bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanol and lipid metabolism Differential effects on liver and brain microsomes
Authors: Sanchez-Amate Maria C. , Zurera Jesús M. , Carrasco Maria P. , Segovia Josefa L. , Marco Carmen
Published: July 2002

We have determined the effect of prolonged ethanol treatment on several enzyme activities related to lipid metabolism in chick‐brain and liver microsomes. Ethanol increased microsome cholesterol levels in both organs. The treatment caused a marked increase in the hepatic HMG‐CoA reductase and ACAT activities while in the brain a clear decrease was found in these enzyme activities. At the same time the activity of reacylation of phospholipids, was clearly modified in both brain and liver. Thus, while in the liver the turnover of acyl moieties of phosphatidylethanolamine, sphingomyelin and phosphatidylinositol was enhanced by ethanol consumption, in the brain only the reacylation of phosphatidylserine increased to any significant extent. These results indicate that ethanol exerts a differential action in brain and liver, namely cholesterol synthesis and esterification decreased in brain and increased in chick liver. Ethanol also induces faster phospholipid metabolism in both brain and liver microsomes.
Posttranscriptional regulation of c‐myc RNA during early development of Xenopus laevis
Authors: Tchang Françoise , Vriz Sophie , Méchali Marcel
Published: July 2002

The remarkable stability of c‐myc during oogenesis contrasts with its degradation during the early developmental period in Xenopus laevis. Three evolutionary conserved motifs found in the 3′‐untranslated region of Xenopus c‐myc RNAs have been analyzed for a possible role in c‐myc RNA degradation. No specific degradation was observed when these sequences were cloned downstream of a reporter gene and the corresponding RNAs were injected into fertilized eggs. The relation between polyadenylation and degradation of c‐myc mRNA has been examined during early development. c‐myc is adenylated during early oogenesis, and a dramatic de‐adenylation occurs in full grown oocytes. Consequently, the de‐adenylation of c‐myc mRNA that occurs in eggs might be a requirement for its degradation after fertilization, but is not sufficient to trigger its degradation.
Concentration‐dependent modulation of basic fibroblast growth factor action on multiplication and locomotion of human teratocarcinoma cells
Authors: Schofield Paul N. , Granerus Marika , Lee Amanda , Ektröm Tomas J. , Engström Wilhelm
Published: July 2002

A human teratoma cell line (Tera 2) was grown in serum‐free medium, and the population multiplication was stimulated by the addition of 1–10 ng basic fibroblast growth factor (bFGF)/ml. The bFGF‐effect was abrogated by the addition of protamin sulphate. When high concentrations of bFGF were added, a preferential effect on cell locomotion was observed. 100 ng bFGF/ml stimulated cell movement but only exerted a marginal effect on cell multiplication. These observed exogenous requirements for multiplication and locomotion were complemented by the expression of bFGF receptors. Scatchard analysis of binding data suggests the existence of a high‐affinity and a low‐affinity class of receptors.
Isolation and characterisation of a new hepatic bilirubin UDP‐glucuronosyltransferase Absence from Gunn rat liver
Authors: Clarke Douglas J. , Keen Jeffrey N. , Burchell Brian
Published: July 2002

A novel UDP‐glucuronosyltransferase that conjugates bilirubin IXα, bilirubin monoglucuronide and an arylalkanoic acid was purified to homogeneity from clofibrate treated Wistar rats. The enzyme displayed a subunit molecular mass of 54 kDa, a pI of 7.6 and was demonstrated to be N‐glycosylated. Sequence analysis of peptides derived by endoproteinase Glu‐C cleavage of the purified enzyme indicated that it was a new member of the recently identified UGT1 subfamily. Immunoblot analysis demonstrated that this enzyme was absent from Gunn rat liver. The molecular derivation of this enzyme and the lack of it in Gunn rats is discussed.
Solution structure of FK506 bound to FKBP‐12
Authors: Lepre Christopher A. , Thomson John A. , Moore Jonathan M.
Published: July 2002

The complex of the immunosuppressant FK506 bound to FKBP‐12 has been studied in solution using 1H and inverse‐detected 13C NMR methods. The resonances of bound, 13C‐labelled FK506 were assigned and a set of 66 intraligand NOE distance restraints were used to calculate the structure of the bound ligand by distance geometry and restrained molecular dynamics methods. The structure of bound FK506 in solution closely resembles that seen in the X‐ray structure [17], except for the allyl region. The differences reflect the influence of intermolecular crystal contacts and have implications for interpretation of the interaction of the FK506/FKBP complex with its putative biological receptor.
Evaluation of the expression of the cationic peptide gene in various types of leukocytes
Authors: Nagaoka Isao , Yomogida Shin , Nakamura Shinji , Someya Akimasa , Iwabuchi Kazuhisa , Yamashita Tatsuhisa
Published: July 2002

To understand the regulation of the production or antimicrobial cationic peptide (CP) in leukocytes, expression of the CP gene was evaluated in various types of leukocytes using guinea pig neutrophils, monocytes, macrophages, cosinophils, lymphocytes and bone marrow cells. Acid‐urea PAGE and SDS‐PAGE/immunoblot analyses showed that CP was present in neutrophils and bone marrow cells, but not in other leukocytes. Northern blot and transcription run‐off analyses revealed that only bone marrow cells expressed CP mRNA and transcribed the CP gene. Interestingly, in situ hybridization analysis using bone marrow cells demonstrated that CP mRNA was expressed in the neutrophil precursor cells, such as promyclocytes and myclocytes, but was not detected in the mature neutrophils and other bone marrow cells. Moreover, immunocytochemical study indicated flat CP present in the neutrophil precursor cells and the mature neutrophils in the bone marrow. Thus, the CP gene appears to be expressed during a limited period of neutrophil maturation, and CP is likely synthesized by the neutrophil precursor cells in the bone marrow.
Differential expression of two sodium channel subtypes in human brain
Authors: Lu Chiung-Mei , Han Jian , Rado Thomas A. , Brown George B.
Published: July 2002

