bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium‐derived vasoconstrictor peptide, endothelin: Identity of human and porcine endothelin
Authors: Itoh Yasuaki , Yanagisawa Masashi , Ohkubo Shoichi , Kimura Chiharu , Kosaka Takuo , Inoue Akihiro , Ishida Norio , Mitsui Youji , Onda Haruo , Fujino Masahiko , Masaki Tomoh
Published: July 2002

A cDNA encoding a human endothelium‐derived vasoconstrictor peptide, endothelin, was isolated from a human placenta cDNA library. The nucleotide sequence of this cDNA clone showed that the primary structure of the human preproendothelin has 212 amino acid residues and is highly homologous to porcine preproendothelin, and that human endothelin is identical with porcine endothelin.
Stereotaxic injection of kainic acid into the striatum of rats induces synthesis of mRNA for heat shock protein 70
Authors: Uney J.B. , Leigh P.N. , Marsden C.D. , Lees A. , Anderton B.H.
Published: July 2002

Stereotaxic injection of the excitotoxin kainic acid into the striatum of rats has been shown to induce the production of messenger RNA to the 70 kDa heat shock protein. This was evident 2 h after injection and was maximal 24 h after injection of kainic acid. No change in the level of messenger RNA to β‐actin was dectected.
Degradation of type IX collagen by matrix metalloproteinase 3 (stromelysin) from human rheumatoid synovial cells
Authors: Okada Yasunori , Konomi Hiroshi , Yada Toshikazu , Kimata Koji , Nagase Hideaki
Published: July 2002

The degradation of type IX collagen, a minor collagen in cartilage, was examined by treatment with three different types of matrix metalloproteinases (MMPs) purified from the culture medium of rheumatoid synovial cells. Neither MMP‐1 (collagenase) nor MMP‐2 (so‐called ‘gelatinase’) could digest type IX collagen, but MMP‐3 (stromelysin) readily degraded it into smaller fragments. This suggests that MMP‐3 may be responsible for the pathological degradation and/or normal turnover of type IX collagen.
Production and purification of a recombinant human 14 kDa β‐galactoside‐binding lectin
Authors: Hirabayashi Jun , Ayaki Hitoshi , Soma Gen-Ichiro , Kasai Ken-Ichi
Published: July 2002

The cDNA for a 14 kDa human β‐galactoside‐binding lectin was inserted into a plasmid carrying a taq promoter, and the lectin protein was expressed in E. coli cells. The recombinant lectin was extracted from the cells and purified to apparent homogeneity by a single‐step chromatography on an asialofetuin‐agarose column. Subunit molecular mass (14 kDa), hemagglutinating activity and antigenicity were indistinguishable from those of the human placental lectin. Though the N‐terminal of the placental lectin is blocked with an acetyl group, the recombinant lectin was found to have a free amino group. However, the N‐terminal amino acid sequences were identical. The recombinant lectin was considered to have the same three‐dimensional structure as the placental lectin.
Protein phosphorylation is required for diazoxide to open ATP‐sensitive potassium channels in insulin (RINm5F) secreting cells
Authors: Dunne M.J.
Published: July 2002

The patch‐clamp open‐cell recording configuration has been used to investigate the effects of non‐hydrolyzable analogues of ATP on the diazoxide‐activation of KATP channels in the insulin‐secreting cell line RINm5F. K+ channels inhibited by 0.1, 0.5 and 1.0 mM ATP were consistently activated by 200 μM diazoxide. During sustained activation of channels, exchange of ATP for either AMP‐PNP, AMP‐PCP or ATPγS abolished the effects of diazoxide. If diazoxide was added to the membrane in the continued presence of AMP‐PNP, AMP‐PCP or ATPγS either no effects were observed or alternatively a small transient activation of channels occurred. This study suggests that protein phosphorylation is necessary for diazoxide to activate ATP‐sensitive potassium channels in insulin‐secreting cells.
The N‐terminal domain I of human lactotransferrin binds specifically to phytohemagglutinin‐stimulated peripheral blood human lymphocyte receptors
Authors: Rochard Elisabeth , Legrand Dominique , Mazurier Joël , Montreuil Jean , Spik Geneviève
Published: July 2002

Human lactotransferrin receptors have been recently characterized on mitogen‐stimulated human lymphocytes [(1989) Eur. J. Biochem. 179, 481–487]. In order to define the lactotransferrin recognition site by these receptors, the binding to lymphocytes of several tryptic fragments, isolated from human lactotransferrin by mild tryptic hydrolysis [(1984) Biochim. Biophys. Acta 787, 90–96], has been investigated. The 30 kDa N‐tryptic fragment (residues 4–281) and the re‐associated N,C‐tryptic complex bind to lactotansferrin lymphocyte receptor with a dissociation constant of 44 nM and 39 nM, respectively, similar to the value obtained for the native lactotransferrin (K
d = 46 nM). However, neither the N‐terminal domain II (residues 91–257) nor the 50 kDa C‐tryptic fragment (residues 282–703) are recognized. These results suggest that the binding site of human lactotransferrin by the lymphocyte receptor is located in the N‐terminal lobe and more precisely in the N‐terminal domain I (residues 4–90 and/or 258–281).
Endothelin increases single‐channel calcium currents in coronary arterial smooth muscle cells
Authors: Silberberg Shai D. , Poder Thomas C. , Lacerda Antonio E.
Published: July 2002

