bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurotrophins and cytokines—intermediaries between the immune and nervous systems
Authors: Otten U. , Gadient R.A.
Published: July 2002

Neurotrophins (NTs), including nerve growth factor (NGF), are multifunctional: in addition to their well‐characterized neurotrophic functions they are known to regulate and to be regulated by cytokines, components of the immune system. In line with this we have found expression of a functional trk proto‐oncogene, constituting the signal transducing‐receptor for NGF, on monocytes/macrophages, lymphocytes and basophils. Moreover, NGF synthesis is regulated by a cytokine cascade including inflammatory mediators such as interleukin‐1 and tumor necrosis factor‐α. The fact that NGF levels are markedly elevated in cerebrospinal fluid of patiens with multiple sclerosis and in serum of patients with systemic lupus erythematosus strongly indicates a role for NGF in immunopathology as well as in normal immune function.
A high affinity nitrate transport system from Chlamydomonas requires two gene products
Authors: Zhou Jing-Jiang , Fernández Emilio , Galván Aurora , Miller Anthony J.
Published: July 2002

A nitrate‐regulated cluster of genes involved in nitrate transport and assimilation has been identified in Chlamydomonas reinhardtii. Mutant strains of the alga, which are defective in some aspect of transport and assimilation have been used to assign functions to these genes. This analysis has suggested that two gene products are necessary to obtain a functional high affinity nitrate system in Chlamydomonas [Quesada et al. (1994) Plant J. 5, 407–419]. In this paper we have tested this hypothesis by injecting Xenopus oocytes with mRNA prepared from these two cDNAs, Nrt2;1 and Nar2, and then assaying the oocytes for nitrate transport activity. Oocytes injected with single types of mRNA did not show any nitrate transport activity. Furthermore, Nar2 mRNA was toxic to oocytes, with nearly 60% of the oocytes dead 3 days after the injection. However, when oocytes were injected with a mixture of two mRNAs prepared from Nrt2;1 and Nar2, a high affinity nitrate transport activity could be measured. However, the K
m for nitrate of this transport system was 28 μM which is higher than the value of 1.6 μM which had been obtained by the analysis of mutant phenotypes. The pH‐dependence of the nitrate‐elicited currents was consistent with a proton‐cotransport mechanism. These results prove that two gene products are required to produce a functional high affinity nitrate transport system and that this process does not involve transcriptional regulation.
Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms
Authors: Borutaite Vilmante , Morkuniene Ramune , Brown Guy C.
Published: July 2002

We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.
Homologous mutations in two diverse sulphate transporters have similar effects
Authors: Khurana Ooma K , Coupland Lucy A , Shelden Megan C , Howitt Susan M
Published: July 2002

Mutations in the human sulphate transporter gene, DTDST, have been implicated in several diseases. Analysis of affected patients has linked disease symptoms to faulty sulphate transporter activity. We have reproduced two of these mutations in SHST1, a homologous member of the family isolated from the tropical legume, Stylosanthes hamata. Both mutations significantly reduce sulphate transport activity of SHST1. These results indicate that conserved residues between distinct members of the family may share essential roles in structure or function. The results also suggest that putative helix 9 may be important for stability and/or trafficking of SHST1 to the plasma membrane.
Inteins invading mycobacterial RecA proteins
Authors: Saves Isabelle , Lanéelle Marie-Antoinette , Daffé Mamadou , Masson Jean-Michel
Published: July 2002

Five new inteins were discovered in a survey of 39 mycobacterial strains that was undertaken to clarify the role of RecA inteins in mycobacteria. They are all inserted at the RecA‐b site of the recA gene of Mycobacterium chitae, M. fallax, M. gastri, M. shimodei and M. thermoresistibile and belong to the MleRecA allelic family. Sequence analysis showed that although only M. tuberculosis harbours an intein at the RecA‐a site the sequence of the RecA‐b site is well conserved between species. Furthermore, the presence of inteins does not correlate with specific characteristics of the species such as pathogenicity or growth rate.
The yeast exchange assay, a new complementary method to screen for Dbl‐like protein specificity: identification of a novel RhoA exchange factor
Authors: De Toledo M. , Colombo K. , Nagase T. , Ohara O. , Fort P. , Blangy A.
Published: July 2002

The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remain to be identified. Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide range screen for GEF specificity. In this assay based on the two‐hybrid system, a wild type GTPase binds to its effector only after activation by a specific GEF. We validated the YEA by activating GTPases by previously reported GEFs. We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F‐actin stress fiber assembly in fibroblasts, characteristic of RhoA activation.
Stable plasma membrane expression of the soluble domain of the human insulin receptor in yeast
Authors: Angermayr Michaela , Strobel Gertrud , Müller Günter , Bandlow Wolfhard
Published: July 2002

The soluble cytoplasmic kinase domain of the human insulin receptor was N‐terminally equipped with either an N‐acetylation or a dual‐acylation motif (MGC box, to allow myristoylation/palmitoylation) and expressed in yeast cells under the control of the inducible CUP1 promoter. Although the cellular concentration was about the same in both instances (reflecting similar stability against proteolysis), only the myristoylated protein was capable of autophosphorylation to a significant extent and was active to phosphorylate endogenous yeast proteins at tyrosine residues in vivo. Cellular subfractionation showed that the insulin receptor was associated with plasma membranes, from where it was not extractable with high salt or alkali, but a significant fraction was also localized in the nuclear fraction. The myristoylated protein is absent from the cytoplasm. No effect of expression of either the acetylated or the myristoylated version on growth and respiration on various carbon sources was detected, suggesting a failure of the active insulin receptor kinase domain to couple to yeast (glucose) signalling cascades.
The cytotoxic lipid peroxidation product, 4‐hydroxy‐2‐nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria
Authors: Millar A.Harvey , Leaver Christopher J
Published: July 2002