Two partial human brain sodium channel cDNA sequences (designated HBSC I and II) have been cloned and mapped to chromosome 2q23–2q24 by chromosome microdissection‐PCR (CMPCR). The distribution of HBSC I and II mRNA in human brain was studied by means of a novel approach based on the ligase detection reaction. These studies demonstrate that HBSC I and II mRNA is heterogeneously distributed in brain. and that the relative ratio or the two forms can vary as much as 7‐fold between different regions.
Structural characterization and expression of a brain specific gene encoding chick kainate binding protein
Authors: Eshhar Nomi , Hunter Chyren , Wenthold Robert J. , Wada Keiji
Published: July 2002

The gene encoding chick kainate‐binding protein (c‐kbp), a memeber of the non‐NMDA ionotropic glutamate receptor family has been isolated and characterized. The c‐KBP gene is at least 13 kilobases long and contains 11 exons interrupted by 10 introns. Primer extension and RNase protection studies identified a major trranscription initiation site located 117 bases upstream from the initiation methionine codon ATG. Consensus TATA and CCAAT sequences were detected in the putative promoter region. The structure of the c‐KBP gene is strikingly different from that of other members of neurotransmitter‐gated ion‐channels (cloned at present) although the topology of c‐KBP consists of four membrane‐spanning domains, a structural characteristics of ionotropic receptor subunits. The c‐KBP gene was found to be expressed at high levels in chick cerebellar Bergmann glia and at extremely low levels in the forebrain. The limited expression of the c‐KBP gene raises important questions concerning the mechanisms governing the regulation of c‐KBP gene transcription.
H+‐translocating ATPase and Na+/H+ antiport activities in the plasma membrane of the marine alga Platymonas viridis
Authors: Popova Larisa G. , Balnokin Yurii V.
Published: July 2002

Proton transport by the vanadate‐sensitive ATPase in plasma membrane (PM) vesicles from the marine unicellular microalga Platymonas viridis was investigated. The ATP‐dependent generation of ΔpH across the membranes of PM vesicles was followed by the changes in the absorbance of the aminoacridine probe. Acridine orange, Na+ caused the decay of ΔpH generated by the ATPase, the rate of the decay being dependent on the concentrations of Na+ added. The phenomenon was specific for Na+. Amiloride inhibited Na+‐dependent ΔpH decay. The experiments support the idea of a Na+‐extruding mechanism (H+‐translocating ATPase plus Na+/H+ antiporter) operating in the PM of marine alga Pl. viridis.
Peptide synthesis by chymotrypsin in frozen solutions
Authors: Tõugu Vello , Meos Helle , Haga Mati , Aaviksaar Aavo , Jakubke Hans-Dieter
Published: July 2002

Nucleophilic efficiency of free amino acids in chymotrypsin‐catalyzed acyl transfer in ice at −18°C using ethyl esters of N‐maleyl‐l‐tyrosine and l‐tyrosine as the acyl group donors has been studied. Although the amino acids did not act as acyl acceptors in liquid water, the high yields of peptides were obtained in frozen solutions at pH 10.5 (before freezing). The efficiency of amino acids in the formation of the corresponding dipeptides depended on the substrate used, and decreased in the order Ser,Thr,Gln>Lys>Cit>Ala>Gly>Asn>Arg>Glu>Val>Orn>Asp (with no peptide formed with His, Leu, He and Pro) for N‐maleyl‐l‐tyrosine ethyl ester and Ser>Lys>Orn>Arg,Cit>Gln>Thr>Asn>Ala>Gly (with no peptide formed with Glu, Val, Asp, His, Leu, Ile and Pro) for l‐tyrosine ethyl ester.
Sarcoplasmic reticulum calcium ATP
Authors: Coan Carol , Jakobs Pia , Ji Ji-Ying , Murphy Alexander J.
Published: July 2002

The sarcoplasmic reticulum Ca2+‐ATPase loses hydrolytic activity and the ability to be phosphorylated by Pi following incubation with EDC [1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide]. 4 nmol of tempamine per mg SR protein can be coupled to either a glu or an asp side chain through the EDC reaction. Mg2+ protects against loss of activity and tempamine labeling with a mid‐point of about 3 mM in the absence of Ca2+. This is similar to the K
d for a Mg2+ that serves as a cofactor in enzyme phosphorylation. The Mg2+ protection constant is lowered by an order of magnitude when Ca2+ is bound to the transport sites. It is suggested that control of the Mg2+ binding site affinity may be part of the mechanism of enzyme activation by Ca2+.
Biosynthesis and secretion of a precursor of nisin Z by Lactococcus lactis, directed by the leader peptide of the homologous lantibiotic subtilin from Bacillus subtilis
Authors: Kuipers Oscar P. , Rollema Harry S. , de Vos Willem M. , Siezen Roland J.
Published: July 2002

The DNA sequence encoding the leader peptide of the lantibiotic subtilin from Bacillus subtilis was fused to the sequence encoding pronisin Z, and this hybrid gene was expressed in a Lactococcus lactis strain that produces nisin A. This strain simultaneously secreted nisin A and a protein of approximately 6 kDa. Amino acid sequencing of the purified 6 kDa protein and structural analysis of its main tryptic fragment by two‐dimensional 1H‐NMR showed that it consists of the unmodified leader peptide of subtilin, without the N‐terminal methionine residue, linked to a fully matured nisin Z part. The hybrid protein and its main tryptic fragment [ITPQ]‐nisin Z, showed at least 200‐fold lower antimicrobial activities than nisin Z against three different indicator strains.
FAD‐linked glycerophosphate dehydrogenase deficiency in pancreatic islets of mice with hereditary diabetes
Authors: Sener Abdullah , Herberg Lieselotte , Malaisse Willy J.
Published: July 2002