Endothelin (ET), a newly identified vasoconstrictor peptide produced by endothelial cells, depends on extracellular calcium for its action [(1988) Nature 332, 411–415]. It is not yet known whether the increase in calcium influx induced by ET results from a direct effect on the Ca2+ channels or is secondary to a reduction in membrane potential. To address this question, we studied the effects of ET on single‐channel calcium currents of freshly dissociated porcine coronary artery smooth muscle cells using the cell‐attached mode of the patch‐clamp technique. We show that ET increases Cal+‐channel activity with no effect on channel open time or conductance. The ability of bath‐applied ET to increase single‐channel calcium currents in the cell‐attached mode is evidence that the peptide acts via a second messenger system.
Inhibition of intraerythrocytic development of Plasmodium falciparum by proteinase inhibitors
Authors: Rockett K.A. , Playfair J.H.L. , Ashall F. , Target G.A.T. , Angliker H. , Shaw E.
Published: July 2002

A group of inactivators of cysteinyl proteinases which function by covalent bond formation have been examined for their ability to inhibit thedevelopment of Plasmodium falciparum within red blood cells. The most effective of these caused inactivation of the parasite near 10−8 M concentration. The range of inhibitory action varied with peptide structure in a manner characteristic of affinity labels for proteinases suggesting that the target of inhibition was an unidentified proteinase, probably of the cysteinyl type, but different from cathepsins B and L.
Inositol trisphosphate‐mediated Ca2+ release in beet mu;somes is inhibited by heparin
Authors: Brosnan James M. , Sanders Dale
Published: July 2002

Microsomes from the storage root of red beet exhibit ATP‐dependent, protonophore‐sensitive Ca2+ accumulation, characteristic of the vacuolar membrane (tonoplast). A portion (20%) of this intravesicular Ca2+ store is specifically released by inositol 1,4,5‐trisphosphate (InsP3) with a K
0.5 = 0.54 μM. InsP3,‐mediated Ca2+ release is eliminated by low concentrations of heparin (K
0.5 = 86 nM). This result highlights the conservation between the animal and plant inositol 1,4,5‐trisphosphate‐mediated Ca2+ release mechanisms, despite the fact that they are located at different intracellular membranes.
Platelet membrane glycoproteins IIb and IIIa are substrates of purified pp60c‐src protein tyrosine kinase
Authors: Findik Duygu , Reuter Christoph , Presek Peter
Published: July 2002

Human platelet glycoproteins IIb and IIIa form the receptor for fibrinogen, von Willebrand factor and fibronectin. Isolated human glycoproteins IIb‐IIIa are phosphorylated by purified pp60c‐src protein tyrosine kinase. Analysis of the phosphorylated proteins on SDS‐PAGE showed that under reducing conditions both phosphoproteins change their relative molecular masses from 135 to 120 kDa and from 97 to 105 kDa, which are characteristic properties of glycoproteins IIb‐IIIa. Phosphorylated proteins could be immunoprecipitated with an antiserum against glycoproteins IIb‐IIIa but not by control serum. Some kinetic properties of the glycoprotein phosphorylations are also investigated. How the glycoprotein IIb‐IIIa complex acquires its receptor activity in stimulated platelets is unknown; however, phosphorylation could be an important mechanism.
Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate
Authors: Sakimura Kenji , Bujo Hideaki , Kushiya Etsuko , Araki Kazuaki , Yamazaki Makoto , Yamazaki Masatoshi , Meguro Hiroyuki , Warashina Akira , Numa Shosaku , Mishina Masayoshi
Published: July 2002

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1‐specific mRNA and the dose‐response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate‐gated ionic channels are hetero‐oligomers of different subunits.
Conformational changes of aminoacyl‐tRNA and unchanged tRNA upon complex formation with polypeptide chain elongation factor Tu
Authors: Haruki Mitsuru , Matsumoto Rieko , Hara-Yokoyama Miki , Miyazawa Tatsuo , Yokoyama Shigeyuki
Published: July 2002

The conformation change of Thermus thermophilus tRNAIle
1 upon complex formation with T. thermophilus elongation factor Tu (EF‐Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2‐thioribothymidine residue in the T‐loop) and at 295 nm (due to the core structure of tRNA). Formation of the ternary complex of isoleucyl‐tRNAIle
1 and EF‐Tu·GTP increased the intensities of these CD bands, indicating stabilization of the association between the T‐loop and the D‐loop and also a significant conformation change of the core region. Upon complex formation of EF‐Tu·GTP and unchanged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized. On the other hand, the binding with EF‐Tu·GDP does not appreciably affect the conformation of isoleucyl‐tRNA or unchanged tRNA. These indicate the importance of the γ‐phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl‐tRNA and EF‐Tu·GTP.
Evidence for co‐regulation of Cu,Zn superoxide dismutase and metallothionein gene expression in yeast through transcriptional control by copper via the ACE 1 factor
Authors: Carri Maria Teresa , Galiazzo Francesca , Ciriolo Maria Rosa , Rotilio Giuseppe
Published: July 2002

Saccharomyces cerevisiae mutant strain DTY26, lacking ACE1, the protein mediator for the induction or metallothionein gene expression, is unable to increase Cu,Zn superoxide dismutase mRNA in response to copper. In the wild‐type strain DTY22 transcription or both Cu,Zn superoxide dismutase and metallothionein genes is induced by copper and silver, as expected on the basis of previous results indicating that ACE1 binds only Ag(I) besides Cu(I). We conclude that at the transcriptional level Cu,ZnSOD is co‐regulation with metallothionein. Furthermore, structural similarities between the two promoters were found, which could explain the co‐regulation effect and the quantitative differences in the response of the two genes to copper.
Comparative membrane locations and activities of human monoamine oxidases expressed in yeast
Authors: Urban P. , Andersen J.K. , Hsu H.-P.P. , Pompon D.
Published: July 2002