4‐Hydroxy‐2‐nonenal (HNE), a cytotoxic product of lipid peroxidation, inhibits O2 consumption by potato tuber mitochondria. 2‐Oxoglutarate dehydrogenase (OGDC), pyruvate dehydrogenase complex (PDC) (both 80% inhibited) and NAD‐malic enzyme (50% inhibited) are its major targets. Mitochondrial proteins identified by reaction with antibodies raised to lipoic acid lost this antigenicity following HNE treatment. These proteins were identified as acetyltransferases of PDC (78 kDa and 55 kDa), succinyltransferases of OGDC (50 kDa and 48 kDa) and glycine decarboxylase H protein (17 kDa). The significance of the effect of these inhibitions on the impact of lipid peroxidation and plant respiratory functions is discussed.
The mitochondrial inner membrane AAA metalloprotease family in metazoans
Authors: Juhola M.Katariina , Shah Zahid H. , Grivell Leslie A. , Jacobs Howard T.
Published: July 2002

Three metalloproteases belonging to the AAA superfamily (Yme1p, Afg3p and Rca1p) are involved in protein turnover and respiratory chain complex assembly in the yeast inner mitochondrial membrane. Analysis of the completed genome sequences of Caenorhabditis elegans and Drosophila melanogaster indicates that this gene family typically comprises 3–4 members in metazoans. Phylogenetic analysis reveals three main branches represented, respectively, by Saccharomyces cerevisiae YME1, human SPG7 (paraplegin) and S. cerevisiae AFG3 and RCA1. mt‐AAA metalloproteases are weak candidates for several previously studied Drosophila mutants. A full elucidation of the cellular and physiological roles of mt‐AAA metalloproteases in metazoans will require the creation of targeted mutations.
Interactions of the ectodomain of HFE with the transferrin receptor are critical for iron homeostasis in cells
Authors: Roy Cindy N. , Carlson Eric J. , Anderson Emily L. , Basava Alivelu , Starnes Steven M. , Feder John N. , Enns Caroline A.
Published: July 2002

Expression of wild type HFE reduces the ferritin levels of cells in culture. In this report we demonstrate that the predominant hereditary hemochromatosis mutation, C282Y2
HFE, does not reduce ferritin expression. However, the second mutation, H63D HFE, reduces ferritin expression to a level indistinguishable from cells expressing wild type HFE. Further, two HFE cytoplasmic domain mutations engineered to disrupt potential signal transduction, S335M and Y342C, were functionally indistinguishable from wild type HFE in this assay, as was soluble HFE. These results implicate a role for the interaction of HFE with the transferrin receptor in lowering cellular ferritin levels.
Homing markers for atherosclerosis: applications for drug delivery, gene delivery and vascular imaging
Authors: Houston Parul , Goodman Jo , Lewis Alan , Campbell Callum J , Braddock Martin
Published: July 2002

Endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. Pro‐inflammatory cytokines such as interleukin‐1β and tumour necrosis factor α activate endothelial cells changing their resting phenotype to become pro‐adhesive, pro‐thrombotic and pro‐atherogenic. Phage display in vivo biopanning has been used to identify peptide sequences that home to diseased regions of the vessel wall in low density lipoprotein receptor (LDLr) knockout mice. In LDLr knockout mice, peptide sequence determinants exhibiting organ specificity have been isolated. These sequences have applications for gene delivery, drug delivery and for improving contrast agents for vascular imaging.
Directed evolution of β‐galactosidase from Escherichia coli by mutator strains defective in the 3′→5′ exonuclease activity of DNA polymerase III
Authors: Stefan Alessandra , Radeghieri Annalisa , Gonzalez Vara y Rodriguez Antonio , Hochkoeppler Alejandro
Published: July 2002

Directed evolution of Escherichia coli β‐galactosidase into variants featuring β‐glucosidase activity was challenged. To this end, mutagenesis of lacZ was performed by replication in E. coli CC954, a mutator strain containing a DNA polymerase III defective in 3′→5′ exonuclease activity. β‐Galactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self‐transmissible episome F′128. Optimal evolution of lacZ can be achieved by propagation of E. coli CC954/F′128 cultures for 15 generations; further growth of mutator cultures for 37 or 55 generations imposes a high mutational load on lacZ and hinders the selection of efficiently evolved clones.
Activation of p38MAPK by TGF‐β in fetal rat hepatocytes requires radical oxygen production, but is dispensable for cell death
Authors: Herrera Blanca , Fernández Margarita , Roncero César , Ventura Juan José , Porras Almudena , Valladares Amparo , Benito Manuel , Fabregat Isabel
Published: July 2002

We have previously found that transforming growth factor‐β (TGF‐β) induces an increase in radical oxygen species (ROS) production that mediates its apoptotic effects in fetal hepatocytes. In this paper we show that TGF‐β activates p38 mitogen‐activated protein kinase (p38MAPK) and ROS may be responsible for this activation. Activation of p38MAPK occurs late, coincident with the maximal production of ROS, it is inhibited by radical scavengers and it is accentuated by the presence of glutathione synthesis inhibitors. However, p38MAPK does not appear to be involved in any of the apoptotic events: loss of Bcl‐xL levels, cytochrome c release, cleavage of caspase substrates and loss of cell viability.
Septide and neurokinin A are high‐affinity ligands on the NK‐1 receptor: evidence from homologous versus heterologous binding analysis
Authors: Hastrup Hanne , Schwartz Thue W.
Published: July 2002