The mitochondrial enzyme FAD‐linked glycerophosphate dehydrogenase plays a key role in the glucose‐sensing device of the insulin‐producing pancreatic B‐cell. Its activity was found to be decreased in islet, but not liver, homogenates of BL/Ks‐db/db mice, in which diabetes mellitus represents an inherited disease. The decreased activity of FAD‐linked glycerophosphate dehydrogenase contrasted with a normal activity of glutamate dehydrogenase and 2‐ketoglutarate dehydrogenase in the islets of db/db mice. It is proposed that a site‐specific defect of FAD‐linked glycerophosphate dehydrogenase in the pancreatic B‐cell might represent a far‐from‐uncommon causal or contributing factor in the pathogenesis of non‐insulin‐dependent diabetes mellitus.
Liver‐enriched HNF‐3α and ubiquitous factors interact with the human transferrin gene enhancer
Authors: Augé-Gouillou Corinne , Petropoulos Isabelle , Zakin Mario M.
Published: July 2002

The human transferrin gene enhancer is organized in two domains. Domain A contains a single enhanson designated 1a. Domain B contains four enhansons named Ib, II, III and IV. We demonstrate here that the liver‐enriched transcription factor HNF‐3α interacts with enhanson Ia and that enhansons Ib and IV are binding sites for members of the NF1 family. In addition, enhansons II and III seem to be respectively the targets for the AP4 protein and for EIII, a factor not yet completely identified. Analysis of mutated enhancer regions establishes that each enhanson is required for full enhancer activity and that the proteins binding to enhansons II, III and IV may interact within a multiprotein complex. This enhancer region presents no activity in the Sertoli cells of testis, where transferrin is also synthesized. We demonstrate that in Sertoli cells, the members of the HNF‐3 family are not expressed; this fact may account for the inactivity of the enhancer in these cells.
Copper pumping ATPases: Common concepts in bacteria and man
Authors: Solioz Marc , Odermatt Alex , Krapf Reto
Published: July 2002

Recently, four genes encoding putative copper pumping ATPases have been cloned from widely different sources: two genes from Enterococcus hirae that are involved in copper metabolism and two human genes that are defective in the copper‐related Wilson and Menkes disease. The predicted gene products are P‐type ATPases. They exhibit extensive sequence similarity and appear to be members of a new class of ATP driven copper pumps involved in the regulation of cellular copper.
Postnatal changes in rhodamine‐123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by atp
Authors: Almeida Angeles , Lopez-Mediavilla Casilda , Orfao Alberto , Medina José M.
Published: July 2002

The incubation of term fetus mitochondria with ATP mimicked in vitro the increase in the respiratory control index and in the percentage of the rhodamine‐123‐low fluorescence population that occurred in vivo immediately after birth, suggesting that both phenomena are closely associated. The administration of streptomycin inhibited the increase in the percentage of the low fluorescence population that occurred immediately after birth, while the administration of cycloheximide even reversed these changes. These results suggest that the in vivo interconversion between mitochondrial forms depends on both cytosolic and mitochondrial protein synthesis.
Na+‐driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H+‐driven ATP synthesis by rhodamine 6G
Authors: Šmigán Peter , Majerník Alan , Greksák Miloslav
Published: July 2002

Rhodamine 6G (3 μM) effectively inhibited ΔpH‐driven ATP synthesis in Methanobacterium thermoautotrophicum while ΔpNA‐driven ATP synthesis was not affected by it. Rhodamine 6G inhibited Mg2+‐stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor. Methanogenesis‐driven ATP synthesis at pH 6.8 of the cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl. On the other hand, the methanogenesis‐driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl. The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl. These results indicate that sodium‐motive force‐driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively at alkaline conditions and it might be the sole ATP synthesizing system when the proton motive force‐supported ATP synthesis is inhibited by rhodamine 6G.
Heat‐shock response in a yeast tps1 mutant deficient in trehalose synthesis
Authors: Argüelles Juan Carlos
Published: July 2002

Exponential cells of the Saccharomyces cerevisiae tps1 mutant underwent a rapid loss of viability upon a non‐lethal heat exposure (from 28 to 42°C). However, a further more severe heat stress (52.5°C 5 min) induced an increase in the fraction of viable cells. This mutant can not synthesize trehalose either at 28° C or at 42°C due to the lack of a functional trehalose‐6P synthase complex. In control experiments carried out with the wild‐type W303‐1 B, heat‐stressed exponential phase cultures grown on YPgal at 28°C acquired thermotolerance to a higher extent than identical cultures grown on YPD, although in both cultures the level of stored trehalose was negligible. These data suggest that the bulk of trehalose accumulated in yeast upon mild heat treaments is not sufficient to account for the acquisition of thermotolerance.
The role of lysine 99 of Thiobacillus versutus cytochrome c‐550 in the alkaline transition
Authors: Ubbink M. , Warmerdam G.C.M. , Campos A.P. , Teixeira M. , Canters G.W.
Published: July 2002