Human monoamine oxidases A and B were expressed under the control of a galactose inducible promoter in Saccharomyces cerevisiae. The two MAO isoenzymes were found located in the yeast mitochrondrial outer membrane, probably in different orientations as suggested by controlled proteolysis experiments. A high level of both human MAO‐A or ‐B activities is measured in intact mitochrondria without the need for any detergent solubilisation step. The substrate and inhibitor selectivities of the membrane‐bound MAOs are highly similar to those of purified human enzymes. The level of MAO‐B activity, however, is selectively lowered when bound to the membrane.
Two antifungal thaumatin‐like proteins from barley grain
Authors: Hejgaard Jørn , Jacobsen Susanne , Svendsen Ib
Published: July 2002

Antifungal activity has been associated with 2 immunochemically distinct proteins, protein R and S (M
r ∼23 kDa; pI 9‐10), which were isolated in pure form from barley grain. The proteins are homologous with thaumatin‐ and pathogenesis‐related proteins of the PR5 family. The proteins inhibit growth of i.a. Trichoderma viride and Candida albicans in microtiter plate assays and act synergistically with barley grain chitinase C. Like maize zeamatin, protein R and S but not chitinase C retarded fungal growth in synergism with nikkomycin Z, a nucleoside‐peptide inhibitor of fungal chitin synthesis. Although no inhibition of α‐amylases or serine proteases could be associated with protein R or S the results indicate that the homologous maize grain bifunctional inhibitor of insect α‐amylase and trypsin is very similar to or identical with maize zeamatin, which was proposed to have permeabilizing activity towards fungal membranes. Thus, in addition to the intensely sweet properties of thaumatin, multiple unrelated defense functions against insect and fungal pests can now be associated with the family of thaumatin‐homologous proteins.
Overproduction of human Mn‐superoxide dismutase modulates paraquat‐mediated toxicity in mammalian cells
Authors: St. Clair Daret K. , Oberley Terry D. , Ho Ye-Shih
Published: July 2002

Manganese superoxide dismutase (MnSOD) is a nuclear encoded mitochondrial matrix enzyme that functions to scavenge superoxide radicals. The human MnSOD cDNA under the transcriptional control of a human β‐actin promoter was introduced into mouse C3H10T cells by cotransfection with a recombinant plasmid containing the NeoR selectable marker. C3H10T transformants (C3H‐SOD) were obtained that expressed high levels of authentic enzymatically active human MnSOD. Overexpression of the MnSOD gene did not affect the protein levels of CuZnSOD, catalase (CAT), or glutathione peroxidase (GPX) in the transformants. Treatment of cells with paraquat was less toxic to the C3H‐SOD cells than to the control cells. These results are consistent with the possibility that superoxide radicals are mediators of paraquat cytotoxicity.
Immunoreactive thyroliberin (TRH) precursor forms in human hypothalamus and anterior pituitary tissues
Authors: Croissandeau G. , Pagésy P. , Grouselle D. , Le Dafniet M. , Peillon F. , Li J.Y.
Published: July 2002

Immunoreactive (IR) proTRH forms were characterized in human hypothalamic tissue with two antisera raised against a hepta‐ and a decapeptide containing the TRH progenitor sequence (‐Gln‐His‐Pro‐Gly‐). A similar study was performed in human normal and adenomatous anterior pituitaries, tissues in which TRH synthesis has been previously suggested. IR‐proTRH was found in all the samples ranging from 42–775 fmol/mg proteins. Size exclusion chromatography identified a major 25–35 kDa form and a minor 4–8 kDa form. The existence of the major form was confirmed by immunoblotting. The results suggest that both human hypothalamic and normal or adenomatous anterior pituitary tissues synthesize similar IR‐proTRH forms.
Both the anaerobic pathway and aerobic desaturation are involved in the synthesis of unsaturated fatty acids in Vibrio sp. strain ABE‐1
Authors: Morita Naoki , Gotoh Masura , Okajima Nobuhiro , Okuyama Hidetoshi , Hayashi auShoichi Higashi Hideyuki , Murata Norio
Published: July 2002

Vibrio sp. strain ABE‐1 is a unique marine bacterium in terms of its ability to synthesize Δ9‐trans‐hexadecenoic acid and Δ7‐cis‐tetradecenoic acid (14:1(7c); Okuyama, H., Sasaki, S., Higashi, S. and Murata, N. (1990) J. Bacteriol. 172, 3515‐3518). The present study, involving labeling with [1‐14C]acetate, demonstrated that 14:1 is synthesized by the anaerobic pathway. When cells of this bacterium were grown in the presence of [1‐14C]myristic acid (14:0), this compound was converted to palmitic (16:0) and hexadecenoic (16:1) acids but not to 14:1, under aerobic conditions. These results suggest that the incorporated 14:0 was elongated to 16:0 and then converted to 16:1 by the aerobic desaturation of 16:0. It appears that the anaerobic pathway and aerobic desaturation are both involved in the synthesis of unsaturated fatty acids during aerobic growth of Vibrio sp. strain ABE‐1.
Involvement of separate domains of the cellulosomal protein S1 of Clostridium thermocellum in binding to cellulose and in anchoring of catalytic subunits to the cellulosome
Authors: Salamitou Sylvie , Tokatlidis Kostas , Béguin Pierre , Aubert Jean-Paul
Published: July 2002