The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK‐1, NK‐2 and NK‐3 receptors. However, NKA also has actions which cannot be mediated through its normal NK‐2 receptor and the synthetic peptide [pGlu6,Pro9]‐Substance P9–11 — called septide — is known to have tachykinin‐like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK‐1 receptor expressed in COS‐7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK‐1 receptor with high affinity: K
d values of 0.51 ± 0.15 nM (NKA) and 0.55 ± 0.03 nM (septide). It is concluded that NKA and septide are high‐affinity ligands for the NK‐1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.
Nuclear factor‐κB activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2
Authors: Conte Enrico , Bonaiuto Corrada , Nesci Caterina , Crimi Nunzio , Vancheri Carlo , Messina Angelo
Published: July 2002

The nuclear factor κB (NF‐κB) is thought to be crucially involved in the gene activation of several cytokines, including tumor necrosis factor α (TNF). Previously, we showed that fibroblast conditioned medium (FCM) is able to inhibit both TNF mRNA accumulation and protein release in peripheral blood‐derived human monocytes (PBM) stimulated with lipopolysaccharide (LPS). In this study we have investigated the effect of FCM on the LPS‐induced DNA‐binding activity of NF‐κB, by means of electrophoretic shift assay (EMSA). We provide evidence that FCM strongly inhibits the LPS‐induced NFκB activation in PBM. Furthermore, we show that exogenous PGE2 mimics the NFκB inhibitory effect of FCM. On the other hand, FCM produced in the presence of indomethacin does not inhibit NF‐κB activation by LPS. Our results lend further support to the hypothesis that inflammatory and immune responses of monocytes/macrophages may be modulated at the molecular level by signals originating from tissue structural cells such as fibroblasts.
Does selective gene activation direct evolution?
Authors: Wright Barbara E
Published: July 2002

Mechanisms may have evolved such that the unique metabolic reaction to a particular environmental stress results in higher mutation rates of those genes most likely to solve the problem. Evidence is presented indicating that the environment in effect directs the evolution of organisms by (1) presenting various kinds of stress resulting in metabolic activities that target particular genes for increased rates of transcription and mutation, and (2) selecting among this specifically enriched mutant population those variants that alleviate the imposed stress. This process should be ongoing and would be expected to accelerate the rate of microbial evolution.
Activation of p21‐activated protein kinase α (αPAK) by hyperosmotic shock in neonatal ventricular myocytes
Authors: Clerk Angela , Sugden Peter H
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Desensitization of the P2X2 receptor controlled by alternative splicing
Authors: Brändle U. , Spielmanns P. , Osteroth R. , Sim J. , Surprenant A. , Buell G. , Ruppersberg J.P. , Plinkert P.K. , Zenner H.-P. , Glowatzki E.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
RNA polymerase III interferes with Ty3 integration
Authors: Connolly Charles M. , Sandmeyer Suzanne B.
Published: July 2002

Ty3, a gypsylike retrotransposon of budding yeast, integrates at the transcription initiation site of genes transcribed by RNA polymerase III (pol III). It was previously shown that integration in vitro requires intact promoter elements and the pol III transcription factors TFIIIB and TFIIIC. In order to test the effect of pol III on integration, increasing amounts of a pol III‐containing fraction were added to Ty3 in vitro integration reactions. The pol III‐containing fraction was inhibitory to integration. These results are consistent with a model where the Ty3 integration complex and pol III recognize similar features of the stable transcription complex and compete with each other for access to the transcription initiation site.
Nucleotide and calcium‐induced conformational changes in histone H1
Authors: Tarkka Tuta , Oikarinen Jouko , Grundström Thomas
Published: July 2002

The principal constituents of chromatin, histone H1 (H1) and the nucleosome have essential roles in regulation of eukaryotic gene expression. However, mechanisms for the H1‐dependent inactivation and for the ATP‐dependent chromatin remodeling upon activation are largely unelucidated. Using circular dichroism (CD) analysis we show that ATP and other nucleotides and Ca2+ induce structural changes in H1. ATP and Ca2+ also induce changes when H1 is interacting with DNA, and the changes in H1 are accompanied by alterations in its DNA interaction. These results suggest that nucleotide and Ca2+ binding may be important for H1‐mediated chromatin changes.
‘Green mice’ as a source of ubiquitous green cells
Authors: Okabe Masaru , Ikawa Masahito , Kominami Katsuya , Nakanishi Tomoko , Nishimune Yoshitake
Published: July 2002

The green fluorescent protein (GFP) is responsible for the green bioluminescence of the jellyfish Aequorea victoria. Many classes of GFP mutants exist that display modified fluorescence spectra and an increased extinction coefficient. We produced transgenic mouse lines with an ‘enhanced’ GFP (EGFP) cDNA under the control of a chicken beta‐actin promoter and cytomegalovirus enhancer. All of the tissues from these transgenic lines, with the exception of erythrocytes and hair, were green under excitation light. The fluorescent nature of the cells from these transgenic mouse lines would facilitate their use in many kinds of cell transplantation experiments.
Bi‐directional movement of actin filaments along long bipolar tracks of oriented rabbit skeletal muscle myosin molecules
Authors: Yamada Akira , Yoshio Maki , Nakayama Haruto
Published: July 2002