The methionine ligand of the heme iron in ferricytochrome c‐550 from Thiobacillus versutus is replaced by another residue at high pH. This transition is similar to the alkaline transition in mitochondrial cytochrome c. To investigate the possible role of lysine 99 in this process, this residue has been mutated to a glutamate. The mutation causes the apparent pKa of the transition to decrease from 11.2 in wild type to 10.8 in Lys99Glu cytochrome c‐550. This destabilization of the native form is ascribed to the absence of the hydrogen bond between the ε‐amine group of Lys99 and the carbonyl of Lys54 in the mutant protein. The 1H‐NMR spectrum of Lys99Glu ferricytochrome c‐550 at alkaline pH still shows resonance positions of the heme methyl peaks that are characteristic of the alkaline form. These results strongly suggest that Lys99 does not act as a ligand in the high‐pH form, contrary to the case of yeast iso‐1‐cytochrome c. Evidence has been presented that in the latter protein the homologous Lys79 can act as a ligand in the alkaline form [1993, J. Am. Chem. Soc. 115, 7507–7508]. In the EPR spectrum of Lys99Glu cytochrome c‐550 the species with Met‐His coordination (gz = 3.27) is replaced by two forms with gz = 3.45 and 3.20 in the alkaline form (pH ≥ 10.6). At pH > 11 yet another form is observed with g‐values 2.87, 2.18 and 1.60, tentatively identified as a species with a lysine‐histidinate coordination of the heme iron.
P‐glycoprotein‐mediated efflux of hydroxyrubicin, a neutral anthracycline derivative, in resistant K562 cells
Authors: Borrel Marie-Nicole , Fiallo Marina , Priebe Waldemar , Garnier-Suillerot Arlette
Published: July 2002

Hydroxyrubin (OH‐Dox), a neutral doxorubicin derivative that is only slightly cross‐resistant to doxorubicin (Dox), can be actively pumped out of resistant K562 cells by P‐glycoprotein (P‐gp). This efflux is saturable and can be inhibited by verapamil. The Michaelis constant is equal to 2 ± 0.5 μM. However, the efficiency of P‐gp in pumping out the drugs is 2.5 times less for OH‐Dox than for Dox. This shows that in order to be pumped out by P‐gp a molecule does not necessarily have to have a basic center. The mean influx coefficient for the drug is 5 times higher for OH‐Dox than for Dox. In conclusion, the degree of resistance of analogs is related not only to their ability to be recognized and transported by P‐gp but also, and probably essentially, to their kinetics of uptake. Both parameters have to be taken into account in the rational design of new compounds capable of overcoming multidrug resistance.
1α‐Hydroxyvitamin D3 inhibits Type II collagen‐induced arthritis in rats
Authors: Tsuji Michiko , Fujii Katsuyuki , Nakano Toshiaki , Nishii Yasuho
Published: July 2002

The effects of 1α‐hydroxyvitamin D3 and 24,25‐dihydroxyvitamin D3 on Type II collagen‐induced arthritis in rats, an experimental model of rheumatoid arthritis, were examined. Oral administration of 1α‐hydroxyvitamin D3 significantly suppressed the incidence of arthritis and inhibited hind paw swelling. The level of anti‐Type II collagen antibodies was decreased in the 1α‐hydroxyvitamin D3 treated‐group. In contrast, 24,25‐dihydroxyvitamin D3, indomethacin, and gold had no effect on either the incidence of arthritis or the antibody levels. These findings demonstrate a beneficial effect of 1α‐hydroxyvitamin D3 on Type II collagen‐induced arthritis in rats and indicate that it has an antirheumatic effect.
Tissue specific membrane association of α1T, a truncated form of the α1 subunit of the Na pump
Authors: Alien Julius C. , Pressley Thomas A. , Odebunmi Timothy , Medford Russell M.
Published: July 2002

We have assessed the Na pump α‐subunit isoform content utilizing site directed antibodies in two vascular smooth muscle (VSM) preparations known to contain functional Na pump sites, VSM microsomal fractions (Na+,K+‐ATPase) and intact primary confluent cells (ouabain inhibited 86Rb uptake). A comparison of isoform content was made with kidney microsomes. Both VSM and kidney microsomes contained a full length α1 subunit (~100 kDa) as well as a truncated subunit, α1T (~66 kDa). SDS treatment of VSM microsomes effected an increase in Na+,K+‐ATPase and a retention of α1T. SDS treated kidney microsomes retained the α1 isoform and Na+,K+‐ATPase. Confluent VSM cells showed no detectable α1T, only α1T. In the absence of detectable full length α1, the α1T protein may represent a functional Na pump component in canine VSM.
Flash‐induced electrogenic reactions in the SA(L223) reaction center mutant in Rhodobacter sphaeroides chromatophores
Authors: Bibikov Sergei I. , Bloch Dmitry A. , Cherepanov Dmitry A. , Oesterhelt Dieter , Semenov Alexey Yu.
Published: July 2002

The charge transfer events in the SA(L223) reaction center mutant Rhodobacter sphaeroides chromatophores were investigated by direct electrometry. Besides the primary charge separation, the small stigmatellin‐sensitive electrogenic reaction due to the electron transfer from the primary to the secondary quinone acceptor in the reaction center complex was observed after the first flash. The second flash‐induced electrogenic phase of the secondary quinone protonation and subsequent electrogenic reactions of the cytochrome bc
1 complex were much slower than those in chromatophores of the wild type. It is suggested that replacement of Ser‐L223 by Ala impairs both specific proton‐conducting pathways leading to the secondary quinone QB.
The three‐dimensional structure of the complex of proteinase K with its naturally occurring protein inhibitor, PKI3
Authors: Pal Gour P. , Kavounis Constantin A. , Jany Klaus D. , Tsernoglou Demetrius
Published: July 2002

Proteinase K forms a 1:1 stable complex with its naturally occurring protein inhibitor, PKI3. The crystal structure of this complex has been determined by a combination of molecular replacement and single isomorphous replacement methods. The model comprises all of the 459 residues: 279 for proteinase K and 180 for PKI3, and it was refined to an R‐factor of 19.2% at a resolution of 2.5 Å. Association of these two molecules in the complex indicates the binding of PKI3 in the substrate recognition site of the enzyme. The active serine residue of proteinase K in this complex possesses a somewhat different configuration to that found in its native structure and hence renders the enzyme inactive.
Processed products of the hevein precursor in the latex of the rubber tree (Hevea brasiliensis)
Authors: Soedjanaatmadja Ukun M.S. , Subroto Toto , Beintema Jaap J.
Published: July 2002