Fragments of the 25OkDa SI subunit of the Clostridium thermocellum cellulosome were obtained by protease‐induced or spontaneous degradation. All detectable fragments, down to a mass of about 30 kDa, retained the ability to bind to 125I‐labelled endoglucanase CelD, one of the catalytic subunits of the cellulosome. Several fragments were able to bind both to cellulose and to CElD. However, some fragments that could still bind to CelD did not have the ability to bind to cellulose. Therefore, S1, a putative scaffolding protein of the cellulosome, is likely to carry two separate types of domains, one of which binds to cellulose, while the other type binds to the various catalytic subunits of the complex.
IL‐2‐dependent in vivo and in vitro tyrosine phosphorylation of IL‐2 receptor γ chain
Authors: Asao Hironobu , Kumaki Satoru , Takeshita Toshikazu , Nakamura Masataka , Sugamura Kazuo
Published: July 2002

We previously reported a molecule, p64, which was tentatively named the γ chain, coprecipitable with the β chain of human interleukin‐2 receptor (IL‐2R). The present study demonstrated that the γ chain, as well as the β chain expressed on IL‐2‐responsive cells, is phosphorylated on tyrosine residues in an IL‐2‐dependent manner in vivo and in vitro. The in vivo tyrosine phosphorylation of both chains was similarly induced within 1 min after IL‐2 stimulation, and their in vitro tyrosine phosphorylation with the anti‐IL‐2Rβ antibody‐directed immunocomplex was also increased by treatment of cells with IL‐2. These results suggest that a tyrosine kinase is associated with the βγ subunit complex, of which activation by IL‐2 may result in transduction of intracellular signals.
Fibronectin type III‐like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates
Authors: Hansen Christian K.
Published: July 2002

Fibronectin type III‐like sequences are present in many proteins from higher eukaryotes and are involved in protein‐protein interactions, heparin binding and cell adhesion. A nine‐member family of bacterial sequences is shown to be significantly homologous to the type III‐like sequences. All the sequences are contained in secreted depolymerases acting on complex, energy‐rich insoluble substrates, in which they apparently do not participate in catalysis or substrate‐binding, their exact function remaining unclear. Furthermore, a new family of sequences, present in some cellulases, is presented.
Structural similarities between the aspartate receptor of bacterial chemotaxis and the trp repressor of E. coli Implications for transmembrane signaling
Authors: Lynch Berkley A. , Koshland Daniel E.
Published: July 2002

A high resolution structure of the N‐terminal ligand‐binding domain of the aspartate receptor which mediates aspartate chemotaxis in Salmonella typhimurium has recently been reported. A least‐squares superposition of the α‐amino nitrogen, α‐carbon, β‐carbon, and α‐carboxylate carbon of the aspartate bound to the aspartate receptor onto the equivalent atoms in the tryptophan bound to the trp repressor provides evidence for similarity between key parts of the active sites that bind to the α‐amino and α‐carboxylates of the respective ligands. Because the N‐terminal domain of the aspartate receptor and the trp repressor also share other structural similarities, we hypothesize that the similarity between the aspartate receptor and the trp repressor derives from a similarity in ligand‐induced conformational changes at the active sites of these proteins. This hypothesis also implies that an important signaling event in the aspartate receptor occurs through tertiary conformational changes within a single subunit.
Controversy on chloroplast origins
Authors: Lockhart Peter J. , Penny David , Hendy Michael D. , Howe Christopher J. , Beanland Timothy J. , Larkum Anthony W.D.
Published: July 2002

Controversy exists over the origins of photosynthetic organelles in that contradictory trees arise from different sequence, biochemical and ultrastructural data sets. We propose a testable hypothesis which explains this inconsistency as a result of the differing GC contents of sequences. We report that current methods of tree reconstruction tend to group sequences with similar GC contents irrespective of whether the similar GC content is due to common ancestry or is independently acquired, Nuclear encoded sequences (high GC) give different trees from chloroplast encoded sequences (low GC). We find that current data is consistent with the hypothesis of multiple origins for photosynthesis organelles and single origins for each type of light harvesting complex.
Decorin is specifically solubilized by heparin from the extracellular matrix of rat skeletal muscles
Authors: Melo Francisco , Brandan Enrique
Published: July 2002

We have previously communicated that heparin co‐solubilizes the asymmetric form of acetylcholinesterase (AChE) and a dermatan sulfate proteoglycan from the extracellular matrix (ECM) of rat skeletal muscles. In this report we unequivocally demonstrate by biochemical and immunological analyses that the proteoglycan that is solubilized by heparin from rat skeletal muscle ECM corresponds to decorin. These results support the concept for the role of decorin in the ECM organization.
Proteasome and cell cycle
Authors: Richter-Ruoff Birgit , Wolf Dieter H.
Published: July 2002

The cell cycle of eukaryotic cells is strictly regulated. This regulation is performed by a serine/threonine kinase. The different functions of this kinase in the cell cycle are modulated by different cyclins, which fluctuate in concentration (‘cycle’) during the different stages of the cell cycle. Using yeast as a model organism we show here that the activity of the multifunctional proteinase, the proteasome, is directly connected to the function of the mitotic cyclin Clb2. Our studies indicate that the proteasome is the proteolytic regulator of this cyclin and thus a central regulator of the cell cycle.
14‐3‐3 proteins bind to histone and affect both histone phosphorylation and dephosphorylation
Authors: Chen Fusheng , Wagner Paul D.
Published: July 2002

