was inhibited by
N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two
N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na
+-dependent [
3H]taurocholate uptake activity with a
Km of
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