The 20 kDa precursor of hevein and its C‐terminal 14 kDa domain have been isolated. Sequence analysis of the C‐terminal tryptic peptides of these proteins and comparison with the cDNA sequence indicate that they represent mature forms from which a C‐terminal propeptide, possibly involved in vacuolar targeting, has been removed. The molar ratio of hevein to the C‐terminal domain in the lutoid‐body fraction of rubber latex is about 30:1. This indicates that not only the pre‐ and propeptides but also the 14 kDa domain are removed by proteolysis or other processes in the latex vessel after the processing of hevein has taken place.
Interleukin‐4 upregulates the heat shock protein Hsp90α and enhances transcription of a reporter gene coupled to a single heat shock element
Authors: Metz Kurt , Ezernieks Juris , Sebald Walter , Duschl Albert
Published: July 2002

Transcription of the heat shock protein Hsp90α was strongly upregulated in human T‐cells by interleukin‐4 (IL‐4) and to a lesser extent by IL‐2, reaching peak levels after 2–3 days of stimulation. Heat shock proteins are induced within minutes under stress conditions, via heat shock factors (HSF), which activate heat shock elements (HSE). IL‐4, IL‐2 and IL‐13 upregulated transcription of a reporter gene coupled to a single HSE site and a minimal promoter. HSE may therefore be involved in cytokine induced heat shock gene transcription in the absence of cellular stress.
Immunolocalization and pharmacological relevance of oligopeptide transporter PepT1 in intestinal absorption of β‐lactam antibiotics
Authors: Sai Yoshimichi , Tamai Ikumi , Sumikawa Hiromi , Hayashi Kiyomi , Nakanishi Takeo , Amano Osamu , Numata Masayuki , Iseki Shoichi , Tsuji Akira
Published: July 2002

A polyclonal antibody (anti‐PepT1/C) was raised against the rabbit intestinal H+‐coupled oligopeptide transporter, PepT1. Anti‐PepT1/C detected 70–80‐kDa protein in crude membranes obtained from rabbit duodenum, jejunum and ileum. PepT1 was localized in the brush‐border of the absorptive epithelial cells by subcellular fractionation of membranes on a sucrose density gradient and by immunohistochemistry using light and electron microscopy. Transport activity for cephalosporins and dipeptide expressed in Xenopus laevis oocytes injected with total mRNA obtained from rabbit small intestine was eliminated completely by prehybridization of the mRNA with antisense oligonucleotide against the 5′‐coding region of rabbit PepT1 cDNA.
The SAAS granin exhibits structural and functional homology to 7B2 and contains a highly potent hexapeptide inhibitor of PC1
Authors: Cameron Angus , Fortenberry Yolanda , Lindberg Iris
Published: July 2002

Prohormone convertases (PCs) 1 and 2 are thought to mediate the proteolytic cleavage of many peptide precursors. Endogenous inhibitors of both PC1 and PC2 have now been identified; the 7B2 protein is a nanomolar inhibitor of PC2, while the novel protein proSAAS was recently reported to be a micromolar inhibitor of PC1 [Fricker et al. (2000) J. Neurosci. 20, 639–648]. We here report evidence that 7B2 and proSAAS exhibit several elements of structural and functional homology. Firstly, 26 kDa human, mouse and rat proSAAS, like all vertebrate 7B2s, contain a proline‐rich sequence within the first half of the molecule and also contain a C‐terminal 40 residue peptide (SAAS CT peptide) separated from the remainder of the protein by a furin consensus sequence. The SAAS CT peptide contains the precise sequence of a hexapeptide previously identified by combinatorial peptide library screening as a potent inhibitor of PC1, and the vast majority of the inhibitory potency of proSAAS can be attributed to this hexapeptide. Further, like the 7B2 CT peptide, SAAS CT‐derived peptides represent tight‐binding competitive convertase inhibitors with nanomolar potencies. Lastly, recombinant PC1 is able to cleave the proSAAS CT peptide to a product with a mass consistent with cleavage following the inhibitory hexapeptide. Taken together, our results indicate that proSAAS and 7B2 may comprise two members of a functionally homologous family of convertase inhibitor proteins.
Changes in intramitochondrial cardiolipin distribution in apoptosis‐resistant HCW‐2 cells, derived from the human promyelocytic leukemia HL‐60
Authors: Garcia Fernandez Maria , Troiano Leonarda , Moretti Laura , Pedrazzi Jessica , Salvioli Stefano , Castilla-Cortazar Inma , Cossarizza Andrea
Published: July 2002

Using a cytofluorimetric approach, we studied intramitochondrial cardiolipin (CL) distribution in HCW‐2 cells, an apoptosis‐resistant clone of human HL‐60 cells. In HL‐60, about 50% of total CL is distributed in the outer leaflet of mitochondrial inner membrane, while in HCW‐2 a significantly higher amount of CL (about 65%) is in that site. In basal conditions, HSW‐2 cells also show a reduced mitochondrial membrane potential even if they are able to proliferate as the parental line. Taking into account the complex functions that CL plays in the regulation of mitochondrial activity, it is likely that HCW‐2 could produce ATP utilizing more glycolytic pathways rather than mitochondrial respiratory chain.
Down‐regulation of uncoupling protein‐3 and ‐2 by thiazolidinediones in C2C12 myotubes
Authors: Cabrero Àgatha , Alegret Marta , Sánchez Rosa M , Adzet Tomás , Laguna Juan C , Vázquez Manuel
Published: July 2002