14‐3‐3 proteins appear to play a critical role in Ca2+‐stimulated secretion in permeabilized chromaffin cells. 14‐3‐3 proteins have been reported to be both stimulators and inhibitors of protein kinase C (PKC). We have found that 14‐3‐3 proteins, isolated on the basis of their ability to enhance secretory activity, stimulated histone phosphorylation by PKC, but they had no effect on myosin light chain phosphorylation by PKC. 14‐3‐3 proteins were also found to inhibit the rate of [32P]histone dephosphorylation but not the rate of [32P]myosin light chain dephosphorylation. Cross‐linking experiments and affinity chromatography demonstrated that 14‐3‐3 proteins bind to histones. These results suggest that at least some of the reported effects of 14‐3‐3 proteins on PKC activity may result from 14‐3‐3 proteins binding to histone.
Mechanism of covalent modification of glyceraldehyde‐3‐phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents
Authors: Mohr Susanne , Stamler Jonathan S. , Brüne Bernhard
Published: July 2002

Previous studies have suggested that glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO•‐induced [32P]NAD+‐dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO• release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS—NO. Peroxynitrite (ONOO−) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO+
2) through the intermediacy of RS—NO. However, the efficiency of this reaction is limited by ONOO−‐induced oxidation of protein SH groups at the active site. ONOO− generation appears to account for the modification of GAPDH by SIN‐1. Thus, S‐nitrosylation of the active site thiol is a prequisite for subsequent post‐translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non‐enzymatic ADP‐ribosylation (like) reactions. In biological systems, endogenous RS—NO are likely to support the NO group transfer to thiol‐containing proteins.
Calcium channels: The β‐subunit increases the affinity of dihydropyridine and Ca2+ binding sites of the α1‐subunit
Authors: Mitterdorfer Jörg , Froschmayr Monika , Grabner Manfred , Striessnig Jörg , Glossmann Hartmut
Published: July 2002

A Ca2+ channel α1‐subunit derived from rabbit heart was transiently expressed in COS‐7 cells. The dihydropyridine (+)‐isradipine had low affinity (K
i = 34.3 nM) for the α1‐subunit in the absence of the β‐subunit due to rapid dissociation (k
−1 = 0.11 min−1). Co‐expression of the β‐subunit resulted in a ⪢ 35‐fold increase in (+)‐isradipine binding affinity (K
i = 0.9 nM) due to decreased dissociation (k
−1 of 0.007 min−1). Higher DHP binding affinity was associated with an increase of the apparent affinity of Ca2+ ions for the channel. Our data suggest that the β‐subunit affects the coordination of Ca2+ ions with sites that are coupled to the dihydropyridine binding domain and by this mechanism increases the affinity for these ligands.
Identification of a novel protein tyrosine phosphatase with sequence homology to the cytoskeletal proteins of the band 4.1 family
Authors: L'Abbé Denis , Banville Denis , Tong Yiai , Stocco Rino , Masson Sylvie , Ma Shuyi , Fantus George , Shen Shi-Hsiang
Published: July 2002

Use of the polymerase chain reaction (PCR) in conjunction with Southern hybridization, using probes corresponding to known phosphatase sequences, resulted in the identification of rat cDNA clones encoding a novel protein tyrosine phosphatase which was termed rPTP2E. The cDNAs comprise 5,543 bp and predict a polypeptide of 1175 amino acids possessing a single catalytic domain at its C‐terminus. The N‐terminal region of the deduced polypeptide displays high sequence homology to the cytoskeleton‐associated proteins of the band 4.1 family. A variant form, termed rPTP2E1, was also identified which contains the catalytic domain only. rPTP2E and rPTP2E1 were expressed in various rat tissues, particularly abundantly in adrenal glands. The catalytic domain of PTP2E was expressed in Escherichia coli and was shown to possess specific protein tyrosine phosphatase activity. The identification of rPTP2E suggests the existence of a subfamily of band 4.1 domain‐containing PTPs which may play an important role in signalling pathway and control of cytoskeletal integrity.
A novel gene delivery system using EGF receptor‐mediated endocytosis
Authors: Chen Jiabing , Gamou Shinobu , Takayanagi Atsushi , Shimizu Nobuyoshi
Published: July 2002

A monoclonal antibody to the human epidermal growth factor (EGF) receptor was conjugated with polylysine and the resulting conjugate was affinity‐linked with DNA (gene). This novel gene delivery system utilizes receptor‐mediated endocytosis and would be especially suitable for gene therapy for EGF receptor‐overproducing squamous cell carcinomas.
Sequence‐specific cleavage of RNA by a hybrid ribonuclease H
Authors: Nakai Chieko , Konishi Aritomo , Komatsu Yasuo , Inoue Hideo , Ohtsuka Eiko , Kanaya Shigenori
Published: July 2002

Site‐specific cleavage of the 22‐, 132‐ and 534‐base RNAs by the DNA/protein hybrid R Nase H were examined. The 22‐base RNA was chemically synthesized, and 132‐ and 534‐base RNAs were prepared by run‐off transcription. The hybrid enzyme cleaves these RNAs, which contain a single target sequence, primarily at the unique phosphodiester bond within the target sequence. The hybrid enzyme performs multiple turnovers, and at a substrate/enzyme ratio of 10:1 the RNAs are almost completely cleaved by the hybrid enzyme at 37°C within 1 h. We propose that hybrid RNase H molecules with various oligodeoxyribonucleotides function as RNA restriction enzymes and are useful for structural and functional studies of RNA.
Fatty acid binding site of the mitochondrial uncoupling protein
Authors: Ježek Petr , Freisleben Hans-Joachim
Published: July 2002

