bluuebunny/crossref_metadata_2025 huggingface dataset:
Inositol 1,4,5‐trisphosphate releases Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae
Authors: Belde P.J.M. , Vossen J.H. , Borst-Pauwels G.W.F.H. , Theuvenet A.P.R.
Published: July 2002

Inositol 1,4,5‐trisphosphate (IP3) induces a release of Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae. The amount released is dependent on IP3 concentration (concentration for half maximal effect, K
m
apparent = 0.4 μM). Myo‐inositol, and inositol 1,4‐bisphosphate up to 50 μM have no effect on Ca2+ levels in the vesicles. The IP3 ‐induced Ca2+ release is blocked by dantrolene and 8‐(N,N‐diethylamino)‐octyl 3,4,5‐trimethoxybenzoate‐HCl (TMB‐8), which are known to block Ca2+ release from Ca2+ stores in animal cells. IP3‐induced release of Ca2+ also occurs when Ca2+ is accumulated by means of an artificial pH gradient, indicating that the effect of IP3 is not due to an effect on the vacuolar H+‐ATPase. The IP3‐induced Ca2+ release is not accompanied by a change in the pH gradient, which indicates that it is not due to a reversal of the Ca2+/nH+ antiport or to a decrease in ΔpH by IP3. The present results suggest that IP3 may act as a second messenger in the mobilization of Ca2+ in yeast cells. As in plant cells, the vacuolar membrane of yeast seems to contain a Ca2+ channel, which can be opened by IP3. In this respect the vacuole could function as an IP3‐regulated intracellular Ca2+ store, equivalent to the endoplasmic‐ and sarcoplasmic reticulum in animal cells, and play a role in Ca2+‐dependent signal transduction in yeast cells.
Naturally occurring deuterium is essential for the normal growth rate of cells
Authors: Somlyai Gábor , Jancsó Gábor , Jákli György , Vass Kornélia , Barna Balázs , Lakics Viktor , Gaál Tamás
Published: July 2002

The role of naturally occurring D in living organisms has been examined by using deuterium‐depleted water (30–40 ppm D) instead of water containing the natural abundance of D (150 ppm). The deuterium‐depleted water significantly decreased the growth rate of the L929 fibroblast cell line, and also inhibited the tumor growth in xenotransplanted mice. Eighty days after transplantation in 10 (59%) out of 17 tumorous mice the tumor, after having grown, regressed and then disappeared. We suggest that the naturally occurring D has a central role in signal transduction involved in cell cycle regulation.
Cloning and nucleotide sequence of cDNA for rhodopsin of the squid Todarodes pacificus
Authors: Hara-Nishimura Ikuko , Kondo Maki , Nishimura Mikio , Hara Reiko , Hara Tomiyuki
Published: July 2002

A cDNA for rhodopsin was isolated from a library constructed from poly(A)+RNA of the squid (Todarodes pacificus) retina. One positive clone with the longest insert of cDNA (3.1 kb) was selected by employing a PCR‐amplified cDNA fragment as a probe. The nucleotide sequence of the cDNA revealed a single open reading frame of 1,344 bp encoding a polypeptide (M
r, 49,833), which covered a complete sequence for the squid opsin. This clone had a very long 3'‐non‐coding region (1.7 kb) including multiple polyadenylation signals, AATAAA, resembling the clones for Todarodes retinochrome and retinal‐binding protein (RALBP). The analysis of hydropathicity demonstrated the presence of seven transmembrane spanning domains, and a possible retinal‐binding site, Lys‐305, was found in the 7th domain. Todarodes rhodopsin contained characteristic sequences of PPQGY repeated in the C‐terminal region, as reported in Loligo and octopus rhodopsins. Structural comparison of those cephalopod rhodopsins is also discussed.
Redox‐linked ligand‐independent cell surface triggering for extensive protein tyrosine phosphorylation
Authors: Rahman S.M.Jamshedur , Pu Mei-yi , Hamaguchi Michinari , Iwamoto Takashi , Isobe Ken-ichi , Nakashima Izumi
Published: July 2002

Exposure of lymphocytes to 0.2–2 mM HgCl2, a thiol‐reactive heavy metal, induced extensive tyrosine phosphorylation of multiple cellular proteins. The phosphorylation started as quickly as 5 s after exposure to HgCl2, and was irreversible. Another 3 thiol‐reactive chemicals also displayed similar, though less marked, actions, whereas dithiothreitol, a reducing agent, antagonized the HgCl2 action. The demonstrated new action of HgCl2 indispensably required membrane‐intact cells as a target. Whereas exposure of lymphocytes to > 0.2 mM HgCl2 caused rapid cell death, 0.01–0.1 mM HgCl2 affected the cells so as to accelerate their c‐fos transcription. These results suggest a novel redox‐linked mechanism of cell surface triggering of intracellular protein kinase activity, which is independent of receptor‐ligand interactions.
A monoclonal antibody raised against an [Na+K+]coupled l‐glutamate transporter purified from rat brain confirms glial cell localization
Authors: Levy Line M. , Lehre Knut P. , Rolstad Bent , Danbolt Niels C.
Published: July 2002

A monoclonal antibody (9C4) shows that an [Na+K+]coupled glutamate transporter protein purified from rat brain runs electrophoretically as a wide band and is localized in neuroglial cell bodies and processes, but not in neurons. This confirms the findings with polyclonal antibodies [Neuroscience 51 (1992) 295‐310], and shows that the apparent heterogeneity in relative molecular mass is accounted for by a single antigenic epitope. By testing several synthetic peptides derived from the deduced amino acid sequences of two cloned rat brain glutamate transporters, the antigenic epitope was identified as residing within the peptide TQSVYDDTKNHRESNSNQC (residues 518–536) of one of these [Nature 360 (1992) 464‐467].
Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura‐2
Authors: Berendes Robert , Burger Alexander , Voges Dieter , Demange Pascal , Huber Robert
Published: July 2002