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. We studied the direct effect of several peroxisome proliferator‐activated receptor (PPAR) ligands on UCP‐3 and UCP‐2 mRNA expression in C2C12 myotubes for 24 h. In the absence of exogenous fatty acids, treatment of C2C12 cells with a selective PPARα activator (Wy‐14,643) or a non‐selective PPAR activator (bezafibrate) did not affect the expression of UCP‐3 mRNA levels, whereas UCP‐2 expression was slightly increased. In contrast, troglitazone, a thiazolidinedione which selectively activates PPARγ, strongly decreased UCP‐3 and UCP‐2 mRNA levels. Another thiazolidinedione, ciglitazone, had the same effect, but to a lower extent, suggesting that PPARγ activation is involved. Further, the presence of 0.5 mM oleic acid strongly increased UCP‐3 mRNA levels and troglitazone addition failed to block the effect of this fatty acid. The drop in UCP expression after thiazolidinedione treatment correlated well with a reduction in PPARα mRNA levels produced by this drug, linking the reduction in PPARα mRNA levels with the down‐regulation of UCP mRNA in C2C12 myotubes after thiazolidinedione treatment.
Adrenergic agonists induce heterologous sensitization of adenylate cyclase in NS20Y‐D2L cells
Authors: Johnston Christopher A , Cumbay Medhane G , Vortherms Timothy A , Watts Val J
Published: July 2002

Adenylate cyclase activity in NS20Y cells expressing D2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both norepinephrine and epinephrine inhibited forskolin‐stimulated cyclic AMP accumulation via D2 receptors. Furthermore, chronic (18 h) activation of D2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (>10‐fold) in subsequent forskolin‐stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of Gαs or adenylate cyclase did not appear to alter noradrenergic agonist‐induced sensitization.
The Drosophila proteins Pelle and Tube induce JNK/AP‐1 activity in mammalian cells
Authors: Bacher Susanne , Großhans Jörg , Dröge Wulf , Schmitz M.Lienhard
Published: July 2002

The mammalian interleukin‐1 (IL‐1) signal transduction pathways display remarkable homology to the Toll signaling cascade in Drosophila. To address the question whether members of the Drosophila Toll pathway are functional in mammalian cells, inactive and constitutively active versions of the protein kinase Pelle and its regulator Tube were expressed in HeLa cells and tested for their impact on IL‐1‐dependent signaling events. The Drosophila proteins failed to induce the IL‐1‐responsive transcription factor, nuclear factor‐κB, but selectively activated the IL‐1‐regulated kinase, c‐Jun N‐terminal kinase (JNK), thus resulting in elevated AP‐1 activity. Activation of JNK/AP‐1 activity was seen upon expression of a Pelle mutant lacking its C‐terminal half or by a membrane‐bound and multimerised Tube protein, showing the functionality of the Drosophila proteins in mammalian cells.
Rat protein tyrosine phosphatase η physically interacts with the PDZ domains of syntenin
Authors: Iuliano Rodolfo , Trapasso Francesco , Samà Irene , Le Pera Ilaria , Martelli Maria Luisa , Lembo Francesca , Santoro Massimo , Viglietto Giuseppe , Chiariotti Lorenzo , Fusco Alfredo
Published: July 2002

The tyrosine phosphatase r‐PTPη is able to suppress the malignant phenotype of rat thyroid tumorigenic cell lines. To identify r‐PTPη interacting proteins, a yeast two‐hybrid screening was performed and an insert corresponding to the full‐length syntenin cDNA was isolated. It encodes a protein containing two PDZ domains that mediates the binding of syntenin to proteins such as syndecan, proTGF‐α, β‐ephrins and neurofascin. We show that r‐PTPη is able to interact with syntenin also in mammalian cells, and although syntenin is a tyrosine‐phosphorylated protein it is not a substrate of r‐PTPη. The integrity of both PDZ domains of syntenin and the carboxy‐terminal region of r‐PTPη are required for the interaction between syntenin and r‐PTPη.
Urotensin II induces hypertrophic responses in cultured cardiomyocytes from neonatal rats
Authors: Zou Yunzeng , Nagai Ryozo , Yamazaki Tsutomu
Published: July 2002

Urotensin II (UII), a cyclic neuropeptide, functions not only in the central nervous system but also in non‐neural systems including cardiovascular systems. In the present study we examined whether UII regulates hypertrophy in cardiomyocytes. The exposure of cultured cardiomyocytes from neonatal rats to UII dose‐dependently activated extracellular signal‐regulated kinases (ERKs), important molecules in the development of cardiac hypertrophy. ERK activation by UII at 100 nM peaked at 8 min after stimulation. UII markedly induced expression of specific genes encoding atrial natriuretic peptide and brain natriuretic peptide, and significantly increased amino acid incorporation into proteins. Incubation of cardiomyocytes with UII increased cell size and myofibril organisation. UII, then, might participate in cardiomyocyte hypertrophy.
Sialic acids in gastropods
Authors: Bürgmayr Sabine , Grabher-Meier Heidi , Staudacher Erika
Published: July 2002

The occurrence of N‐acetylneuraminic acid and N‐glycolylneuraminic acid residues in preparations of the slug Arion lusitanicus (Gastropoda) was determined by sodium dodecyl sulphate electrophoresis of the proteins followed by lectin blots stained with the sialic acid specific lectin from Maackia amurensis, by the sensitivity of this binding to sialidase from Clostridium perfringens, by specific fluorescent labelling of sialic acids with 1,2‐diamino‐4,5‐methylenedioxybenzene, by the determination of the sensitivity to sialate‐pyruvate‐lyase, by co‐migration with standards on high performance anion exchange chromatography with pulsed amperometric detection and by identification of the typical masses in the fragmentation patterns of the trimethylsilyl derivatives after gas chromatography. It is the first time sialic acids are identified in gastropods.
Role of Rac GTPase in the nuclear signaling by EGF
Authors: Kim Byung-Chul , Kim Jae-Hong
Published: July 2002