In actomyosin in vitro motility assays, orientation of myosin molecules affects their interaction with actin. We obtained long tracks of myosin molecules with uniform orientation. Bipolar filaments about 50 μm long were made from myosin rod prepared from molluscan smooth muscles, to which rabbit skeletal‐muscle myosin bound, creating long synthetic thick‐filaments. Movement of F‐actin toward their center was much faster (4.7±0.6 μm s−1) than in the opposite direction (1.9±0.2 μm s−1), indicating that myosin molecules were arranged in the same orientation along each half of the bipolar filament. These complex thick‐filaments permit measurement of actin movement over 20 μm of oriented skeletal myosin tracks facilitating mechanistic studies of actomyosin motility.
Evidence for a 65 kDa sulfonylurea receptor in rat pancreatic zymogen granule membranes
Authors: Braun Matthias , Anderie Ines , Thévenod Frank
Published: July 2002

In rat pancreatic zymogen granules (ZG), a K+ selective conductance which can be blocked by ATP has been characterized. Here we show that this pathway can be specifically blocked by glibenclamide. Using a rapid filtration assay, we also found specific binding of [3H]glibenclamide to a low‐affinity site (K
d 5.6±1.1 μM) in rat pancreatic zymogen granule membranes (ZGM). In photoaffinity labeling experiments with [3H]glibenclamide, a 65±1.5 kDa polypeptide was specifically labeled. Previously, a ∼65 kDa mdr1 gene product has been demonstrated to be involved in the regulation of the K+ selective conductance of ZG. We conclude that this protein may be a subunit of, or associated with, a ZG KATP channel.
Nitric oxide inhibits enterocyte mitochondrial phospholipase D
Authors: Madesh M , Balasubramanian K.A
Published: July 2002

Mitochondrial damage is one of the prominent features of cell death in oxidative stress and related pathological conditions. Alteration in membrane lipid composition may be responsible for the mitochondrial damage. In this study, we have shown that intestinal mitochondria contain an active phospholipase D (PLD) which is activated by oxidants, Ca2+ or polyamines and this results in degradation of phosphatidylethanolamine (PE) and formation of phosphatidic acid (PA). This PLD activity is inhibited by nitric oxide (NO) which prevents the lipid alteration in mitochondria when exposed to these agents. This can be reversed by the NO scavenger, haemoglobin. This suggests that alteration of mitochondrial membrane lipid composition by activation of PLD in certain pathological condition such as oxidative stress may be prevented by the simultaneous presence of nitric oxide.
Cleavage of Gag precursor is required for early replication phase of HIV‐1
Authors: Kawamura Meiko , Shimano Reika , Inubushi Ritsuko , Amano Kazushi , Ogasawara Takashi , Akari Hirofumi , Adachi Akio
Published: July 2002

A mutant of human immunodeficiency virus type 1 (HIV‐1), which is deficient for Gag precursor cleavage and non‐infectious, was characterized with respect to its defective step in the viral replication phase. Upon transfection, the mutant produced a normal level of progeny virions as monitored by electron microscopy and RNA hybridization. Single‐round replication assay demonstrated, in contrast, that the mutant was defective at the early phase of the replication cycle. Furthermore, no viral DNA was detected in the cells infected with the mutant. Taken together, it is concluded that maturation of Gag precursor protein of HIV‐1 is required for an early event(s) before or during a coupled process of uncoating/reverse transcription.
Translational control of terminal oligopyrimidine mRNAs requires a specific regulator
Authors: Loreni Fabrizio , Amaldi Francesco
Published: July 2002

Terminal oligopyrimidine (TOP) mRNAs are a group of messengers translationally regulated according to the growth status of the cell. Two hypotheses have been proposed for the mechanism of the regulation: (i) there is a specific translational regulator which can reversibly alter TOP‐mRNA structure, (ii) a component of the general translational apparatus can specifically affect the translation of TOP‐mRNAs. To verify one of the two hypotheses we induced a partial inhibition of translation initiation in Xenopus cultured cells and analyzed the effect on TOP‐mRNA translation. Our results suggest that a specific regulator is necessary to explain the translational control of these of mRNAs.
Effect of the chaperone‐like alpha‐crystallin on the refolding of lysozyme and ribonuclease A
Authors: Raman B , Ramakrishna T , Rao Ch.Mohan
Published: July 2002

Alpha‐crystallin exhibits chaperone‐like properties in preventing aggregation of proteins. We have studied the effect of alpha‐crystallin on the refolding of denatured‐disulfide intact and denatured‐reduced lysozyme and RNase A. Alpha‐crystallin does not have any effect on the refolding of both the denatured‐disulfide intact enzymes. However, it inhibits the aggregation and oxidative renaturation of denatured‐reduced lysozyme. Interestingly, it has no effect on the refolding of denatured‐reduced RNase A. In order to probe the molecular basis of this differential behavior of alpha‐crystallin towards lysozyme and RNase A, we have carried out circular dichroism and fluorescence studies on the refolding of denatured‐reduced RNase A. It exhibits an extended conformation with little difference in the exposed hydrophobicity during the refolding process. We have earlier shown the presence of an aggregation‐prone, refolding‐competent, molten‐globule‐like intermediate on the refolding pathway of lysozyme. Alpha‐crystallin binds to this intermediate, prevents its aggregation and inhibits its oxidative refolding. It was earlier believed that alpha‐crystallin, unlike other chaperones, does not recognize intermediates on the refolding pathway but only recognizes intermediates on the unfolding pathway of proteins. Our present study clearly shows that it recognizes the refolding intermediates as well.
Metal‐induced metallothionein gene expression can be inactivated by protein kinase C inhibitor
Authors: Yu Chih-Wen , Chen Je-Hsin , Lin Lih-Yuan
Published: July 2002

