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Volume 136, Issue 1
Displaying 1-15 of 15 articles from this issue
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Hiroshi Kanda, Masayuki Miura2004 Volume 136 Issue 1 Pages 1-6
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSProgrammed cell death or apoptosis is the regulatory mechanism for removing unneeded cells during animal development and in tissue homeostasis. Perturbation of the cell death mechanisms leads to various disorders, including neurodegenerative diseases, immunodeficiency diseases, and tumors. c-Jun N-terminal kinase (JNK) has crucial roles in the regulation of cell death in response to many stimuli. Since JNK is highly conserved from yeast to mammals, genetic studies using model animals are helpful in understanding the principal cell death mechanisms regulated by JNK. For example, loss-of-function studies using the targeted disruption of murine genes have established the genetic framework of the mechanisms of the cell death induced by UV radiation. Also, in Drosophila, many cell death-related genes have been identified by genetics. Genetic studies of JNK-dependent cell death mechanisms should shed light on the regulation of both physiological and pathological cell death.View full abstractDownload PDF (1662K) -
Aisa Sakaguchi, Kunihiro Matsumoto, Naoki Hisamoto2004 Volume 136 Issue 1 Pages 7-11
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that are activated by diverse stimuli such as growth factors, cytokines, neurotransmitters and various cellular stresses. MAPK cascades are generally present as three-component modules, consisting of MAPKKK, MAPKK and MAPK. The precise molecular mechanisms by which these MAPK cascades transmit signals is an area of intense research, and our evolving understanding of these signal cascades has been facilitated in great part by genetic analyses in model organisms. One organism that has been commonly used for genetic manipulation and physiological characterization is the nematode Caenorhabditis elegans. Genes sequenced in the C. elegans genome project have furthered the identification of components involved in several MAPK pathways. Genetic and biochemical studies on these components have shed light on the physiological roles of MAPK cascades in the control of cell fate decision, neuronal function and immunity in C. elegans.View full abstractDownload PDF (1956K) -
Takashi Adachi-Yamada, Michael B. O'Connor2004 Volume 136 Issue 1 Pages 13-17
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSVarious cell differentiation signals are tightly linked with apoptotic signals. For example, as a result of somatic mutations, cells within a developing field occasionally receive an altered level of morphogenetic signaling that gives rise to an abnormal cell type. However, these developmentally abnormal cells are frequently removed by activating apoptotic signals. Although such phenomena are crucial for assuring normal development and maintaining a healthy state of various organs, the molecular mechanisms that sense aberrant signals and activate the apoptotic pathway (s) have not fully been investigated. In this review, we discuss recent progress in this area. Cell competition and morphogenetic apoptosis are two kinds of cell death, both of which are mediated by abnormal signaling of Dpp, a member of the TGF-beta superfamily that functions in Drosophila as a morphogen, mitogen and survival factor. Cell competition results in autonomous apoptosis induced by reduced reception of the extracellular survival factor Dpp, while morphogenetic apoptosis is nonautonomous, and is induced by contact of cells receiving different levels of Dpp signaling.View full abstractDownload PDF (4772K) -
Xiang-Bin Wang, Bing Zhou, Chang-Cheng Yin, Qing Lin, Hua-Liang Huang2004 Volume 136 Issue 1 Pages 19-28
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAntibody reshaping is an effective way to reduce the immunogenicity while maintaining or improving the affinity of murine antibodies. This paper describe a new in vitro approach for rapidly reshaping murine antibodies by combining DNA shuffling with ribosome display. With the new method, a reshaping anti-4-1 BB single-chain antibody (scFv), Re-4B4-1 scFv, which bound to its antigen (4-1 BB) specifically and strongly, was selected from a reshaping library. These results proved definitely the feasibility of the new designed approach for antibody reshaping.View full abstractDownload PDF (2390K) -
Yasuhiro Kumaki, Katsutoshi Nitta, Kunio Hikichi, Takeshi Matsumoto, N ...2004 Volume 136 Issue 1 Pages 29-37