The role of Rac in epidermal growth factor (EGF)‐induced c‐fos serum response element (SRE) activation was examined in Rat‐2 fibroblast cells. By reporter gene analysis following transient or stable transfections with pEXV‐RacN17 encoding a dominant‐negative mutant of Rac, EGF‐induced activation of c‐fos SRE‐luciferase gene was shown to be selectively inhibited, suggesting that Rac activity is necessary for the full activation of SRE by EGF. Our further study to analyze the downstream mediator of Rac in EGF‐signaling cascade demonstrated that there is a functional link between Rac and phospholipase A2 (PLA2) activation and further that PLA2 mediates, at least partly, the Rac signaling to SRE. Together, our results point to a critical role of Rac and Rac‐activated PLA2 in the EGF‐signaling cascade to c‐fos SRE. We propose that `Rac‐PLA2' cascade is one of the major signaling pathways by which EGF stimulates c‐fos SRE.
Scavengase p20: a novel family of bacterial antioxidant enzymes
Authors: Wan Xiao-Yu , Zhou Yuan , Yan Zi-Ying , Wang Hai-Lin , Hou Yun-De , Jin Dong-Yan
Published: July 2002

A novel antioxidant enzyme designated scavengase p20 was identified in various pathogenic bacteria through database searching for sequences strikingly homologous to a recently discovered Escherichia coli thiol peroxidase p20. The direct biochemical evidence for the existence of scavengase p20 in Haemophilus influenzae, Streptococcus pneumoniae and Helicobacter pylori was provided by protein microsequencing and by in vitro assays for antioxidant activities. Overlapping genes encoding scavengase p20 and superoxide dismutase were recognized in H. pylori and their functional implications are discussed.
Mutations in the carboxy‐terminal part of IS30 transposase affect the formation and dissolution of (IS30)2 dimer
Authors: Olasz Ferenc , Farkas Tibor , Stalder Rolf , Arber Werner
Published: July 2002

The transposase of IS30 catalyses different transpositional rearrangements via the dimer (IS30)2 intermediate structure. Mutation analysis provides evidence that the C‐terminal part of IS30 transposase is required for the formation and dissolution of (IS30)2 dimer. C‐terminal mutants are also defective in transpositional fusion; however, this deficiency can be `suppressed' by addition of the final product of site‐specific dimerisation, the core (IS30)2 intermediate structure. The transposase part studied shows significant homologies in three highly conserved regions to proteins of IS30‐related mobile elements.
Improved enzymatic synthesis of a highly potent oligosaccharide antagonist of L‐selectin
Authors: Salminen Heidi , Ahokas Katja , Niemelä Ritva , Penttilä Leena , Maaheimo Hannu , Helin Jari , Costello Catherine E , Renkonen Ossi
Published: July 2002

The polylactosamine sLexβ1–3′(sLexβ1–6′)LacNAcβ1–3′(sLexβ1–6′)LacNAcβ1–3′(sLexβ1–6′)LacNAc (7) (where sLex is Neu5Acα2–3Galβ1–4(Fucα1–3)GlcNAc and LacNAc is Galβ1–4GlcNAc) is a nanomolar L‐selectin antagonist and therefore a potential anti‐inflammatory agent (Renkonen et al. (1997) Glycobiology, 7, 453). Here we describe an improved synthesis of 7. The octasaccharide LacNAcβ1–3′LacNAcβ1–3′LacNAcβ1–3′LacNAc (4) was converted into the triply branched undecasaccharide LacNAcβ1–3′(GlcNAcβ1–6′)LacNAcβ1–3′(GlcNAcβ1–6′)LacNAcβ1–3′(GlcNAcβ1–6′)LacNAc (5) by incubation with UDP‐GlcNAc and the midchain β1,6‐GlcNAc transferase activity of rat serum. Glycan 5 was enzymatically β1,4‐galactosylated to LacNAcβ1–3′(LacNAcβ1–6′)LacNAcβ1–3′(LacNAcβ1–6′)LacNAcβ1–3′(LacNAcβ1–6′)LacNAc (6). Combined with the enzymatic conversion of 6 to 7 (Renkonen et al., loc. cit.) and the available chemical synthesis of 4, our data improve the availability of 7 for full assessment of its anti‐inflammatory properties.
Regulation of insulin‐stimulated tyrosine phosphorylation of Shc and IRS‐1 in the muscle of rats: effect of growth hormone and epinephrine
Authors: Thirone Ana C.P , Paez-Espinosa Enma V , Carvalho Carla R.O , Saad Mario J.A
Published: July 2002

Insulin receptor substrate‐1 (IRS‐1) and Shc protein have the same binding site at the insulin receptor and compete in their association with the phosphorylated receptor. The present study demonstrates that a decrease in the level of muscle insulin receptor phosphorylation induced by chronic growth hormone (GH) treatment or acute epinephrine infusion is accompanied by a reduction in the level of IRS‐1 phosphorylation and in the association with phosphatidylinositol 3‐kinase. In contrast, no change is observed in insulin‐stimulated Shc tyrosine phosphorylation, or in the association of this substrate with Grb2. These data suggest that a reduction in insulin receptor phosphorylation may affect post‐receptor processes differentially by preserving the phosphorylation of some substrates and pathways, but not of others.
Genomic organization and chromosomal localization of the human peroxisomal membrane protein‐1‐like protein (PXMP1‐L) gene encoding a peroxisomal ABC transporter1
Authors: Holzinger Andreas , Roscher Adelbert A , Landgraf Pablo , Lichtner Peter , Kammerer Stefan
Published: July 2002