A new assay for the calcium channel activity of annexin V was developed. The calcium‐sensitive fluorescence indicator, fura‐2, was incorporated into large unilamellar vesicles (LUV). After establishing a calcium gradient across the liposomal membranes, native or mutated annexin V was added. The resulting calcium influx into the LUV detected through the fluorescence changes of fura‐2 was used as a qualitative test for the electrophysiological properties of annexin V.
Interaction of NBD‐talin with lipid monolayers
Authors: Dietrich C. , Goldmann W.H. , Sackmann E. , Isenberg G.
Published: July 2002

Fluorescently labelled smooth muscle talin like native talin interacts with negatively or partly negatively charged lipid monolayers. This was measured in time/area diagrams using the film balance technique combined with fluorescence imaging after double photolabelling of talin and phospholipids.
P‐glycoprotein‐mediated transcellular transport of MDR‐reversing agents
Authors: Saeki Tohru , Ueda Kazumitsu , Tanigawara Yusuke , Hori Ryohei , Komano Tohru
Published: July 2002

Understanding of the interactions between P‐glycoprotein and multidrug resistance (MDR) reversing agents is important in designing more effective MDR modulators. We examined transcellular transport of several MDR modulators by using a drug‐sensitive epithelial cell line, LLC‐PK1 and its transformant cell line, LLC‐GA5‐COL300, which expresses human P‐glycoprotein on the apical surface. Basal‐to‐apical transports of azidopine and diltiazem across the LLC‐GA5‐COL300 monolayer were increased and apical‐to‐basal transports were decreased compared to those across the LLC‐PK1 monolayer, indicating that P‐glycoprotein transports azidopine and diltiazem. Movements of nitrendipine and staurosporine across the epithelial monolayer were not affected by P‐glycoprotein. These results suggests that some MDR modulators exert their inhibitory effect not only by blocking the initial binding of anticancer drugs but throughout the course of the transport process.
Chemical modification of rat liver cytosolic NADP+‐linked isocitrate dehydrogenase by N‐ethylmaleimide Evidence for essential sulphydryl groups
Authors: Fatania Hasmukh R. , Eid Al-Nassar Khalid , Thomas Nancy
Published: July 2002

Incubation of rat liver cytosolic isocitrate dehydrogenase with N‐ethylmaleimide (NEM) resulted in the inactivation of the enzyme following pseudo‐first order kinetics. Isocitrate affords considerable protection against inactivation whereas NADP+ enhances modification of the enzyme, suggesting localization of the modified group at the active site. Correlation of loss of activity with incorporation of [14C]NEM indicated that two sulphydryl residues/sub‐unit are modified of which only one is shown to be involved in catalysis. pH dependence of the inactivation process implicates a reactive group of pK
a 8.1 in catalysis. We conclude that a unique cysteine residue is essential for maximal catalytic activity of isocitrate dehydrogenase.
Metabolism of exogenous ganglioside GM1 in cultured cerebellar granule cells The fatty acid and sphingosine moieties formed during degradation are re‐used for lipid biosynthesis
Authors: Riboni Laura , Bassi Rosaria , Conti Mauro , Tettamanti Guido
Published: July 2002

Cerebellar granule cells, differentiated in vitro, were parallelly fed with [Sph‐3H]GM1 and [stearoyl‐14C]GM1, under identical conditions (10−6 M ganglioside; pulse, from 1–4 h; chase, up to 24 h after 4 h pulse) and the salvage pathways of sphingosine and stearic acid were investigated. It was observed that both sphingosine and stearic acid, liberated during the intralysosomal degradation of ganglioside, are metabolically recycled, along distinct pathways. Sphingosine is used for the biosynthesis of a number of sphingolipids, particularly ceramide, glucosyl‐ceramide, gangliosides and sphingomyelin; stearic acid is utilized for the biosynthesis of sphingolipids, and to a greater extent, glycero‐phospholipids, especially those endogenously richer in stearic acid (phosphatidyl‐ethanolamine and phosphatidyl‐choline). No evidence was provided for a salvage pathway for ceramide.
Anti‐CD9 monoclonal antibody elicits staurosporine inhibitable phosphatidylinositol 4,5‐bisphosphate hydrolysis, phosphatidylinositol 3,4‐bisphosphate synthesis, and protein‐tyrosine phosphorylation in human platelets
Authors: Yatomi Yutaka , Ozaki Yukio , Satoh Kaneo , Kume Shoji
Published: July 2002

Phosphoinositide metabolism elicited by anti‐CD9 monoclonal antibody, a well‐characterized platelet activator, was studied using acetylsalicylic acid‐treated human platelets. TP82, which is an anti‐CD9 monoclonal antibody, induced classical phosphatidylinositol 4,5‐bisphosphate hydrolysis, as monitored by intracellular Ca2+ mobilization and phosphatidic acid production, and synthesis of phosphatidylinositol 3,4‐bisphosphate, which is a major component of newly‐described 3‐phosphorylated inositol phospholipids produced during platelet activation. These changes were severely inhibited by 1 μM staurosporine, a potent, though non‐selective, protein kinase inhibitor, which also abolished TP82 induction of tyrosine phosphorylation of multiple platelet proteins. Protein‐tyrosine phosphorylation appears necessary to initiate both the classical phosphoinositide turnover and synthesis of the newly‐described 3‐phosphorylated inositol phospholipids in anti‐CD9 monoclonal antibody‐induced platelet activation.
Metabolic channelling and control of the flux
Authors: Kholodenko Boris N. , Westerhoff Hans V.
Published: July 2002

