bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of RII‐binding proteins in the mollusc Mytilus galloprovincialis
Authors: Cao Jesús , Fernández Montserrat , Ramos-Martínez J.Ignacio , Antonio Villamarín J
Published: July 2002

Several proteins with M

r
70 kDa from various tissues of the sea mussel Mytilus galloprovincialis were specifically recognized in vitro by the regulatory subunit (type RIIα) of cAMP‐dependent protein kinase (cAPK) from porcine heart. However, none of these proteins interacted with the regulatory subunit of cAPK from the mollusc itself. The results suggest that, unlike mammalian RII, regulatory subunit from mussel lacks the specific residues responsible for interaction with R‐binding proteins. Consequently, the identified molluscan RIIα‐binding proteins should play a distinct role from cAPK anchoring.
Basal phosphorylation of μ opioid receptor is agonist modulated and Ca2+‐dependent
Authors: Wang Zaijie , Arden James , Sadee Wolfgang
Published: July 2002

The μ opioid receptor was shown to be phosphorylated at a basal rate in the absence of agonist, measured in permeabilized HEK293 cells transfected with an epitope tagged μ receptor (EE‐μ) [Arden, J., Segredo, V., Wang, Z., Lameh, J. and Sadée, W. (1995) J. Neurochem. 65, 1636–1645]. In the present study, basal phosphorylation was found to be Ca2+ dependent; however, several inhibitors of protein kinase C and Ca2+‐cahnodulin dependent kinases failed to affect basal μ receptor phosphorylation. Thus, the basal μ receptor phosphorylating activity differed from the main kinases involved in receptor regulation. The general kinase inhibitor H7 (100 μM) suppressed basal μ receptor phosphorylation. Pretreatment with the agonist morphine, followed by drug removal, resulted in a sustained increase of basal It receptor phosphorylation. The gradual agonist dependent modulation of basal μ receptor phosphorylation suggests a novel regulatory mechanism which may play a role in narcotic tolerance and dependence.
Regulation of the gluconeogenic phosphoenolpyruvate carboxykinase and the glycolytic aldolase A gene expression by O2 in rat hepatocyte cultures. Involvement of hydrogen peroxide as mediator in the response to O2
Authors: Kietzmann Thomas , Freimann Stefanie , Bratke Jutta , Jungermann Kurt
Published: July 2002

Heme proteins acting as oxidases which produce H2O2 have been proposed to function as O2 sensors. In order to find out whether the modulation by O2 of PCK gene activation and the stimulation of the ALD A gene by venous O2 operate via H2O2, the effects of different concentrations of H2O2 and catalase as H2O2 scavenger were studied in rat hepatocyte cultures under different O2 tensions. Primary hepatocytes were treated with 0.1 nM glucagon, 50 μM H2O2 and/or 100 μg/ml catalase each at arterial O2 or venous pO2. PCK mRNA was induced by glucagon maximally under arterial O2 and only half maximally under venous O2. ALD A mRNA was induced only by venous O2. H2O2 enhanced the induction of PCK mRNA to similar levels under both O2 tensions and the induction of ALD A mRNA under venous O2 was completely inhibited. Addition of catalase antagonized the actions of H2O2 completely. These findings support the hypothesis that an H2O2‐generating heme protein is involved in the O2 sensing system regulating gluconeogenic and glycolytic gene expression in response to O2.
Salvage of catabolic products in ganglioside metabolism: a study on rat cerebellar granule cells in culture
Authors: Riboni Laura , Bassi Rosaria , Prinetti Alessandro , Tettamanti Guido
Published: July 2002

Cerebellar granule cells in culture were subjected to a pulse (0.5–4 h)‐chase (0–4 h) of 10−6 M [3H]ganglioside GM1 carrying the radioactive label at the level of NeuAc ([3H‐NeuAc]GM1), Sph ([3H‐Sph]GM1) or Gal ([3H‐GallGMl) and the formed [3H]metabolites were determined. With all forms of [3H]GM1, there was formation of [3H]catabolites, including [3H]H2O and [3H]biosynthetic products obtained by recycling of [3H]NeuAc, [3H]Sph and [3H]Gal released during intralysosomal ganglioside degradation (salvage processes). Much higher amounts of [3MH2O were produced from [3H‐Gal]GM1 than [3H‐Sph]GM1 and [3H‐NeuAc]GM1; conversely, more products from salvage processes (polysialogangliosides GD1a, GDlb, GTlb, O‐acetylated GT1b, protein‐bound radioactivity) were obtained with [3H‐NeuAc]GM1; than the two other forms of [3H]GM1, Liberated [3H]NeuAc produced 10‐fold less tritiated water and 10‐fold higher salvage products than [3H]Gal. Using [3H‐NeuAc]GM1; granule cells appeared to metabolize 7.7% of membrane‐incorporated exogenous GM1 per hour with a high degree of NeuAc recycling and the calculated metabolic half‐life was 6.5 h.
Contraceptive use during lactational amenorrhea
Authors: Hight‐Laukaran V. , Rutstein S.O. , Labbok M.H. , Ballard E.
Published: July 2002