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPlant glycine-rich RNA-binding proteins (GRRBPs) contain a glycine-rich region at the C-terminus whose structure is quite unknown. The C-terminal glycine-rich part is interposed with arginine and tyrosine (arginine/glycine/tyrosine (RGY)-rich domain). Comparative sequence analysis of forty-one GRRBPs revealed that the RGY rich domain contains multiple repeats of Tyr-(Xaa)h-(Arg)k-(Xaa)l, where Xaa is mainly Gly, “k” is 1 or 2, and “h” and “1” range from 0 to 10. Two peptides, 1 (G1G2Y3G4G5G6R7R8D9G10) and 2 (G1G2R3R4D5G6G7Y8G9G10), corresponding to sections of the RGY-rich domain in Zea mays RAB 15, were selected for CD and NMR experiments. The CD spectra indicate a unique, positive band near 228 nm in both peptides that has been ascribed to tyrosine residues in ordered structures. The pH titration by NMR revealed that a side chain-side chain interaction, presumably an H-Nε…O=Cγ hydrogen bonding interaction in the salt bridge, occurs between Arg (i) and Asp (i+2). 1 D GOESY experiments indicated the presence of NOE between the aromatic side chain proton and the arginine side chain proton in the two peptides suggesting strongly that the Arg (i) aromatic side chain interacts directly with the Tyr (i±4 or i±5) side chain.View full abstractDownload PDF (3239K) -
Masao Miki, Hong Hai, Kimiko Saeki, Yuji Shitaka, Ken-Ichi Sano, Yuich ...2004 Volume 136 Issue 1 Pages 39-47
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSFluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104-1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the Cterminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241-261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S 1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration.View full abstractDownload PDF (1170K) -
Qin Guo, Feng Zhao, Zhi Guo, Xicheng Wang2004 Volume 136 Issue 1 Pages 49-56
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSEquilibrium studies of guanidine hydrochloride (GdnHCI)-induced unfolding of dimeric arginine kinase (AK) from sea cucumber have been performed by monitoring by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphthalene-8 sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking. The unfolding is a multiphasic process involving at least two dimeric intermediates. The first intermediate, I1, which exists at 0-0.4M GdnHCl, is a compact inactive dimer lacking partial global structure, while the second dimeric intermediate, I2, formed at 0.5-2.0M GdnHCl, possesses characteristics similar to the globular folding intermediates described in the literature. The whole unfolding process can be described as follows: (1) inactivation and the appearance of the dimeric intermediate h; (2) sudden unwinding of I1 to another dimeric intermediate, 12; (3) dissociation of dimeric intermediate I2 to monomers U. The refolding processes initiated by rapid dilution in renaturation buffers indicate that denaturation at low GdnHCl concentrations (below 0.4M GdnHCl) is reversible and that there seems to be an energy barrier between the two intermediates (0.4-0.5M GdnHCl), which makes it difficult for AK denatured at high GdnHCl concentrations (above 0.5M) to reconstitute and regain its catalytic activity completely.View full abstractDownload PDF (2003K) -
Shinsaku Maruta, Yasuo Uyehara, Tomoki Aihara, Eisaku Katayama2004 Volume 136 Issue 1 Pages 57-64
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMyosin forms stable ternary complexes with ADP and phosphate analogues of fluorometals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin-ADPfluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167-7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM)•ADP•fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM•ADP•BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HAM-ADP-fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.View full abstractDownload PDF (2511K) -
Oliver Zschenker, Devrim Oezden, Detlev Ameis2004 Volume 136 Issue 1 Pages 65-72
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSLysosomal acid lipase (LAL; EC 3. 1. 1. 13) hydrolyzes intracellular triglycerides and cholesterol esters taken up by various cell-types. Previously, LAL purified from human liver tissue was described as a preproprotein with a 27 amino acid signal peptide and a 49 amino acid propeptide. Three mutants of the putative proregion of LAL were produced and expressed in Spodoptera frugiperda insect cells. Pulse-chase experiments demonstrated that LAL undergoes proteolytical processing. The deletion of the 49 amino acids led to a complete loss of the LAL activity. The two other mutants were produced at the C-terminus of the pro-region, at positions 49 and 50, by site-directed mutagenesis. Mutant K49R showed wild-type LAL activity, but mutant G50A showed significantly reduced enzyme activity compared to wild-type LAL and a greater reduction in culture medium than in detergent cell extracts. Kinetic data suggest that mutant G50A is less stable than wild-type LAL and mutant K49R. In contrast to K 49, the highly conserved amino acid residue G 50 seems to be in a very important position and its mutation influences both secretion and enzyme activity of LAL. A three-dimensional model of LAL shows that K 49 and G 50 are localized in the loopregion between two beta-sheets, highly accessible for proteolytic enzymes. These data together indicate that LAL is indeed a preproprotein, in which the pro-region is essential for its folding and stability, secretion, and enzyme activity.View full abstractDownload PDF (4615K) -