The effects of protein kinase C (PKC) inhibitors on the metallothionein (MT) gene expression induced by metals were investigated. When PKC inhibitor (H7 or chelerythrine) was administered to Cd resistant, MT gene‐amplified Chinese hamster ovary (CdR) cells, the induction of MT mRNA by Cd or Zn was blocked. Treating the CdR cells with a PKA‐specific inhibitor, HA1004, did not cause an inhibition of metal‐induced MT gene transcription. The inhibitory effect was effectuated by adding inhibitors within 40 min of exposing the cells to Cd. Apparently, AP1 was not involved in this down‐regulatory effect of PKC inhibitor on MT gene expression since the inducibility of MT promoter was blocked by H7 even in the absence of the AP1‐binding sequence. For Cd‐treated cells, Cd accumulation in the cell was similar with or without H7 treatment. However, H7 markedly reduced cellular Zn accumulation when the cells were treated with Zn. Cycloheximide treatment increased the level of MT mRNA. This elevation can also be blocked by treating the cell with PKC inhibitor. Results in this study suggest that PKC participates in the process of metal‐induced MT gene expression.
Ionic currents underlying HTRP3 mediated agonist‐dependent Ca2+ influx in stably transfected HEK293 cells
Authors: Hurst Raymond S , Zhu Xi , Boulay Guylain , Birnbaumer Lutz , Stefani Enrico
Published: July 2002

hTrp3 is a human homologue of the Drosophila gene responsible for a transient receptor potential (trp) mutation. When stably expressed in HEK293 cells, hTrp3 formed ion channels that were active under resting conditions but could be further stimulated by carbachol or ATP via endogenous muscarinic or purinergic receptors, respectively. Agonist evoked currents reversed polarity near 0 mV in physiological ionic conditions and were associated with a significant increase in the current variance. These results suggest the involvement of a non‐selective cation channel with relatively large unitary amplitude. Consistent with this, resolved unitary events had a conductance of approximately 60 pS in the negative voltage range and an extrapolated reversal potential near 0 mV.
Fuel oxidation in skeletal muscle is increased by nitric oxide/cGMP – evidence for involvement of cGMP‐dependent protein kinase
Authors: Young Martin E , Leighton Brendan
Published: July 2002

The cyclic guanosine‐3′,5′‐monophosphate (cGMP) analogue, 8‐bromo‐cGMP (1 mM), increased glucose oxidation in isolated soleus muscle. The nitric oxide (NO) donor, sodium nitroprusside (SNP) (15 mM), increased glucose, pyruvate, palmitate and leucine oxidation. Removal of extracellular Ca2+ did not affect SNP‐stimulated glucose oxidation (or other glucose utilization parameters), thus eliminating the influx of Ca2+ as a mechanism for the increases. The guanylate cyclase inhibitor, LY‐83583 (10 μM), inhibited SNP‐stimulated palmitate oxidation and activation of cGMP‐dependent protein kinase (PKG). Activation of PKG might supersede any inhibitory effects of NO on respiration to stimulate metabolic fuel oxidation in skeletal muscle.
Role of MAP kinase cascades in inducing arginine transporters and nitric oxide synthetase in RAW264 macrophages
Authors: Caivano Matilde
Published: July 2002

Bacterial lipopolysaccharide (LPS) in the presence of interferon gamma (IFNγ) stimulates the synthesis of the cationic amino acid transporter 2B (CAT‐2B) and inducible nitric oxide synthetase (iNOS) in RAW264 macrophages, which are thought to underlie the increased rate of arginine uptake into these cells and its conversion to nitric oxide, respectively. Here I demonstrate that the LPS‐ and IFNγ‐induced increase in arginine uptake into RAW264 cells is partially suppressed in the presence of PD 98059, partially suppressed in the presence of SB 203580, and completely inhibited by both drugs. In contrast, the LPS‐ and IFNγ‐induced synthesis of CAT‐2B mRNA and iNOS protein is unaffected by PD 98059 and SB 203580. The results indicate that the MAPK/ERK and SAPK2/p38 cascades are both rate‐limiting for LPS‐ and IFNγ‐stimulated arginine uptake, but not for iNOS synthesis. They also suggest that PD 98059 and SB 203580 suppress CAT‐2B synthesis at a post‐transcriptional level.
A single missense mutant of Smad3 inhibits activation of both Smad2 and Smad3, and has a dominant negative effect on TGF‐β signals
Authors: Goto Daisuke , Yagi Ken , Inoue Hirofumi , Iwamoto Itsuo , Kawabata Masahiro , Miyazono Kohei , Kato Mitsuyasu
Published: July 2002