Fatty acid binding site on isolated mitochondrial uncoupling protein (UcP) is demonstrated using EPR spectroscopy of 5‐DOXYL‐stearic acid (5‐SASL), which also activated H+ transport in proteoliposomes containing UcP. In the presence of UcP the EPR spectrum showed reproducible broadening of the low field peak as well as an increase in h
+1I/h
+1M ratio, rotational correlation time and in order parameter. The half‐height width of the low field peak was even doubled in the presence of another UcP ligand, GDP. Palmitic acid reversed the effect of 5‐SASL and non‐ionizable 5‐DOXYL‐decane did not exhibit it.
Induction of glyoxylate cycle enzymes in rat liver upon food starvation
Authors: Popov Vasily N. , Igamberdiev Abir U. , Schnarrenberger Claus , Volvenkin Sergei V.
Published: July 2002

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112‐fold by ammonium sulfate precipitation, and chromotography on DEAE‐cellulose and Toyopearl HW‐65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K
m(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4‐fold by ammonium sulfate precipitation. The enzyme had a K
m(acetyl‐CoA) of 0.2 μM, a K
m(glyoxylate) of 3 mM and a pH optimum of 7.6.
The impulsivity rating scale (IRS): preliminary results
Authors: Lecrubier Y , Braconnier A , Said S , Payan C
Published: July 2002

Until now, the instruments for the clinical evaluation of impulsivity have relied essentially on either personality inventories or to a lesser extent on specific questionnaires. Therefore this paper presents the preliminary results of a new rating scale: the Impulsivity Rating Scale (IRS). This is a 7-item hetero-evaluation of impulsivity based on the behaviour of the patient in usual life situations. The administration of the scale is easy and short: 15 minutes. The IRS was tested in five different population samples: 31 impulsive inpatients, 36 adults with major depressive episode, 15 healthy control subjects, 56 smokers before and after one week of tobacco withdrawal, and 47 adolescent depressed inpatients before and four weeks after antidepressant treatment. The results show good construct validity, good concurrent validity, good inter-rater reliability and sensitivity to change. A threshold of 8 for the total score (range 0 to 21) gives good specificity and sensitivity. The principal component analysis shows the existence of a main factor composed of all items, with lower correlation for two items which may belong to a second factor needing further investigations.
Functional Fv fragment of an antibody specific for CD28: Fv‐mediated co‐stimulation of T cells
Authors: Takemura Shin-ichi , Asano Ryutaro , Tsumoto Kouhei , Arai Takashi , Sakurai Naoki , Kodama Hideaki , Yoshida Hiroshi , Katayose Yu , Suzuki Masanori , Matsuno Seiki , Kudo Toshio , Kumagai Izumi
Published: July 2002

The most predominant co‐stimulation pathway, which is critical for T cell activation and proliferation, is the CD28‐B7 pathway. The anti‐CD28 monoclonal antibody (mAb) also provides a co‐stimulatory signal to T cells. In order to construct a functional Fv fragment (complex of VH and VL domains) of anti‐CD28 antibody using a bacterial expression system, cDNA encoding the variable regions of immunoglobulin from 15E8 hybridoma cells was cloned and expressed in Escherichia coli. The Fv fragment was obtained as a soluble protein from the periplasmic fraction and showed a binding pattern similar to parental IgG. The Fv fragment induced proliferation of peripheral blood mononuclear cells in the presence of anti‐CD3 or anti‐CD2 mAb and enhanced anti‐tumor activity of anti‐MUC1×anti‐CD3 bispecific antibody when tested with lymphokine‐activated killer cells with T cell phenotype. Thus, the anti‐CD28 Fv fragment will be promising not only for the study of co‐stimulation, but also for cancer immunotherapy.
Disruption of SMN function by ectopic expression of the human SMN gene in Drosophila
Authors: Miguel-Aliaga Irene , Chan Yick Bun , Davies Kay E. , van den Heuvel Marcel
Published: July 2002

Spinal muscular atrophy is a neurodegenerative disorder caused by mutations or deletions in the survival motor neuron (SMN) gene. We have cloned the Drosophila ortholog of SMN (DmSMN) and disrupted its function by ectopically expressing human SMN. This leads to pupal lethality caused by a dominant‐negative effect, whereby human SMN may bind endogenous DmSMN resulting in non‐functional DmSMN/human SMN hetero‐complexes. Ectopic expression of truncated versions of DmSMN and yeast two‐hybrid analysis show that the C‐terminus of SMN is necessary and sufficient to replicate this effect. We have therefore generated a system which can be utilized to carry out suppressor and high‐throughput screens, and provided in vivo evidence for the importance of SMN oligomerization for SMN function at the level of an organism as a whole.
Leukemia inhibitory factor enhances mast cell growth in a mast cell/fibroblast co‐culture system through Stat3 signaling pathway of fibroblasts
Authors: Hiragun Takaaki , Morita Eishin , Mihara Shoji , Tanaka Toshihiko , Gyotoku Eiichi , Kameyoshi Yoshikazu , Yamamoto Shoso
Published: July 2002