Objective: The study uses data from nationally representative sample surveys in developing countries to estimate the overlap between lactational amenorrhea and contraceptive use during the first 6 months postpartum. Method: Secondary analyses of survey data were used to tabulate the proportion of the population in lactational amenorrhea among contraceptive users for all women, for postpartum women and for the country as a whole. Results: Among postpartum women, the proportion in lactational amenorrhea was particularly high in Africa and the Near East and lower in Latin America and the Caribbean where breast‐feeding practices have declined. The median duration of use for oral contraceptives is also presented as an aid to interpreting the significance of the findings. Conclusions: The significance of the findings is considered in the context of planning reproductive health services in the postpartum period. Decisions about timing of contraceptive use for postpartum women, while arrived at on an individual basis, also result from program strategies that focus counseling immediately postpartum or at a later interval, such as when menses resume. On a national level the impact of postpartum contraception policies on use of commodities may be substantial.
Effect of carbamazepine on agitation and emotional lability associated with severe dementia
Authors: Lemke MR
Published: July 2002

In an open prospective study, the stabilizing effects of carbamazepine on agitation, impulsivity and affective lability were investigated in 15 severely demented patients, who had failed to respond to prior neuroleptic medication. Because of leukopenia and allergic reactions in two subjects, carbamazepine treatment was discontinued. In nine patients a clinically relevant improvement was observed.
N‐terminal and core‐domain random mutations in human topoisomerase II α conferring bisdioxopiperazine resistance
Authors: Jensen Lars H , Wessel Irene , Møller Marianne , Nitiss John L , Sehested Maxwell , Jensen Peter B
Published: July 2002

Random mutagenesis of human topoisomerase II α cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF‐187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer >37, 37, 18, 14, and 19 fold resistance towards ICRF‐187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF‐187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non‐responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.
Catalytic activities of membrane‐type 6 matrix metalloproteinase (MMP25)
Authors: English William Roger , Velasco Gloria , Stracke Jan Olaf , Knäuper Vera , Murphy Gillian
Published: July 2002

This study describes the biochemical characterisation of the catalytic domain of membrane‐type 6 matrix metalloproteinase (MT6‐MMP, MMP25, leukolysin). Its activity towards synthetic peptide substrates, components of the extracellular matrix and inhibitors of MMPs was studied and compared with MT1‐MMP, MT4‐MMP and stromelysin‐1. We have found that MT6‐MMP is closer in function to stromelysin‐1 than MT1 and MT4‐MMP in terms of substrate and inhibitor specificity, being able to cleave type‐IV collagen, gelatin, fibronectin and fibrin. However, it differs from stromelysin‐1 and MT1‐MMP in its inability to cleave laminin‐I, and unlike stromelysin‐1 cannot activate progelatinase B. Our findings suggest that MT6‐MMP could play a role in cellular migration and invasion of the extracellular matrix and basement membranes and its activity may be tightly regulated by all members of the TIMP family.
Inhibition of endothelial cell migration by cerivastatin, an HMG‐CoA reductase inhibitor: contribution to its anti‐angiogenic effect
Authors: Vincent Loı̈c , Chen Wenming , Hong Li , Mirshahi Farrokh , Mishal Zohair , Mirshahi-Khorassani Tooran , Vannier Jean-Pierre , Soria Jeannette , Soria Claudine
Published: July 2002