Metabolic control theory is extended to include channelled metabolism in general. A simple relationship between the flux control by the enzymes and the degree of metabolite channelling is derived. This relationship suggests experiments in which modulation of gene expression allows one to quantify channelling.
X‐Arrestin: a new retinal arrestin mapping to the X chromosome
Authors: Murakami Akira , Yajima Toshihiro , Sakuma Hitoshi , McLaren Margaret J. , Inana George
Published: July 2002

We have been using a differential cDNA cloning approach to isolate human retina‐specific and retina‐enriched genes [1]. A 1,314 bp cDNA was isolated by this approach, representing a highly retina‐specific message encoding a 388 amino acid protein showing 58%, 50%, and 49% homology to bovine β‐arrestin, and bovine and human retinal arrestin (S‐antigen), respectively. Chromosomal mapping localized this new arrestin genc to the proximal long arm of the X chromosome, hence it was named X‐arrestin. In situ hybridization demonstrated its expression in the inner and outer segments and the inner plexiform regions of the retina.
Localization of Kex2‐like processing endoproteases, furin and PC4, within mouse testis by in situ hybridization
Authors: Torii Seiji , Yamagishi Toshiyuki , Murakami Kazuo , Nakayama Kazuhisa
Published: July 2002

By in situ hybridization analysis, we show here the localization of furin and PC4, which are both members of a growing family of endoproteases structurally related to the yeast precursor processing protease Kex2, within mouse testis. Furin transcript was detected in both germ and somatic cells, while PC4 transcript was found only in round spermatids. Proenkephalin transcript was also localized in round spermatids. These observations suggest that, within testis, PC4 is involved in processing of peptide precursors such as proenkephalin and may play a role in regulation of sperm maturation. while furin may serve as a more general processing endoprotease.
On the activation of phosphodiesterase by a 26 kDa protein
Authors: Nikonov S.S. , Filatov G.N. , Fesenko E.E.
Published: July 2002

The effects of a 26 kDa protein isolated from vertebrate retina rod outer segments (ROS) and its reconstituted analog on the phosphodiesterase (PDE) activity and cGMP‐dependent conductance have been studied [Nature 313 (1985) 310–313]. Using the patch‐clamp technique it was shown that the 26 kDa protein in concentrations up to 1 μM accelerates hydrolysis of cGMP by near‐membrane PDE by 1–2 orders of magnitude. This process is suggested to be mediated by some intracellular agent. At the same concentrations the 26 kDa protein was shown to inhibit cGMP‐dependent conductance of the photoreceptor membrane. A possible role of these effects in the processes of phototransduction and adaptation is discussed.
Cloning of the rat heart Na+ ‐Ca2+ exchanger and its functional expression in HeLa cells
Authors: Low Walter , Kasir Judith , Rahamimoff Hannah
Published: July 2002

A functional rat heart Na+ ‐Ca2+ exchanger gene has been obtained by splicing and ligating two partially overlapping clones isolated from a rat heart λZAP cDNA library. The deduced primary structure of the protein encoded by the open reading frame corresponds to 971 amino acids, that can be organized into 12 transmembrane helices. The cloned gene was functionally expressed in HeLa cells. Maximal expression was detected 18 h after transfection, after which transport activity rapidly declined. The electrogenic properties of the cloned transporter were demonstrated following reconstitution of the expressed exchanger protein into a tightly sealed phospholipid membrane.
Purification and sequence determination of heat‐stable enterotoxin elaborated by a cholera toxin‐producing strain of Vibrio cholerae O1
Authors: Yoshino Ken-ichi , Miyachi Miki , Takao Toshifumi , Bag Prasanta K. , Xiaozhe Huang , Nair G.Balakrish , Takeda Tae , Shimonishi Yasutsugu
Published: July 2002

Four molecular species of heat‐stable enterotoxins elaborated by a cholera toxin‐producing strain of Vibrio cholerae O1 were isolated from its culture supernatant. The amino acid sequence of one of the enterotoxins was determined to be Phe‐Ile‐Lys‐Gln‐Val‐Asp‐Glu‐Asn‐Gly‐Asn‐Leu‐Ile‐Asp‐Cys‐Cys‐Glu‐Ile‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Phe‐Gly‐Cys‐Leu‐Asn with three intramolecular disulfide linkages. The other enterotoxins had shorter amino acid sequences in the N‐terminal regions, but possessed the same sequence in their C‐terminal regions including the three disulfide linkages. The enterotoxins with the shorter N‐terminal sequences showed more potent toxicities, and the minimum effective dose of the longest one with 28 amino acid residues was 10‐folds of that of the shortest one.
On the fractal nature of cytoplasm
Authors: Aon Miguel A. , Cortassa Sonia
Published: July 2002

On the basis of a quantification of the fractal dimension, D, in micrographs of cytoskeleton components or microtrabecular lattice, we propose that the cellular cytoplasm can be described as a percolation cluster, a sort of ‘random fractal’. Our hypothesis deals with: (i) the existence of the percolation threshold — a remarkable property of percolation processes; and (ii) the reactivity increase — when enzymes, or targets, and substrates, or effectors, coexist in the same topological dimension.
Identification of the U‐937 membrane‐associated proteinase interacting with the V3 loop of HIV‐1 gp12O as cathepsin G
Authors: Avril Laurence-Emmanuelle , Di Martino-Ferrer Michèle , Pignede Georges , Séman Michel , Gauthier Francis
Published: July 2002