Leukemia inhibitory factor (LIF) enhanced mast cell growth in a mast cell/3T3 fibroblast co‐culture system, however the precise mechanisms have not been defined. Western blot analysis showed that bone marrow‐derived mast cells failed to express both LIF receptor (LIFR) and gp130, whereas 3T3 fibroblasts expressed both LIFR and gp130. This result indicates that the activity of LIF for mast cell growth is mediated by 3T3 fibroblasts. Signal transducer and activator of transcription (Stat) 3‐transfected 3T3 fibroblasts enhanced mast cell growth. In addition, dominant‐negative Stat3‐transfected fibroblasts blocked LIF‐mediated mast cell growth in the co‐culture system. In conclusion, LIF‐induced mast cell growth in the co‐culture system is mediated by an indirect pathway via 3T3 fibroblasts through activating Stat3 signaling pathway in 3T3 fibroblasts.
Developing germ cells in mouse testis express pheromone receptors
Authors: Tatsura Hiroyuki , Nagao Hiroshi , Tamada Atsushi , Sasaki Shoichi , Kohri Kenjiro , Mori Kensaku
Published: July 2002

Pheromone receptors are expressed in the accessory olfactory system, which is vital for non‐specific chemical communication and for sexual behavior. Under the hypothesis that some of the pheromone molecules released from female reproductive organs might regulate sperm chemotaxis or chemokinesis, we examined whether the V1R type pheromone receptor mRNAs are expressed in developing germ cells. By a reverse transcription‐PCR method, we obtained nine kinds of cDNA fragments belonging to the receptor family. In situ hybridization analysis in testicular sections using probes of testicular pheromone receptors (TVRs) revealed that TVR mRNAs were expressed by spermatids. TVRs were also expressed in the accessory olfactory organ. In the testis, hybridization signals were localized in subsets of the seminiferous tubules, suggesting that TVRs were expressed by selective subsets of the spermatids. In situ hybridization study suggests also that each sperm expresses multiple pheromone receptors. The testicular pheromone receptors might have an important role in the maturation and/or migration of sperm.
An internal region of the RpoH heat shock transcription factor is critical for rapid degradation by the FtsH protease
Authors: Bertani Dino , Oppenheim Amos B. , Narberhaus Franz
Published: July 2002

The proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (σ32) is highly unstable. Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP‐dependent protease FtsH. Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH1 is highly stable. To determine which regions of E. coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins. Truncation of N‐terminal amino acids had no, and deletion of C‐terminal amino acids only a minor effect on stability of RpoH. A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36–122) roughly comprising the sigma factor region 2. This is the first demonstration of an internal RpoH region being responsible for FtsH‐mediated degradation.
Molecular and functional characterization of a 5‐HT4 receptor cloned from human atrium
Authors: Blondel Olivier , Vandecasteele Grégoire , Gastineau Monique , Leclerc Stéphanie , Dahmoune Yamina , Langlois Michel , Fischmeister Rodolphe
Published: July 2002

5‐Hydroxytryptamine (5‐HT) has been shown to exert positive inotropic, chronotropic, and lusitropic effects and to stimulate the L‐type calcium channel current (I
Ca) in human atrial tissue through activation of the pharmacologically defined 5‐HT4 receptor subtype. However, the molecular nature of the receptor(s) involved in these effects is still unknown. In the present study, we report the molecular nature of a 5‐HT4 receptor cloned from human atrium, h5‐HT4A. Sequence analysis reveals that h5‐HT4A displays a 93% protein identity with the short form of the 5‐HT4 receptor recently isolated from rat brain. h5‐HT4A mRNA is expressed in human atrium but not ventricle, and is also found in brain and GI tract. h5‐HT4A transiently expressed in COS‐7 cells displays a classical 5‐HT4 pharmacological profile. However, affinities of the h5‐HT4A receptor for agonists such as ML10302, BIMU1, renzapride or zacopride were 4–10‐fold lower than the ones found in brain. Moreover, the stimulatory patterns of cAMP formation by h5‐HT4A in response to the 5‐HT4 agonists ML10302 and renzapride were very similar to the patterns of stimulation of I
Ca obtained in response to these compounds in human atrial myocytes. We conclude that h5‐HT4A likely mediates the effects of 5‐HT in human atrium and may differ from 5‐HT4 receptor isoforms present in the brain and GI tract.
Activation of transcription factor NF‐κB by phagocytic stimuli in human neutrophils
Authors: McDonald Patrick P , Cassatella Marco A
Published: July 2002

Phagocytosis represents an important physiological trigger for the inducible expression of several genes in human neutrophils. Here, we report that a DNA‐binding activity primarily consisting of the classical NF‐κB heterodimer, p50/RelA, is induced in phagocytosing neutrophils. Under these conditions, NF‐κB activation was found to be a rapid and transient response, reaching a maximum by 10–15 min, and returning to near‐basal levels by 30 min. In neutrophils undergoing the phagocytosis of opsonized yeasts, the onset of NF‐κB activation was paralleled by a decline in immunoreactive IκB‐α protein levels, and the cellular IκB‐α pool was replenished by 30 min, in agreement with our gel shift data. We conclude that NF‐κB activation could constitute one of the mechanisms whereby the expression of κB‐responsive genes is enhanced in phagocytosing neutrophils. To our knowledge, this represents the first demonstration that phagocytic stimuli can induce NF‐κB activation in human neutrophils.
Contribution of NO synthases to neutrophil infiltration in the gastric mucosal lesions in rats with water immersion restraint stress
Authors: Nishida Keiji , Ohta Yoshiji , Ishiguro Isao
Published: July 2002