Recent studies have suggested that inhibitors of 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase (statins) can play a role in protection against vascular risk, which is independent of cholesterol reduction. It could act by inhibiting the synthesis of isoprenoids (farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP)), which are respectively essential for membrane attachment and biological activity of GTPases Ras and RhoA. This study demonstrates that a statin (cerivastatin) inhibits angiogenesis. This effect was due to a decrease in endothelial cell locomotion which was reversed by GGPP. It was mainly related to delocalization of RhoA from cell membrane to cytoplasm, responsible for the disorganization of actin stress fibers. Furthermore, a decrease in MMP‐2 secretion, involved in cell invasion, was also observed. This effect is rather due to Ras inhibition as it was reversed by FPP. This anti‐angiogenic activity could explain the beneficial effect of statins on atherosclerosis and on cancer prevention as shown by clinical studies.
Angiotensin II type 1 receptor expression in human pancreatic cancer and growth inhibition by angiotensin II type 1 receptor antagonist
Authors: Fujimoto Yoshifumi , Sasaki Tamito , Tsuchida Akira , Chayama Kazuaki
Published: July 2002

We investigated the expression of angiotensin II type 1 receptor (AT1) in pancreatic cancer. Both AT1 mRNA and protein were expressed in human pancreatic cancer tissues and cell lines. Binding assays showed that pancreatic cancer cells have specific binding sites for angiotensin II and that binding could be eliminated by treatment with a selective AT1 antagonist in a dose‐dependent fashion. Surprisingly, the growth of cancer cells was significantly suppressed by treatment with antagonist, also in a dose‐dependent manner. These observations suggest AT1 plays an important role in pancreatic cancer growth. Furthermore, ligand‐induced inhibition of AT1 may be a useful therapeutic strategy.
LytB, a novel gene of the 2‐C‐methyl‐D‐erythritol 4‐phosphate pathway of isoprenoid biosynthesis in Escherichia coli
Authors: Altincicek Boran , Kollas Ann-Kristin , Eberl Matthias , Wiesner Jochen , Sanderbrand Silke , Hintz Martin , Beck Ewald , Jomaa Hassan
Published: July 2002

The mevalonate‐independent 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.
Exon 3 of the α folate receptor gene contains a 5′ splice site which confers enhanced ovarian carcinoma specific expression
Authors: Galmozzi Enrico , Tomassetti Antonella , Sforzini Sabrina , Mangiarotti Fabio , Mazzi Mimma , Nachmanoff Kiki , Elwood Patrick C. , Canevari Silvana
Published: July 2002

The human folate receptor (FR) is overexpressed in ovarian carcinoma. FR transcripts are heterogeneous due to the use of two promoters, P1 and P4, and alternative splicing of exon 3. RNase protection assay and RT‐PCR revealed higher levels of the transcripts that include exon 3 in lines and specimens from ovarian carcinoma. A P1–chloramphenicol acetyltransferase (CAT) construct containing exon 3 demonstrated efficient reporter expression only in ovarian carcinoma. 5′ and 3′ deleted variants of the P1–CAT construct were analyzed by RT‐PCR of the exogenous transcripts and reporter activity. A 5′ splice site and 35 bp downstream intronic region of exon 3 appeared to regulate enhanced FR expression in ovarian carcinoma.
Direct production of an activated matrix metalloproteinase‐9 (gelatinase B) from mammalian cells
Authors: Watanabe Yoshifumi , Hirakawa Koichiro , Haruyama Takahiro , Akaike Toshihiro
Published: July 2002

Matrix metalloproteinase‐9 (MMP‐9) is produced by the inactive proform and activated by a proteolytic process. However, it has not been reported to produce the active form directly from cells, which has hindered the research to elicit the physiological roles of this enzyme. In this study, we prepared mutant MMP‐9 containing the furin‐recognizing sequence in the prodomain and showed that the mutant MMP‐9 was secreted as the active form directly from CHO‐K1 cells and primary hepatocytes after the gene was transfected. The secreted MMP‐9 showed proteolytic activity without further activation and degraded collagen IV in vitro. In addition, the transfection of the gene into the liver resulted in the efficient expression of active MMP‐9 in the liver and the serum in vivo.
Complicated character of the M decay pH dependence in the D96N mutant is due to the two pathways of the M conversion
Authors: Radionov Alexey N. , Kalaidzidis Inna V. , Kaulen Andrey D.
Published: July 2002