Guangyu Chen, Shanyun Peng, Minji Zou, Honglian Xu, Donggang Xu, Jiaxi ...2004 Volume 136 Issue 1 Pages 73-79
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSB lymphocyte stimulator (BLyS) is a novel member of tumor necrosis factor (TNF) lig- and family that is important in B cell maturation and survival. Previous studies were almost related to the function or mechanism of its wild type. Here, we constructed two site-directed mutants of the recombinant human soluble BLyS, the BY-A and BY V, and found that BY-V ranked the highest whenever in the process of promoting proliferation of B lymphocytes in vitro or stimulating total serum IgG and IgM secretion in vivo. Besides, assays for the biological responses of human leukemic cell lines to BLyS, BY-A and BY-V demonstrated that they could suppress the proliferation of Raji cells but promote the growth of THP-1. The discovery of BY-V with high activity will help come to a conclusion that the mutation of Cys 146 to Val 146 might improve the biological activity of BLyS.View full abstractDownload PDF (2181K) -
Kiyotoshi Maruyama, Takashi Ohuchi, Kenji Yoshida, Yasushi Shibata, Fu ...2004 Volume 136 Issue 1 Pages 81-87
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPeroxynitrite (ONOO-) is thought to be involved in the neurodegenerative process. To screen for neuroprotective compounds against ONOO--induced cell death, we developed 96-well based assay procedures for measuring surviving cell numbers under oxidative stress caused by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1), a generator of ONOO-, and sodium N, N-dietyldithiocarbamate trihydrate (J) DO, an inhibitor of Cu/Zn superoxide (O2-) dismutase. Using these procedures, we obtained a microbial metabolite that rescued primary neuronal cells from SIN-1-induced damage, but not from DDC-induced damage. By NMR analysis, the compound was identified as neoechinulin A, an antioxidant compound that suppresses lipid oxidation. We found that the compound rescues neuronal cells such as primary neuronal cells and differentiated PC 12 cells from damage induced by extracellular ONOO-. However, non-neuronal cells, undifferentiated PC 12 cells and cells of the fibroblast cell line 3Y1 were not rescued. Neoechinulin A has scavenging, neurotrophic factor-like and antiapoptotie activities. This compound specifically scavenges ONOO-, but not O2- or nitric oxide (NO). Similar to known neuroprotective substances such as nerve growth factor and extracts of Gingko biloba leaves, neoechinulin A inhibits the SIN-1-induced activation of caspase-3-like proteases and increases NADH-dehydrogenase activity. These results suggest that neoechinulin A might be useful for protecting against neuronal cell death in neurodegenerative diseases.View full abstractDownload PDF (748K) -
Kun Shi, Katsuya Egawa, Hiroshi Maegawa, Takaaki Nakamura, Satoshi Ugi ...2004 Volume 136 Issue 1 Pages 89-96
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPhosphorylated platelet-derived growth factor (PDGF) receptor becomes internalized and then is dephosphorylated by protein-tyrosine phosphatase (PTP) 1 B at the endoplasmic reticulum (ER). However, it remains unclear where PTP1B dephosphorylates insulin receptor and inhibits its activity. To clarify how and where PTP1B could interact with insulin receptor, we overexpressed a phosphatase-inactive mutant, PTPIBC/S, in 3T3-L 1 adipocytes. Although PDGF receptor was maximally associated with PTP1BC/S at 30min after PDGF stimulation, the maximal association of insulin receptor with PTPIBC/S was attained at 5min after insulin stimulation. Furthermore, dansylcadaverine, a blacker of receptor internalization, inhibited this PDGF-induced association of PTP1BC/S with its receptor. However, dansylcadaverine did not affect the insulin-stimulated association of PTPIBC/S with insulin receptor, as well as dephosphorylation of insulin receptor by PTP1B. These results indicate that PTP1B might interact with insulin receptor and deactivate it without internalization. Finally, we overexpressed the wild-type and cytosolic-form of PTP1B to determine the role of ER-anchoring of PTP1B, and found that both inhibited insulin signaling equally. Thus, our data indicate that localization of PTP1B at the ER is not needed for insulin receptor dephosphorylation by PTP1B.View full abstractDownload PDF (5811K) -
Yoshito Abe, Tomohito Chaen, Xiu Ri Jin, Tomohiro Hamasaki, Naotaka Ha ...2004 Volume 136 Issue 1 Pages 97-106