We have purified a serine proteinase from the membrane of U‐937 cells that was inhibited in a tight‐binding manner by recombinant gp120 and by peptides mimicking the V3 loop of gp12O [(1993) FEBS Lett. 317, 167–172]. This proteinase has now been characterized, both structurally and functionally. It has a dual trypsin‐ and chymotrypsin‐like specificity, and N‐terminal sequence analysis of the first 32 residues indicates complete identity with leukocyte cathepsin G. Cathepsin G‐like material was located at the surface of U‐937 cells using a monoclonal antibody directed against leukocyte cathepsin G, and polyclonal anti‐cathepsin G antibodies precipitated the purified proteinase. However, the U‐937 enzyme differs slightly from commercial leukocyte cathepsin G in its apparent M
r because of different glycosylation. No other protein structurally related to cathepsin G was found upon screening a U‐937 cDNA library using several oligonucleotide probes constructed from the membrane proteinase N‐terminal amino acid sequence. The possible interaction of a cathepsin G‐like proteinase at the surface of U‐937 cells with the V3 loop of HIV‐1 gp120 is discussed.
TIMP‐1 protein expression is stimulated by IL‐1β and IL‐6 in primary rat hepatocytes
Authors: Roeb Elke , Graeve Lutz , Müllberg Jürgen , Matern Siegfried , Rose-John Stefan
Published: July 2002

Degradation of extracellular matrix proteins is performed by metalloproteinases which are inhibited by tissue inhibitors of metalloproteinases (TIMP). We expressed the murine TIMP‐1 protein in E. coli and prepared a polyclonal antiserum against the recombinant protein. Using this antiserum we studied the biosynthesis and glycosylation of murine TIMP‐1 protein in COS‐7 cells transfected with a TIMP‐1 expression plasmid by metabolic labeling and indirect immunofluorescence studies. In primary rat hepatocytes we show for the first time that TIMP‐1 protein expression is up‐regulated upon stimulation with IL‐1β and IL‐6. Since TIMP‐1 is induced during the acute phase reaction it could possibly be involved in the pathogenesis of liver fibrosis.
Hydroxyl radical generation in skeletal muscle atrophied by immobilization
Authors: Kondo Hisao , Nishino Kohsuke , Itokawa Yoshinori
Published: July 2002

Male Wistar rats (15 weeks old), the ankle joints of one hindlimb of which were immobilized in the extended position for 12 days, were injected with salicylate as a trapping agent for hydroxyl radicals before sacrifice. Atrophied and contralateral soleus, typical slow red muscles were collected and their levels of 2,3‐dihydroxybenzoic acid (2,3‐DHB), one of the main reaction products formed by the attack of hydroxyl radical on salicylate, were determined using high‐performance liquid chromatography with an electrochemical detector. There was a significant increase in 2,3‐DHB in the atrophied muscle. This result strongly suggests the enhanced generation in vivo of hydroxyl radicals in atrophied muscle.
Infrared studies of interaction between metal ions and Ca2+‐binding proteins Marker bands for identifying the types of coordination of the side‐chain COO− groups to metal ions in pike parvalbumin (pI = 4.10)
Authors: Nara Masayuki , Tasumi Mitsuo , Tanokura Masaru , Hiraoki Toshifumi , Yazawa Michio , Tsutsumi Akihiro
Published: July 2002

Metal‐ligand interactions in the Ca2+‐binding sites of pike parvalbumin (pI = 4.10) have been examined by Fourier‐transform infrared spectroscopy. The region of the COO− antisymmetric stretch provides useful information on the types of coordination of the COO− groups to the metal ions in the Mg2+‐, Mn2+‐, and Ca2+‐bound forms. In the spectrum of the Ca2+‐bound form, two bands are observed at 1,582 and 1,553 cm−, whereas, in the spectra of the Mg2+‐ and Mn2+‐bound forms, bands are observed only in the region around 1,582 cm−1 and no band is found in the region around 1,553 cm−1. The 1,553‐cm−1 band of the Ca2+‐bound form reflects the bidentate coordination of the COO− groups of both Glu‐62 in the CD site and Glu‐101 in the EF site to the Ca2+ ions, which has been made clear by X‐ray analysis as a feature of the Ca2+‐bound form. Absence of such a band in the spectrum of the Mn2+‐bound form is consistent with the X‐ray structure of this form where both of the two COO− groups are unidentate. These unidentate COO− groups of Glu‐62 and Glu‐101 in the Mn2+‐bound form seem to give rise to a band at 1,577‐1,574 cm−. The spectrum of the Mg2+‐bound form is also consistent with the ‘pseudo‐bridging’ coordination of the COO− group of Glu‐101 reported in the X‐ray structure of a form where the Mg2+ ion occupies only the EF site, and the same spectrum is further indicative of the ‘pseudo‐bridging’ coordination of the COO− group of Glu‐62.
Existence of a molecular ruler in proteasomes suggested by analysis of degradation products
Authors: Wenzel Thorsten , Eckerskorn Christoph , Lottspeich Friedrich , Baumeister Wolfgang
Published: July 2002

Analysis of the degradation products from two proteins, the insulin B‐chain and human hemoglobin, generated by archaebacterial Thermoplasma acidophilum 20 S proteasomes, revealed an unexpectedly broad specificity. In spite of the vast number of different peptides found, they fell into a rather narrow size range. This suggests that a molecular ruler exists which determines the length of the cleavage products.
Gq and G11 are concurrently activated by bombesin and vasopressin in Swiss 3T3 cells
Authors: Offermanns Stefan , Heiler Eva , Spicher Karsten , Schultz Günter
Published: July 2002

The α‐subunits of the widely expressed G‐proteins Gq and G11 indistinguishably activate β‐isoforms of phospholipase C. In this report we have tested whether differences exist in the activation of both G‐proteins via phospholipase C‐linked receptors. We found that bombesin and vasopressin, with very similar potencies and time dependencies, induce the activation of both Gq and G11 in Swiss 3T3 cells, suggesting that these G‐proteins, at least in part, serve interchangeable functions.
Serine‐202 is the putative precursor of the active site dehydroalanine of phenylalanine ammonia lyase Site‐directed mutagenesis studies on the enzyme from parsley (Petroselinum crispum L.)
Authors: Schuster Birgid , Rétey János
Published: July 2002