A missense mutation of Smad2 identified in cancer cells was reconstructed on the corresponding residue of Smad3. This mutant, Smad3D407E, was not phosphorylated by the constitutively active form of type I receptor for transforming growth factor‐β (TGF‐β), and inhibited the phosphorylation of co‐expressed wild‐type Smad2 and Smad3. This mutant also had a dominant negative effect on the growth inhibition of HaCaT cells and on the expression of p3TP‐lux reporter gene induced by TGF‐β. However, it did not alter the phosphorylation of Smad1 induced by the constitutively active form of the bone morphogenetic protein type IA receptor. These findings showed that a single missense mutation in Smad3 could specifically block TGF‐β signals by preventing activation of both Smad2 and Smad3.
A novel aspect of calpain activation
Authors: Suzuki Koichi , Sorimachi Hiroyuki
Published: July 2002

Calpain, a Ca2+‐dependent biomodulator, alters the properties of substrate proteins by cleaving them at a limited number of specific sites. Recent studies of the structure‐function relationship of calpain and X‐ray analysis of its Ca2+‐binding domain have revealed hitherto unknown features of the regulation of calpain activity. A novel dissociation/autolysis mechanism for the activation of calpain at the membrane is proposed, which incorporates recent findings from structure‐function studies of calpain, and its implications are discussed.
Activation of protein kinase C is required for protection of cells against apoptosis induced by singlet oxygen
Authors: Zhuang Shougang , Lynch Mary C , Kochevar Irene E
Published: July 2002

We evaluated the role of protein kinase C (PKC) in the regulation of apoptosis triggered by singlet oxygen. Activation of PKC by short‐term 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) treatment inhibited apoptosis, whereas inhibition of PKC with several inhibitors potentiated this process. The antiapoptotic effect of TPA was accompanied by phosphorylation of extracelluar signal‐regulated kinase 1/2 (ERK1/2). Pretreatment of cells with MEK inhibitor, PD98059, inhibited TPA‐induced phosphorylation of ERK1/2 and the cytoprotective ability of TPA. These results suggest that activation of PKC in HL‐60 cells confers protection against apoptosis induced by singlet oxygen and that ERK1/2 mediates antiapoptotic signaling of PKC.
Kinetics of oxidation of aliphatic and aromatic thiols by myeloperoxidase compounds I and II
Authors: Burner Ursula , Jantschko Walter , Obinger Christian
Published: July 2002

Myeloperoxidase (MPO) is the most abundant protein in neutrophils and plays a central role in microbial killing and inflammatory tissue damage. Because most of the non‐steroidal anti‐inflammatory drugs and other drugs contain a thiol group, it is necessary to understand how these substrates are oxidized by MPO. We have performed transient kinetic measurements to study the oxidation of 14 aliphatic and aromatic mono‐ and dithiols by the MPO intermediates, Compound I (k
3) and Compound II (k
4), using sequential mixing stopped‐flow techniques. The one‐electron reduction of Compound I by aromatic thiols (e.g. methimidazole, 2‐mercaptopurine and 6‐mercaptopurine) varied by less than a factor of seven (between 1.39±0.12×105 M−1 s−1 and 9.16±1.63×105 M−1 s−1), whereas reduction by aliphatic thiols was demonstrated to depend on their overall net charge and hydrophobic character and not on the percentage of thiol deprotonation or redox potential. Cysteamine, cysteine methyl ester, cysteine ethyl ester and α‐lipoic acid showed k
3 values comparable to aromatic thiols, whereas a free carboxy group (e.g. cysteine, N‐acetylcysteine, glutathione) diminished k
3 dramatically. The one‐electron reduction of Compound II was far more constrained by the nature of the substrate. Reduction by methimidazole, 2‐mercaptopurine and 6‐mercaptopurine showed second‐order rate constants (k
4) of 1.33±0.08×105 M−1 s−1, 5.25±0.07×105 M−1 s−1 and 3.03±0.07×103 M−1 s−1. Even at high concentrations cysteine, penicillamine and glutathione could not reduce Compound II, whereas cysteamine (4.27±0.05×103 M−1 s−1), cysteine methyl ester (8.14±0.08×103 M−1 s−1), cysteine ethyl ester (3.76±0.17×103 M−1 s−1) and α‐lipoic acid (4.78±0.07×104 M−1 s−1) were demonstrated to reduce Compound II and thus could be expected to be oxidized by MPO without co‐substrates.
Involvement of tyrosine phosphorylation in HMG‐CoA reductase inhibitor‐induced cell death in L6 myoblasts
Authors: Mutoh Tatsuro , Kumano Takanori , Nakagawa Hiroto , Kuriyama Masaru
Published: July 2002

Our previous studies have shown that the HMG‐CoA reductase (HCR) inhibitor (HCRI), simvastatin, causes myopathy in rabbits and kills L6 myoblasts. The present study was designed to elucidate the molecular mechanism of HCRI‐induced cell death. We have demonstrated that simvastatin induces the tyrosine phosphorylation of several cellular proteins within 10 min. These phosphorylations were followed by apoptosis, as evidenced by the occurrence of internucleosomal DNA fragmentation and by morphological changes detected with Nomarski optics. Simvastatin‐induced cell death was prevented by tyrosine kinase inhibitors. The MTT assay revealed that the addition of mevalonic acid into the culture medium partially inhibited simvastatin‐induced cell death. Thus, these results suggested that protein tyrosine phosphorylation might play an important role in the intracellular signal transduction pathway mediating the HCRI‐induced death of myoblasts.
Differential association of cytoplasmic signalling molecules SHP‐1, SHP‐2, SHIP and phospholipase C‐γ1 with PECAM‐1/CD31
Authors: Pumphrey Nicholas J. , Taylor Vanessa , Freeman Sylvie , Douglas Michael R. , Bradfield Paul F. , Young Stephen P. , Lord Janet M. , Wakelam Michael J.O. , Bird Ian N. , Salmon Michael , Buckley Christopher D.
Published: July 2002