At high ionic strength, the pH dependence of the M intermediate decay in a photocycle of the D96N mutant bacteriorhodopsin shows a complicated behavior which is found to be due to the coexistence of two pathways of the M conversion. The M decay which dominates at pH <5 is coupled to the proton uptake from the cytoplasmic surface and proceeds probably through the N intermediate. This pathway is inhibited by glutaraldehyde, the potent inhibitor of M decay in the wildtype bacteriorhodopsin and of the azide‐facilitated M decay in the D96N mutant. Another pathway of the M decay is predominant at pH > 5. This pathway is insensitive to glutaraldehyde and some other similar inhibitors (lutetium ions, sucrose and glycerol). On the other hand, it is sensitive to the pK changes of the group X (Glu‐204) in the outward proton pathway. Possibly, the M decay through this pathway represents a reverse H+ transport process (the proton uptake from the external surface) and proceeds via the L intermediate.
Coexistence of somatostatin receptor subtypes in the human neuroblastoma cell line LA‐N‐2
Authors: Nilsson Lars , Folkesson Ronnie
Published: July 2002

Five distinct human somatostatin receptor subtypes have recently been cloned and characterized. Previous studies have suggested that these receptor subtypes might display coexistent localization, based on in situ hybridization or immunoblockage experiments. Here we provide evidence for coexistence of somatostatin receptor subtypes 2 and 5 in the human neuroblastoma cell line LA‐N‐2, using a combined approach with RT‐PCR and receptor binding studies with somatostatin analogues. Somatostatin receptor subtypes simultaneously localized to a single cell might serve distinct functions in terms of targeting to different intraneuronal compartments or subtype specificity against so far unidentified somatostatin‐related peptides.
Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase
Authors: Ohta Shozo , Usami Satoru , Ueki Jun , Kumashiro Takashi , Komari Toshihiko , Burnell Jim N
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Characterisation of neprilysin (EC 3.4.24.11) S2′ subsite
Authors: Dion Natalie , Cohen Paul , Crine Philippe , Boileau Guy
Published: July 2002

Neprilysin is a neutral peptidase that cleaves small peptide substrates on the amino‐side of hydrophobic amino acid residues. In the present study, we have used inhibition of non‐mutated and mutated enzymes with dipeptide inhibitors and hydrolysis of the substrate [Leu5, Arg6]enkephalin in order to evaluate the contribution of the S2′ subsite to substrate and inhibitor binding. Our results suggest that (1) Arg‐102 and Asn‐542 provide major contributions to the interaction of the enzyme with the P2′ residue of the substrate, (2) the S2′ subsite is vast and can accommodate bulky side chains, and (3) Arg‐102 restricts access to the S2′ subsite to some side chains such as arginine.
In vivo translational efficiency of different hepatitis C virus 5′‐UTRs
Authors: Buratti E. , Gerotto M. , Pontisso P. , Alberti A. , Tisminetzky S.G. , Baralle F.E.
Published: July 2002

Initiation of translation in hepatitis C virus (HCV) is dependent on the presence of an internal ribosome entry site (IRES) contained in its 341‐nt‐long 5′‐untranslated region (UTR). This region is very conserved among different isolates and has been used to classify HCV isolates in six different genotypes. These genotypes differ in nucleotide sequence that generally preserve the IRES structure. However, the small differences seen may be biologically and clinically significant as the HCV strains seem to differ from each other in several important ways, such as the behaviour of the viral infection and the response to interferon therapy. Therefore, differences in translational initiation efficiency amongst the various genotypes could provide an explanation for these phenomena. Using a bicistronic expression system we have compared the in vivo translational ability of the three most common European genotypes of HCV (1, 2, and 3). The results show that genotype 3 is less able than 1 and 2 to promote translation initiation. In addition, by site‐directed mutagenesis of the sequence of the IRES domain III apical stem loop structure, we have shown that the conservation of the primary nucleotide sequence and not only the structure, is important for the conservation of IRES function.
Relevance of Na,K‐ATPase to local extracellular potassium homeostasis and modulation of synaptic transmission
Authors: Gloor Sergio M
Published: July 2002

The ion gradients generated by the Na,K‐ATPase are essential for Na+‐coupled transport systems, osmoregulation and restoration of ion concentrations in excitable tissues. Indirectly, the sodium pump controls intracellular Ca2+ concentration through the Na/Ca exchanger. In the nervous system various neurotransmitters can modulate Na,K‐ATPase activity. The great diversity of Na,K‐ATPase subunit isoforms, their complex spatial and temporal regulation of expression and their cellular localisation imply a functional role of the sodium pump in different regulatory pathways. Among these, potassium homeostasis and modulation of synaptic transmission are discussed here.
pmg‐1, a novel gene specifically expressed during the invasive growth phase of the mammary gland at puberty
Authors: Kuhn Franziska , Andres Anne-Catherine
Published: July 2002