To investigate the possible role of serine as a precursor of dehydroalanine at the active site of phenylalanine ammonia lyase, two serines, conserved in all known PAL and histidase sequences, were changed to alanine by site‐directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7.7 and the gene products were assayed for PAL activity. Mutant PALMutS209A showed the same catalytic property as wild‐type PAL, whereas mutant PALMutS202A was devoid of catalytic activity, indicating that serine‐202 is the most likely precursor of the active site dehydroalanine.
Metastatic potential of transformed rat 3Y1 cell lines is inversely correlated with lysosomal‐type sialidase activity
Authors: Miyagi Taeko , Sato Kaoru , Hata Keiko , Taniguchi Shun'ichiro
Published: July 2002

We have investigated sialidase activities in transformed rat 3Y1 cells of different metastatic potential. Only lysosome‐type sialidase was apparent in the particulate fractions of 3Y1 cells and their transformants. As compared with control 3Y1 cells, src‐transformed cells exhibited decreased sialidase activity, and v‐fos transfer to these latter induced even more severe decrease in the sialidase activity with acquisition of high lung metastatic ability. Various lysosomal enzymes other than sialidase were hardly affected by the transformation. Sialic acid transfer to N‐linked glycoproteins was slightly elevated in the transformants, but not in parallel with their metastatic potential.
Glucagon‐like peptide 1: A potent glycogenic hormone
Authors: Valverde Isabel , Morales Mónica , Clemente Felipe , López-Delgado Maria I. , Delgado Elena , Perea Almudena , Villanueva-Peñacarrillo Maria L.
Published: July 2002

GLP‐1(7–36)amide is an insulinotropic peptide derived from the intestinal post‐translational proglucagon process, the release of which is increased mainly after a carbohydrate meal; also, its anti‐diabetogenic effect in normal and diabetic states has been reported. In this study, GLP‐1(7–36)amide stimulates the formation of glycogen from glucose in isolated rat hepatocytes, such a glycogenic effect being achieved with physiological concentrations of the peptide. The GLP‐1(7–36)amide‐induced glycogenesis is abolished by glucagon, and it is accompanied by stimulation of the glycogen synthase a activity and by a decrease in the basal and glucagon‐stimulated cyclic AMP content. These findings could explain, at least in part, the GLP‐1(7–36)amide insulin‐independent plasma glucose lowering effect.
The cyclic AMP response element plays an essential role in the expression of the human prostaglandin‐endoperoxide synthase 2 gene in differentiated U937 monocytic cells
Authors: Inoue Hiroyasu , Nanayama Toyomichi , Hara Shuntaro , Yokoyama Chieko , Tanabe Tadashi
Published: July 2002

The promoter activity of 1432 bp upstream of the human prostaglandin‐endoperoxide synthase 2 gene (PTGS2) was examined in differentiated U937 monocytic cells expressing prostaglandin‐endoperoxide synthase 2 mRNA. Transient transfection experiments were performed using these cells and reporter vectors containing the upstream region of the gene with deletions or site‐specific mutations and the luciferase gene. The deletion or destruction of the cyclic AMP response element (nucleotides −59 to −53) markedly reduced the promoter activity of this gene. Electrophoretic mobility shift assays showed that a nuclear protein(s) binding to the cyclic AMP response element was induced during monocytic differentiation of U937 cells. These results indicate that expression of the human prostaglandin‐endoperoxide synthase 2 gene in differentiated U937 monocytic cells is regulated by the cyclic AMP response element.
Structural features of plant chitinases and chitin‐binding proteins
Authors: Beintema Jaap J.
Published: July 2002

Structural features of plant chitinases and chitin‐binding proteins are discussed. Many of these proteins consist of multiple domains, of which the chitin‐binding hevein domain is a predominant one. X‐ray and NMR structures of representatives of the major classes of these proteins are available now, and are used to describe the structures of the other ones. Conserved positions of Cys residues can be taken as evidence for identically located disulfide bridges or cysteine residues. The current classification of chitinases is unsatisfactory and needs to be replaced by an evolutionarily more correct one. As the currently known three‐dimensional structures of chitinases are those from barley and the rubber tree, Hevea brasiliensis, it is proposed to adopt the designation b‐type (classes I, II and IV) and h‐type (classes III and V) chitinases, respectively.
Brefeldin A inhibits protein synthesis through the phosphorylation of the α‐subunit of eukaryotic initiation factor‐2
Authors: Mellor Harry , Kimball Scot R. , Jefferson Leonard S.
Published: July 2002

Brefeldin A is a fungal metabolite which disrupts protein traffic through the Golgi apparatus and thereby inhibits protein secretion. Recently, it has been shown that Brefeldin A also causes a marked decrease in the rate of protein synthesis in cells in culture [1992, FEBS Lett. 314, 371–374]. We show here that treatment of rat GH3 pituitary cells with Brefeldin A leads to an inhibition of protein synthesis at the level of peptide‐chain initiation through a mechanism involving the phosphorylation of the α‐subunit of eukaryotic initiation factor‐2 (eIF‐2α).
Localization and structural characterization of an oligosaccharide O‐linked to bovine PDC‐109 Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa
Authors: Calvete Juan J. , Raida Manfred , Sanz Libia , Wempe Frank , Scheit Karl-Heinz , Romero Antonio , Töpfer-Petersen Edda
Published: July 2002