Recent studies have shown that, in addition to its role as an adhesion receptor, platelet endothelial cell adhesion molecule 1/CD31 becomes phosphorylated on tyrosine residues Y663 and Y686 and associates with protein tyrosine phosphatases SHP‐1 and SHP‐2. In this study, we screened for additional proteins which associate with phosphorylated platelet endothelial cell adhesion molecule 1, using surface plasmon resonance. We found that, besides SHP‐1 and SHP‐2, platelet endothelial cell adhesion molecule 1 binds the cytoplasmic signalling proteins SHIP and PLC‐γ1 via their Src homology 2 domains. Using two phosphopeptides, NSDVQpY663TEVQV and DTETVpY686SEVRK, we demonstrate differential binding of SHP‐1, SHP‐2, SHIP and PLC‐γ1. All four cytoplasmic signalling proteins directly associate with cellular platelet endothelial cell adhesion molecule 1, immunoprecipitated from pervanadate‐stimulated THP‐1 cells. These results suggest that overlapping immunoreceptor tyrosine‐based inhibition motif/immunoreceptor tyrosine‐based activation motif‐like motifs within platelet endothelial cell adhesion molecule 1 mediate differential interactions between the Src homology 2 containing signalling proteins SHP‐1, SHP‐2, SHIP and PLC‐γ1.
Conversion of an inactive cardiac dihydropyridine receptor II‐III loop segment into forms that activate skeletal ryanodine receptors
Authors: Zhu Xinsheng , Gurrola Georgina , Tao Jiang Ming , Walker Jeffery W. , Valdivia Hector H.
Published: July 2002

A 25 amino acid segment (Glu666–Pro691) of the II‐III loop of the α1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp788–Pro814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II‐III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II‐III loop (681RKRRK685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu800 in the group of negatively charged residues 798EEEEE802 of the cardiac II‐III loop may serve to prevent the binding of the activation domain.
Activation of Ras by phorbol esters in cardiac myocytes
Authors: Montessuit Christophe , Thorburn Andrew
Published: July 2002

The relationship between protein kinase C (PKC) activation and Ras function was investigated in cardiac cells. Ras function was required for ERK activation by phorbol esters in cardiac myocytes, but not in cardiac fibroblasts. Accordingly, treatment with phorbol esters resulted in GTP loading of Ras in cardiac myocytes, but not fibroblasts. Ras activation by phorbol esters was abolished by a PKC specific inhibitor, but was insensitive to tyrosine kinase inhibitors. Ras activation was mediated by stimulation of guanine nucleotide exchange. These results suggest the existence of a novel pathway for Ras activation, specific to cardiac myocytes, with implications for myocardial hypertrophy.
Cold‐induced ependymin expression in zebrafish and carp brain: implications for cold acclimation
Authors: Tang Shye-Jye , Sun Kuang-Hui , Sun Guang-Huan , Lin Guang , Lin Wen-Wen , Chuang Mei-Jan
Published: July 2002

Cold acclimation has been suggested to be mediated by alternations in the gene expression pattern in the cold‐adapted fish. To investigate the mechanism of cold acclimation in fish brain at the molecular level, relevant subsets of differentially expressed genes of interest were identified and cloned by the PCR‐based subtraction suppression hybridization. Characterization of the selected cold‐induced cDNA clones revealed one encoding ependymin. This gene was shown to be brain‐specific. The expression of ependymin was induced by a temperature shift from 25°C to 6°C in Cyprinus carpio or 12°C in Danio rerio. Activation of ependymin was detected 2 h after cold exposure and peaked at more than 10‐fold at 12 h. This peak level remains unchanged until the temperature returns to 25°C. Although the amount of soluble ependymin protein in brain was not changed by cold treatment, its level in the fibrous insoluble polymers increased 2‐fold after exposure to low temperature. These findings indicate that the increase in ependymin expression is an early event that may play an important role in the cold acclimation of fish.
Evidence for a role of C‐terminus in Ca2+ inactivation of skeletal muscle Ca2+ release channel (ryanodine receptor)
Authors: Nakai Junichi , Gao Ling , Xu Le , Xin Chunlin , Pasek Daniel A. , Meissner Gerhard
Published: July 2002

Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca2+ release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca2+ dependence in [3H]ryanodine binding and single channel measurements. The results indicate that the C‐terminal one‐fourth has a major role in Ca2+ activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [3H]ryanodine binding affinity while maintaining channel activity.
Effect of cigarette smoke on human placenta morphometry
Authors: Rocha J.E.S , Matheus M , Sala M.A
Published: July 2002

Objective: To evaluate the placental alterations in smoking mothers. Method: A case‐control stereologic study of 10 placentas from smokers and 10 from non‐smokers. Result: The authors found a significant increase in stromal volume density, villus size and trophoblast thickness, and a significant decrease in villus and vessel surface densities, total surfaces, and number/mm2 histological field in the smokers' placenta. Conclusions: The alterations found in the smokers' placenta greatly contribute to impairing the exchanges between maternal and fetal blood.
Maternal syphilis and vertical perinatal transmission of human immunodeficiency virus type‐1 infection
Authors: Lee M.‐J. , Hallmark R.J. , Frenkel L.M. , Del Priore G.
Published: July 2002