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIt is difficult to understand the functional mechanisms of integral membrane proteins without having protein chemical information on these proteins. Although there have been many attempts to identify functionally important amino acids in membrane proteins, chemically and enzymatically cleaved peptides of integral membrane proteins have been difficult to handle because of their hydrophobic properties. In the present study, we have applied an analytical method to transmembrane proteins combining amino acid sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography with electro-spray ionization (LCIESI) mass spectrometry. We could analyze most (97%) of the tryptic fragments of the transmembrane domains of band 3 as well as other minor membrane proteins. The peptide mapping of the transmembrane domain of band 3 was completed and the peptide mapping information allowed us to identify the fragments containing lysine residues susceptible to 4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonic acid (SITS) and to 2, 4-dinitrofluorobenzene (DNFB). This method should be applicable to membrane proteins not only in erythrocyte membranes but also in other membranes.View full abstractDownload PDF (1271K) -
Akira Inoue, Takao Ojima, Kiyoyoshi Nishita2004 Volume 136 Issue 1 Pages 107-114
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAkazara scallop striated muscle tropomyosin mutants without a fused amino acid (nf-Tm), and with Ala- (A-Tm) or Asp-Ala. (DA-Tm) fused at the N-terminus were expressed in Escherichia coli cells. Among them, of-Tm alone has an initial methionine. The native Akazara scallop tropomyosin and DA-Tm showed similar α-helix contents and intrinsic viscosity, but nf-Tm and A-Tm exhibited lower values than those of the native tropomyosin. According to the relative viscosity, all the expressed tropomyosins appear to have lost head-to-tail polymerization ability. Though nf-Tm has extremely low actin-binding ability, the ability was almost completely recovered with a two amino acid fusion but incompletely with a one amino acid fusion. On the other hand, an amino acid fusion, irrespective of the number, seemed to inhibit the Mg-ATPase activity of actomyosin. However, the bacterially expressed tropomyosins together with Akazara scallop troponin do not confer the full Ca2+-regulation ability of Mg-ATPase activity of actomyosin. These results support that N-terminal blocking probably by an acetyl group of Akazara scallop tropomyosin plays an important role not only in head-to-tail polymerization and actin-binding, as known for vertebrate tropomyosin, but also in maintaining the secondary or higher structure and Ca2+-reg-ulation together with troponin.View full abstractDownload PDF (1888K) -
Hiroyuki Suzuki, Masaki Nojiri, Nobuo Kamiya, Takumi Noguchi2004 Volume 136 Issue 1 Pages 115-121
Published: July 01, 2004
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSNitrile hydratase (NHase) from Rhodococcus N-771 is a novel enzyme that is inactive in the dark due to an enodogenous nitric oxide (NO) molecule bound to the non-heme iron center, and is activated by its photodissociation. FTIR spectra in the NO stretching region of the dark-inactive NHase were recorded in the temperature range of 270-80 K. Two NO peaks were observed at 1854 and 1846cm-1 at 270 K, and both frequencies upshifted as the temperature was lowered, retaining the peak separation of 8-9cm-1. The relative intensity of the lower-frequency peak increased with decreasing temperature up to _??_120 K, whereas it was mostly unchanged below this temperature. This observation indicates that two distinct conformations with slightly different NO structures are thermally equilibrated in the dark-inactive NHase above _??_120 K, and the interconversion is frozen-in at lower temperatures. The intensity ratio of the NO bands changed gradually upon increasing the pH from 5.5 to 11.0, but no specific pKa value was found. This result, together with the comparison of the light-induced FTIR difference spectra measured at pH 6.5 and 9.0, suggests that the protonation/deprotonation of a specific amino acid group in the active site of NHase is not a direct cause of the occurrence of the two conformations, although several protonatable groups in the protein may influence the energetics of the two conformers. From the previous observation that the isolated a subunit of NHase exhibited a single broad NO peak, it is suggested that interaction of the β subunit forming the reactive cavity is essential for the double-minimum potential of the active-site structure. The frequencies and widths of the two NO bands changed upon addition of propionamide, 1, 4-dioxane, and cyclohexyl isocyanide, indicating that these compounds are bound to the active pocket and change the interactions of the iron center or the dielectric environments around the NO molecule. Thus, the NO bands of NHase can also be a useful probe to monitor the binding of substrates and their analogues to the active pocket.View full abstractDownload PDF (856K)
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