PDC‐109 (13 kDa) is the most abundant component, and the major heparin‐binding protein, of bovine (Bos taurus) seminal plasma. Here, we show that PDC‐109 contains a single O‐linked oligosaccharide (NeuNAcα(2–6)‐Galβ(1–3)‐GalNAc‐) attached to Thr11. Immunoquantitation of PDC‐109 indicates that its concentration in seminal plasma is 15–20 mg/ml. Though PDC‐109 is not present on epididymal sperm, ejaculated spermatozoa on average are coated with (9.5 ± 0.3) × 106 molecules of PDC‐109/cell. This value remained constant in swim‐up sperm and decreased to (7.7 ± 0.4) × 106/spermatozoon after incubation for 24 h in capacitation medium at 39°C. These data substantiate the hypothesis that PDC‐109 may be one of the seminal plasma components that enhance the fertilizing capacity of bull spermatozoa upon interaction with heparin‐like glycosaminoglycans present in the female genital tract.
Ion channel regulation by calmodulin binding
Authors: Saimi Yoshiro , Kung Ching
Published: July 2002

While many ion channels are modulated by phosphorylation, there is growing evidence that they can also be regulated by Ca2+‐calmodulin, apparently through direct binding. In some cases, this binding activates channels; in others, it modulates channel activities. These phenomena have been documented in Paramecium, in Drosophila, in vertebrate photoreceptors and olfactory receptors, as well as in ryanodine receptor Ca2+‐release channels. Furthermore, studies on calmodulin mutants in Paramecium have shown a clear bipartite distribution of two groups of mutations in the calmodulin gene that lead to opposite behavioral and electrophysiological phenotypes. These results indicate that the N‐lobe of calmodulin specifically interacts with one class of ion‐channel proteins and the C‐lobe with another.
Overexpression of TGN38/41 leads to mislocalisation of γ‐adaptin
Authors: Reaves Barbara , Banting George
Published: July 2002

TGN38 and TGN41 are isoforms of a monotopic integral membrane protein which recycles between the trans Golgi network (TGN) and the cell surface, but which, at steady state, is predominantly located in the TGN. Full‐length and truncated versions of rat TGN38/41 have been expressed in monkey (COS) and human (Heb7a) cells under the control of the heavy metal inducible Metallothionein IIA promoter. This has allowed the regulated expression of TGN38/41 protein constructs to different levels in the transfected cells. These studies show that (i) controlled overexpression of TGN38/41 results in mislocalisation to parts of the endocytic pathway, (ii) a truncated version of TGN38/41, lacking the cytoplasmic domain, remains in the TGN, and (iii) there is a direct or indirect interaction between the cytoplasmic domain of TGN38/41 and γ‐adaptin.
Induction of calbindin‐D 28K gene and protein expression by physiological stimuli but not in calcium‐mediated degeneration in rat PC12 pheochromocytoma cells
Authors: Vyas Sheela , Michel Patrick P. , Copin Marie-Christine , Biguet Nicole Faucon , Thomasset Monique , Agid Yves
Published: July 2002

To understand the role of calbindin‐D 28K in neuronal degeneration, we examined its expression in differentiated PC12 cells in response to calcium intoxication, using the ionophore A23187 treatment, that results in cell degeneration and death. We first established that calbindin‐D 28K is expressed in PC12 cells. The amounts of calbindin‐D 28K mRNA and protein were increased by the differentiation factors, NGF and retinoic acid, but not by vitamin D3. Calbindin‐D 28K expression was also significantly up‐regulated by stimuli (depolarization, low concentrations of Ca2+ ionophore A23187) which increase intracellular calcium levels within the physiological range. In contrast, the calbindin‐D 28K mRNA and protein concentrations were not modulated by high concentrations of A23187, which resulted in cell degeneration and death. Experiments with the antisense oligonucleotides showed that, although the calbindin‐D 28K protein levels were decreased significantly, the progression of degenerative changes induced by calcium via A23187, was not altered.
The kinetics of HIV‐1 long terminal repeat transcriptional activation resemble those of hsp70 promoter in heat‐shock treated HeLa cells
Authors: Kretz-Remy Carole , Arrigo Andre-Patrick
Published: July 2002

The long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV‐1) is activated under different conditions including heat shock. By using transient transfection assays, we have compared the thermal activation of HIV‐1 LTR to that of the promoter of the gene encoding the human stress protein hsp70 which is under the control of the heat shock transcription factor HSF. In these assays, the chloramphenicol acetyl transferase (Cat) gene was used as a reporter gene. Several parameters of the heat stress were analyzed such as the temperature, the duration of heat stress and that of the recovery period. Under every condition tested, we have found that the kinetics of activation of both promoters were very similar. In addition, both showed a similar inhibition by actinomycin D. These results were compared to those obtained with a DNA construct containing the early promoter of SV‐40 virus coupled to the Cat gene. In this case, no heat‐mediated accumulation of CAT protein was observed, indicating that the transcriptional activation of HIV‐1 LTR by heat shock is specific. HIV‐1 LTR contains two NF‐κB binding elements, involved in the activation of this promoter during oxidative stress, which are sequence related to the heat shock element HSE. However, under all the heat shock conditions tested, we have been unable to detect the binding of any protein to κB elements, suggesting that this site is not directly involved in the thermal activation of HIV‐1 LTR. These results indicate that the thermal transcriptional activation of HIV‐1 LTR and hsp70 promoters occurs through different mechanisms that are triggered by similar heat shock conditions.
Ascorbic acid synthesis is stimulated by enhanced glycogenolysis in murine liver
Authors: Braun László , Garzó Tamás , Mandl József , Bánhegyi Gábor
Published: July 2002