The cDNA of the peroxisomal membrane protein‐1‐like protein (PXMP1‐L, synonyms: PMP69, P70R), a novel peroxisomal ATP binding cassette transporter of yet unknown function, has recently been cloned. The best known peroxisomal member of this protein family is the adrenoleukodystrophy protein, defects of which are the underlying cause of X‐linked adrenoleukodystrophy (X‐ALD). Here we describe the complete exon‐intron structure (19 exons and 18 introns covering 16.0 kb) of the human PXMP1‐L gene, transcript variants, the localization on chromosome 14q24 by cytogenetic analysis and sequencing of the putative promoter region. PXMP1‐L has been proposed to play a role as a modifier in determining the phenotypic variations observed in X‐ALD. The data presented will enable sequence analysis of the PXMP1‐L gene in X‐ALD patients and facilitate the analysis of PXMP1‐L function.
The shrimp hyperglycemic hormone‐like neuropeptide is encoded by multiple copies of genes arranged in a cluster1
Authors: Gu P.-L. , Chan S.-M.
Published: July 2002

The crustacean hyperglycemic hormone (CHH) plays an important role in the regulation of glucose metabolism. We have cloned and sequenced several cDNAs encoding the preproCHH‐like of the shrimp, Metapenaeus ensis. The preproCHH‐like peptide of the shrimp consists of a signal peptide, a CHH precursor‐like peptide (CPRP) and the CHH‐like peptide. Comparative analysis revealed that the signal peptide and the CPRP of the shrimp peptide are the shortest among all the CHHs reported. MeCHH‐like is expressed in the eyestalk, but it is not expressed in the heart, hepatopancreas, muscle, nerve cord and pre‐hatch embryo. To study the structural organization of the shrimp CHH‐like gene, we have screened the genomic DNA library constructed from one shrimp. Three groups of overlapping genomic clones have been isolated. The results from both genomic Southern blot analysis and library screening indicate that the shrimp genome contains at least six copies of the CHH‐like genes arranged in a cluster on the chromosome. The size of the CHH‐like genes is 1.5–2.1 kb. DNA sequence determinations indicate that the CHH‐like genes share 98–100% amino acid sequence identity. There are three exons and two introns in each CHH‐like gene. The first intron separates the signal peptide and the second intron separates the mature peptide in the coding region. The 150–200 bp of the upstream 5′ flanking region of the CHH‐like genes contains promoters with characteristics similar to most eukaryotic genes. Several putative cis‐acting elements are also identified in the first 400 bp 5′ end upstream region. The organization of the shrimp CHH‐like genes is similar to that of the molt inhibiting hormone gene of the same shrimp and the crab, Charybdis feriatus.
A 69 bp fragment in the pyrroline‐5‐carboxylate reductase promoter of Arabidopsis thaliana activates minimal CaMV 35S promoter in a tissue‐specific manner
Authors: Hua Xuejun , van de Cotte Brigitte , Van Montagu Marc , Verbruggen Nathalie
Published: July 2002

The Arabidopsis thaliana gene that encodes pyrroline‐5‐carboxylate reductase (At‐P5R), the last enzyme in proline biosynthesis in A. thaliana, is developmentally regulated and is highly expressed in cells that divide rapidly or undergo changes in osmotic potential. A 69 bp region (P69; −120 to −51) has previously been identified in a 5′ deletion analysis of the At‐P5R promoter to be necessary for the basal expression. Here, the essential role of P69 for tissue‐specific expression of At‐P5R is demonstrated by loss‐ and gain‐of‐function experiments.
Regulation of mouse somatostatin receptor type 2 gene expression by glucocorticoids
Authors: Kraus Jürgen , Wöltje Michael , Höllt Volker
Published: July 2002

Somatostatin is a regulatory peptide with important inhibitory functions. Its effects are mediated by five receptors, sst1–sst5. Previous studies revealed that sst2 contains three alternative, tissue specific promoters. Here, molecular mechanisms for the regulation of sst2 transcription by glucocorticoids were investigated. Reporter gene assays in NG 108‐15 cells and electrophoretic mobility shift assays revealed that a glucocorticoid responsive element at position −1044 on the second promoter mediates dexamethasone induction. These findings, the existence of a cAMP response element and the tissue dependent activity (brain, pituitary and gastrointestinal tissues) indicate the importance of the second sst2 gene promoter.
Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in Synechocystis PCC 6803
Authors: Hisbergues Michael , Jeanjean Robert , Joset Françoise , Tandeau de Marsac Nicole , Bédu Sylvie
Published: July 2002

In Synechocystis PCC 6803 as in other cyanobacteria, involvement of protein PII in the co‐regulation of inorganic carbon and nitrogen metabolism was established based on post‐translational modifications of the protein resulting from changes in the carbon/nitrogen regimes. Uptake of bicarbonate and nitrate in response to changes of the carbon and/or nitrogen regimes is altered in a PII‐null mutant, indicating that both processes are under control of PII. Modulation of electron flow by addition of methyl viologen with or without duroquinol, or in a NAD(P)H dehydrogenase‐deficient mutant, affects the phosphorylation level of PII. The redox state of the cells would thus act as a trigger for PII phosphorylation.
Hypoosmolarity influences the activity of transcription factor NF‐κB in rat H4IIE hepatoma cells
Authors: Michalke Manuela , Cariers Alexandra , Schliess Freimut , Häussinger Dieter
Published: July 2002

The influence of anisoosmolarity on NF‐κB binding activity was studied in H4IIE rat hepatoma cells. Hypoosmolarity induced a sustained NF‐κB binding activity whereas the hyperosmotic NF‐κB response was only minor. Hypoosmotic NF‐κB activation was accompanied by degradation of the inhibitory IκB‐α. Protein kinase C, PI3‐kinase, reactive oxygen intermediates and the proteasome apparently participate in mediating the hypoosmotic effect on NF‐κB. Hypoosmolarity plus PMA induced, amplified and prolonged IκB‐α degradation and NF‐κB binding activity. Transforming growth factor β‐induced apoptosis was diminished by hypoosmolarity. However, this anti‐apoptotic effect was probably not related to NF‐κB activation.