Objectives: To identify risk factors for vertical HIV transmission that may provide opportunities for the prevention of future pediatric HIV infection. Study Design: A prospective cohort of 44 pregnant HIV(+) women and their 53 offspring was followed between July 1989 and July 1996. Results: Secondary and latent maternal syphilis infection was diagnosed in four pregnancies with VDRL titers ≥ 1:128. Concurrent maternal syphilis infection was associated with 100% vertical HIV transmission (4/4), compared to 21% (3/14) deliveries that occurred in 13 women with only a history of treated sypillis (P = 0.01) and 14% (5/35) deliveries in women with no history of syphilis (P = 0.0015). Among non‐Zidovudine exposed pregnancies, 100% (3/3) patients with concurrent syphilis transmitted HIV to their infants vs. 0% (0/8) patients with only a history of syphilis (P = 0.006). A history of treated syphilis did not increase subsequent transmission risk compared to women with no history of syphilis (3/14 vs. 5/35, P = 0.41). One patient delivered twice during the study and received Zidovudine prophylaxis during both pregnancies. Her first pregnancy VDRL titer was ≥ 1:128, during which she delivered an HIV infected child. Following successful treatment for syphilis (VDRL titer, 1:1), she conceived again and delivered an uninfected infant 11 months later. Additional risk factors significantly associated with vertical HIV transmission were prematurity and low birth weight. Variables studied but not statistically‐related to HIV transmission included: other sexually transmitted diseases, maternal age, race, intravenous drug of cocaine use, prenatal care, route or delivery, duration of HIV(+) status, and CD4 count. Conclusion: Among HIV(+) women, concurrent syphilis infection, but not a history of syphilis, is significantly associated with vertical perinatal HIV transmission. Frequent screening and prompt treatment of syphilis in non‐pregnant HIV(+) women may help prevent subsequent HIV infected pregnancies.
Fibre‐type distribution and subcellular localisation of α and β enolase in mouse striated muscle
Authors: Keller Angélica , Demeurie Jeanine , Merkulova Tatyana , Géraud Gérard , Cywiner‐Golenzer Charlotte , Lucas Marguerite , Châtelet François‐Patrick
Published: July 2002

Enolase is a dimeric glycolytic enzyme exhibiting tissue specific isoforms. During ontogenesis, a transition occurs from the embryonic αα towards the specific αβ, and ββ isoforms in striated muscle. Immunocytochemical analyses on transverse sections of adult mouse gastrocnemius muscle, allowed us to compare the expression of α and β subunits to that of myosin heavy chain (MHC) isoforms. Levels of β immunoreactivity followed the order IIB > IIX > IIA > I. This gradient parallels the ATPase activity associated to MHC isoforms, indicating that the expression of β enolase in myofibres is finely regulated as a function of energetic requirements. By contrast, variations in α immunolabelling intensity appeared independent of fibre types.

Longitudinal muscle sections exhibited a striated pattern of α immunoreactivity. Confocal microscopy analyses demonstrated that α was localised at the M band. Most β immunoreactivity was diffuse all over the sarcoplasm. However, some β immunoreactivity was striated and localized at both Z and M bands. Thus, ββ enolase could participate to multi‐enzyme complexes present at the I band, and involved with local ATP production.

Our results support the notion that isozymes differ in their ability to interact with other macromolecules, thus segregating to different subcellular sites where they would respond to specific functional demands.
Suicide attempts in the Hungarian adult population. Their relation with DIS/DSM-III-R affective and anxiety disorders
Authors: Szádóczky E. , Vitrai J. , Rihmer Z. , Füredi J.
Published: July 2002

summary

Prevalence of suicide attempts and their relationship with DIS anxiety and affective disorder diagnoses were investigated in a Hungarian adult community sample. Despite the high suicide mortality rate, the rate of suicide attempts was similar to that reported in other studies using similar methods. Suicide attempts occurred more frequently among women and previously married persons. Although the presence of any lifetime anxiety and/or affective disorder increased the rate of reported suicide attempts, the effect of co-morbidity, recurrence and chronicity might be considered significant predictors. The highest odds of an attempt were related to the diagnoses of dysthymic or bipolar disorders. Agitation was the only depressive symptom, which increased the odds of a suicide attempt.
A review of the efficacy and tolerability of venlafaxine
Authors: Dierick M
Published: July 2002

Venlafaxine is a selective serotonin norepinephrine reuptake inhibitor with no activity at muscarinic, histaminergic or adrenergic receptors. The antidepressant activity of venlafaxine has been demonstrated in placebo-controlled and active comparator-controlled clinical trials. Venlafaxine was effective in outpatients and hospitalized patients with major depression and in those with melancholia, agitated or retarded symptoms, and refractory or treatment-resistant depression. Venlafaxine was at least as effective as comparative antidepressants and was more effective than fluoxetine or imipramine in some trials. A positive dose-response relationship has been shown with venlafaxine. When doses of venlafaxine are titrated rapidly upward, an onset of antidepressant action has been detected within one week in some studies. Venlafaxine is well tolerated during short- and long-term treatment. The most common adverse effects are nausea, somnolence and dry mouth. The overall tolerability of venlafaxine appears to exceed that of tricyclic antidepressants and compares favorably with that of selective serotonin reuptake inhibitors. Venlafaxine is a novel antidepressant that is appropriate for first-line therapy in patients with major depression.