Ascorbic acid synthesis was stimulated by glucagon, dibutyryl cyclic AMP, as well as phenylephrine, vasopressin or okadaic acid, in hepatocytes prepared from fed mice. However, no such effect was observed in glycogen‐depleted cells from starved animals, either in the presence or absence of glucose. The rate of ascorbate synthesis showed close correlation with the glucose release by hepatocytes. In mice the injection of glucagon increased plasma ascorbate concentration fifteenfold, and caused a sixfold elevation of the ascorbate content of the liver. These results show that hepatic ascorbate synthesis is dependent on glycogenolysis, and indicate a regulatory role of ascorbate released by the liver.
Zonation of the action of glucagon on gluconeogenesis studied in the bivascularly perfused rat liver
Authors: Constantin Jorgete , Ishii-Iwamoto Emy , Suzuki-Kemmelmeier Fumie , Bracht Adelar
Published: July 2002

We have measured the action of glucagon, infused into the hepatic artery, on gluconeogenesis from lactate in the rat liver, bivascularly perfused in both the anterograde and retrograde modes. Concerning glucose production and oxygen uptake per unit cell space, the response of the periportal cells reached via the hepatic artery in retrograde perfusion to glucagon is superior to the response of the cells reached via the same vessel in anterograde perfusion. This phenomenon, however, most probably reflects zonation of gluconeogenesis rather than zonation of the hormonal action. The latter conclusion is based on the observation that the fractional change caused by the hormone is the same for all liver cells.
Dystroglycan is a binding protein of laminin and merosin in peripheral nerve
Authors: Yamada Hiroki , Shimizu Teruo , Tanaka Takeshi , Campbell Kevin P. , Matsumura Kiichiro
Published: July 2002

α‐Dystroglycan, a 156 kDa dystrophin‐associated glycoprotein, binds laminin in skeletal muscle. Here we demonstrate that α‐dystroglycan is a binding protein of laminin (A/B1/B2) and merosin (M/B1/B2) in peripheral nerve. Immunocytochemical analysis demonstrates the localization of α‐dystroglycan and merosin surrounding myelin sheath of peripheral nerve fibers. Biochemical analysis demonstrates that the 120 kDa peripheral nerve α‐dystroglycan binds merosin as well as laminin. The binding of laminin and merosin is Ca2+ dependent and is inhibited by NACl and heparin. Recently, merosin was shown to be deficient in the peripheral nerve of dy mice which have defects in myelination. The interaction between α‐dystroglycan and merosin may play a role in the regulation of Schwann cell myelination and/or maintenance of myelin sheath.
The cyclic structure of the enterococcal peptide antibiotic AS‐48
Authors: Samyn Bart , Martinez-Bueno Manuel , Devreese Bart , Maqueda Mercedes , Gálvez Antonio , Valdivia Eva , Coyette Jacques , Van Beeumen Jozef
Published: July 2002

The complete primary structure of the peptide antibiotic AS‐48 produced by Enterococcus faecalis has been determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. As far as we know, this is the first example of an antibiotic protein cyclised by a tail—head peptide bond formation and not by branching of the polypeptide side chains.
Comparative modelling of barley‐grain aspartic proteinase: A structural rationale for observed hydrolytic specificity
Authors: Guruprasad Kunchur , Törmäkangas Kirsi , Kervinen Jukka , Blundell Tom L.
Published: July 2002

A model of the barley‐grain aspartic proteinase (HvAP; Hordeum vulgare aspartic proteinase) has been constructed using the rule‐based comparative modelling approach encoded in the COMPOSER suite of computer programs. The model was based on the high resolution crystal structures of six highly homologous aspartic proteinases. Results suggest that the overall three‐dimensional structure of HvAP (excluding the plant‐specific insert; 104 residues in HvAP) is closer to human cathepsin D than other aspartic proteinases of known three‐dimensional structure. Comparisons of the complexes with the substrate modelled in the active site of HvAP with those of the same substrate modelled in the active site of other aspartic proteinases of known three‐dimensional structure and specificity, define residues that may influence hydrolytic specificity of the barley enzyme. We have identified residues in the S4 (Ala12), S3 (Gln13, Thr111), S2 (Ala222, Thr287, Met289), S′
1 and S′
3 (Ile291), S′
2 and S′
3 (Gln74), S′
2 (Arg295), and S′
3 (Pro292) pockets, that may account for the observed trends in the kinetic behaviour and specificity when compared to other aspartic proteinases. The plant‐specific inserted sequence, which may play a role in the transport of HvAP to plant vacuoles (lysosomes), is similar to the saposins and is predicted to be a mixed α‐helical and β‐strand domain.
Herbivore‐induced volatiles: The emission of acyclic homoterpenes from leaves of Phaseolus lunatus and Zea mays can be triggered by a β‐glucosidase and jasmonic acid
Authors: Hopke Jörn , Donath Jens , Blechert Siegfried , Boland Wilhelm
Published: July 2002

The treatment of healthy, undamaged plants of the Lima bean Phaseolus lunatus with solutions of a β‐glucosidase from bitter almonds (at 5 U·ml−1) through the petiole results in an enhanced emission of volatiles to the environment. The compounds are identical with those emitted in response to infestation with the red spotted spider mite Tetranychus urticae. Dominant products are the two acyclic homoterpenes 4,8‐dimethyl‐1,3E,7‐dimethylnonatriene (homoterpene I) and 4,8,12‐trimethyl‐1,3E,7E,11‐tridecatetraene (homoterpene II) which are of sesquiterpenoid and diterpenoid origin. Therefore, a β‐glucosidase of the herbivore may be considered as the true elicitor for the odor induction. Homoterpene I and most other of the herbivore‐induced volatiles can also be triggered by treatment of the plant with solutions of jasmonic acid (JA) at 100 nmol·ml−1 to 10 μmol·ml−1. The C16 homoterpene II is not significantly induced by JA. The time—course of the enzymatic‐ and the JA‐triggered induction of the volatiles is identical. The dose—response to JA parallels previous reports on alkaloid induction in cell cultures. In corn plants (Zea mays) JA triggers the emission of all volatiles which are known to be emitted in response to the damage by the beet army worm Spodoptora exigua. In summary, the emission of volatiles after damage by a herbivore resembles the production of phytoalexins in response to an attacking microorganism and uses similar elicitors and internal transduction pathways.
T cell epitope analysis with peptides simultaneously synthesized on cellulose membranes: Fine mapping of two DQ dependent epitopes
Authors: Adler Sabine , Frank Ronald , Lanzavecchia Antonio , Weiss Siegfried
Published: July 2002