A decrease in constitutive NO synthase (cNOS) activity and an increase in inducible NO synthase (iNOS) activity occurred with an increase in myeloperoxidase (MPO) activity, an index of neutrophil infiltration, in the gastric mucosa of rats with water immersion restraint (WIR) stress. This increase in gastric mucosal MPO activity was enhanced by pretreatment with N
G‐monomethyl l‐arginine, a non‐selective NOS inhibitor, but was prevented with maintenance of gastric mucosal cNOS activity by pretreatment with aminoguanidine, a selective iNOS inhibitor. The MPO activity was negatively correlated with the cNOS activity in all WIR‐stressed rats used (r=−0.723). These results suggest that a decrease in cNOS activity could contribute to an increase in neutrophil infiltration in the gastric mucosa of WIR‐stressed rats.
Stress‐responsive expression of genes for two‐component response regulator‐like proteins in Arabidopsis thaliana
Authors: Urao Takeshi , Yakubov Bakhtiyor , Yamaguchi-Shinozaki Kazuko , Shinozaki Kazuo
Published: July 2002

Four cDNAs that encode two‐component response regulator‐like proteins were cloned from Arabidopsis thaliana. Putative proteins (ATRR1–4) contain a receiver domain with a conserved aspartate residue – a possible phosphorylation site – at the N‐terminal half. ATRR2 lacks the C‐terminal half; the others contain a C‐terminal domain abundant in acidic amino acids or proline residues. ATRR1 and ATRR2 are expressed more in roots than in other tissues and are induced by low temperature, dehydration and high salinity. Levels of ATRR3 and ATRR4 were not affected by stress treatments. These results suggest that ATRRs play distinct physiological roles in Arabidopsis, and that some are involved in stress responses.
Molecular cloning, tissue distribution and androgen regulation of rat protein C inhibitor
Authors: Wakita Toshiaki , Hayashi Tatsuya , Yuasa Hiroyuki , Nishioka Junji , Kawamura Juichi , Suzuki Koji
Published: July 2002

Protein C inhibitor (PCI) is the plasma serine protease inhibitor of activated protein C, the active enzyme of the anticoagulant protein C pathway. Recently, PCI was also detected in human seminal plasma and reproductive organs (testis, seminal vesicle and prostate) suggesting that PCI may also play an important role in the reproductive system. In this study, we cloned the full length of rat PCI cDNA, and determined its amino acid sequence and tissue distribution. We also evaluated the effect of androgen on PCI mRNA expression in seminal vesicles and testes. The isolated 2074‐bp rat PCI cDNA was composed of a 47‐bp 5′‐non‐coding region, a 1218‐bp coding region of a 406‐amino acid precursor protein, a stop codon and a 806‐bp 3′‐non‐coding region. The deduced amino acid sequence of rat PCI showed 85.7%, 64.1% and 62.2% homology with that of mouse, rhesus monkey and human PCIs, respectively. Northern blot analysis showed that the rat PCI mRNA is expressed strongly in the seminal vesicle, moderately in the testis, but not in the liver. PCI mRNA expression in seminal vesicles and testes was found to increase during the process of development, suggesting that it is under androgen control. Subsequently, we examined the effect of castration and/or treatment with 17β‐estradiol or testosterone on PCI mRNA expression in the mature rat seminal vesicles. The PCI mRNA expression in seminal vesicles was significantly decreased after castration or 17β‐estradiol treatment. Testosterone itself did not affect PCI mRNA expression, but treatment in castrated rats significantly enhanced its mRNA expression. These findings suggest that the PCI gene expression in rat seminal vesicles is regulated by androgen.
Activation of phospholipase D1 by direct interaction with ADP‐ribosylation factor 1 and RalA
Authors: Kim Jae Ho , Lee Sang Do , Han Jung Min , Lee Taehoon G , Kim Yong , Park Jong Bae , Lambeth J.David , Suh Pann-Ghill , Ryu Sung Ho
Published: July 2002

Phospholipase D1 (PLD1) is known to be activated by ADP‐ribosylation factor 1 (ARF1). We report here that ARF1 co‐immunoprecipitates with PLD1 and that the ARF1‐dependent PLD activation is induced by the direct interaction between ARF1 and PLD1. We found that RalA, another member of the small GTP‐binding proteins, synergistically enhances the ARF1‐dependent PLD activity with an EC50 of about 30 nM. Using in vitro binding assay, we show that ARF1 and RalA directly interact with different sites of PLD1. The results suggest that the independent interactions of RalA and ARF1 with PLD1 are responsible for the synergistic activation.
pADPRT‐2: a novel mammalian polymerizing(ADP‐ribosyl)transferase gene related to truncated pADPRT homologues in plants and Caenorhabditis elegans1
Authors: Berghammer Heinrich , Ebner Maria , Marksteiner Rainer , Auer Bernhard
Published: July 2002

Until recently, poly(ADP‐ribosyl)ation was supposed to be confined only to polymerizing(ADP‐ribosyl)transferase/(ADP‐ribose)polymerase (E.C. 2.4.2.30). Here, we present novel polymerizing(ADP‐ribosyl)transferase homologues from mouse and man that lack all of the N‐terminal DNA binding and BRCA1 C‐terminus domains and will be designated polymerizing(ADP‐ribosyl)transferase‐2 as distinguished from the classical polymerizing(ADP‐ribosyl)transferase (polymerizing(ADP‐ribosyl)transferase‐1). The murine polymerizing(ADP‐ribosyl)transferase‐2 gene shares three identical intron positions with its Caenorhabditis elegans (EMBL nucleotide sequence database Z47075) and one with the Arabidopsis thaliana homologue (‘APP’, GenBank database AF069298). Expression of the murine polymerizing(ADP‐ribosyl)transferase‐2 gene was elevated in spleen, thymus and testis and the corresponding poly(ADP‐ribosyl)ation activity might account for most of the residual poly(ADP‐ribosyl)ation observed in polymerizing(ADP‐ribosyl)transferase‐1−/− mice.