Several MHC class II restricted, CD4+ human T cell clones from three donors were induced with soluble matrix protein of influenza virus. The epitopes recognized by these clones were mapped using a complete set of overlapping 15‐mer peptides synthesized with the newly developed SPOT method which allows the simple simultaneous synthesis of numerous peptides on cellulose membranes. Fine analysis of two clones by truncation of the stimulatory peptide by single subsequent amino acids from either the NH2‐ or the COOH‐terminus revealed the minimal stimulatory determinants of two DQ dependent T cell epitopes.
Analysis of RNase A refolding intermediates by electrospray/mass spectrometry
Authors: Claudia Torella , Margherita Ruoppolo , Gennaro Marino , Piero Pucci
Published: July 2002

Electrospray/mass spectrometry (ESIMS) was extensively used to obtain information on disulphide‐containing intermediates formed during refolding of bovine pancreatic ribonuclease A. The analysis showed the existence of an equilibrated population of disulphide bonded intermediates, and indicates that intermediates containing two intramolecular S–S are predominant until late stages of the refolding process. Mixed disulphides with exogenous glutathione were also detected, supporting previous evidence of conformational restrictions on the ability of RNase A to form intramolecular disulphides. The results indicate that ES/MS is a suitable technique to detect and characterize refolding intermediates.
Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell‐permeable activator of AMP‐activated protein kinase
Authors: Sullivan Jane E. , Brocklehurst Katy J. , Marley Anna E. , Carey Frank , Carling David , Beri Raj K.
Published: July 2002

In vivo, hormone‐sensitive lipase (HSL) is known to be phosphorylated on two sites termed the regulatory and basal sites. However, the intracellular role of the basal site or the identity of the protein kinase phosphorylating this site has not been established. We show that 5‐amino‐4‐imidazolecarboxamide ribonucleoside (AICAR) markedly activates cellular AMP‐activated protein kinase (AMPK) in a time‐ and dose‐dependent manner. As expected for an agent that activates AMPK intracellularly, AICAR had no effect on the basal activity of HSL. However, preincubation of adipocytes with AICAR led to a reduced response of these cells to the lipolytic agent isoprenaline. AICAR was also shown to profoundly inhibit lipogenesis through increased phosphorylation of acetyl‐CoA carboxylase (ACC). Thus it appears that in addition to regulating lipogenesis, AMPK also plays an important antilipolytic role by regulating HSL in rat adipocytes.
Molecular cloning and chromosomal localization of the human thrombopoietin gene
Authors: Sohma Yoshiaki , Akahori Hiromichi , Seki Naohiko , Hori Tada-aki , Ogami Kinya , Kato Takashi , Shimada Yoshihiro , Kawamura Kazuo , Miyazaki Hiroshi
Published: July 2002

The complete gene for human thrombopoietin (TPO) has been cloned by screening a human genomic library using human TPO cDNA as a probe. This gene is 6.2 kb in length and contains six exons and five introns. It is shown that the human genome contains a single copy of the human TPO gene according to Southern blotting analysis. The transcription initiation site was determined by S1 nuclease mapping. The human TPO gene expressed TPO activity when transfected into COS‐1 cells. The human TPO gene has been mapped to chromosome 3q27 by in situ hybridization using a biotin‐labeled probe.
Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions
Authors: Glab Nathalie , Labidi Brahim , Qin Li-Xian , Trehin Christophe , Bergounioux Catherine , Meijer Laurent
Published: July 2002

The cdc2/cdk2 protein kinases play key roles in the cell cycle at two control points: the G1/S transition and the entry into mitosis. Olomoucine, a specific inhibitor of these kinases, was tested in two plant cell systems: Petunia mesophyll protoplasts induced to divide and Arabidopsis thaliana cell suspension cultures. The cell cycle status was analysed from DNA histograms or through continuous labelling of cells with 5‐bromodeoxyuridine (BrdUrd) followed by double staining with bis‐benzimide (Hoechst 33258) and propidium iodide (PI). Such analyses resolve cells from several generations according to the extent of their DNA replication. Olomoucine was shown to reversibly arrest differentiated Petunia cells induced to divide at G1 phase and cycling Arabidopsis cells in late G1 and G2. A comparison of the effects of aphidicolin, oryzalin and olomoucine suggests that in the Arabidopsis cell suspension culture, a cdc2/cdk2‐like kinase is activated at a restriction point in late G1.
Structure of the gene for human uracil—DNA glycosylase and analysis of the promoter function
Authors: Haug Terje , Skorpen Frank , Lund Henning , Krokan Hans E.
Published: July 2002

The gene for human uracil‐DNA glycosylase (UNG) contains 4 exons and has an approximate size of 13 kb. The promoter is very GC rich and lacks a TATA box. Nested deletions of the promoter demonstrated that two SPI elements and a putative c‐MYC element proximal to the transcription initiation region were sufficient to support some 27% of the promoter activity, while a clone that in addition contained the elements E2F/SP1/CCAAT increased expression to almost 90% of the full‐length construct. A region upstream of these elements appears to exert a negative control function.