The acquisition of invasive properties is a crucial event during carcinogenesis, determining the clinical outcome. The mammary gland at puberty provides an ideal model for investigating the induction and control of invasive growth. During this growth phase, the mammary epithelium participates in a normal, hormonally controlled invasive penetration into the stroma. We have applied the differential display method to search for genes specifically activated during this developmental stage. We have identified and molecularly characterized a novel
p
ubertal
m
ammary
g
land specific gene, pmg‐1. This gene is conserved in mammals and encodes a protein of 19.9 kDa. Northern blotting and in situ hybridization revealed that pmg‐1 expression was exquisitely restricted to the epithelium at early puberty. To our knowledge this represents the first isolation of a gene specifically associated with the induction of mammary epithelial invasiveness at puberty.
Proteolytic cleavage of Pseudomonas aeruginosa exotoxin A in the presence of lipid bilayers of different composition
Authors: Nordera Paola , Menestrina Gianfranco
Published: July 2002

Pseudomonas aeruginosa exotoxin A (ETA) must be proteolytically nicked by furin at Arg279 before being translocated into the cytosol of target cells. A similar cleavage can also be obtained with trypsin. Using this assay we could show that the interaction with lipid bilayers can strongly influence the extent of nicking. We found that in the presence of vesicles containing negatively charged lipids ETA is cleaved into its two fragments A and B at enzyme concentrations ∼50 times lower, or at pH values higher by 1.5 units, than in the absence of lipids. We suggest that the interaction with the lipid bilayer of the positively charged loop containing Arg279 provides the energy for its partial unfolding and makes it more accessible for proteolysis.
Regulation of osteopontin expression in rat mesangial cells
Authors: Pröls Felicitas , Heidgress Dorothe , Rupprecht Harald D , Marx Martin
Published: July 2002

Hypercellularity and accumulation of extracellular matrix are common responses of renal glomeruli to inflammatory stimuli. Using the differential display approach, we compared the gene expression patterns of proliferating and differentiating rat mesangial cells in two‐ and three‐dimensional cultures. Osteopontin, an extracellular matrix protein, was found to be transcribed, synthesized, and secreted by rat mesangial cells. Osteopontin transcription was not associated with cell proliferation and was found to be FCS‐inducible in proliferating cells. Osteopontin expression was independent of exogenously supplied FCS in differentiating cells. The presented data indicate that osteopontin is differentially regulated in proliferating and differentiating mesangial cells.
The cytochrome subunit structure in the photosynthetic reaction center of Chromatium minutissimum
Authors: Chamorovsky S.K , Zakharova N.I , Remennikov S.M , Sabo Ya , Rubin A.B
Published: July 2002

Gel‐electrophoretic assay revealed that the photosynthetic reaction center (RC) of Chromatium minutissimum, in contrast to the well‐known RC Rhodopseudomonas viridis, consists of five rather than four subunits with molecular masses of 37, 34, 25, 19, and 17 kDa. The 37‐ and 19‐kDa subunits are stained with tetramethylbenzidine for the cytochrome c hemes. Absorption spectra show that the concentration of reduced cytochromes in the C. minutissimum RC poised at redox potential of −150 mV (fully reduced pool of hemes) is about three times more than in the C. minutissimum RC poised at redox potential of +260 mV (only high‐potential hemes are reduced). The results of redox titration of absorption changes at the cytochrome c α‐band are most appropriately approximated by a six‐component theoretical curve with the midpoint potentials of E
m1=390 mV, E
m2=320 mV, E
m3=210 mV, E
m4=100 mV, E
m5=20 mV, and E
m6=−50 mV. Possible functions of the cytochromes with the midpoint potentials 210 and 100 mV, which have not been found in purple bacteria before, are discussed.
Mutant T7 RNA polymerase is capable of catalyzing DNA primer extension reaction
Authors: Rusakova E.E , Tunitskaya V.L , Memelova L.V , Kochetkova S.V , Kostyuk D.A , Kochetkov S.N
Published: July 2002

The mutant T7 RNA polymerase (T7 RNAP), containing two substitutions (Y639F, S641A) was earlier shown to utilize both rNTP and dNTP in a transcription‐like reaction. In this report the ability of the enzyme to catalyze DNA primer extension reaction was demonstrated. The efficiency of the reaction essentially depended on the type of the primer sequence, and was significantly higher if the primer coincided with the T7 promoter non‐coding sequence. In this case the primer extension reaction proceeded along with de novo RNA synthesis. The length of the product did not exceed 8 nucleotides, indicating that the primer extension reaction proceeds according to the mechanism of the T7 RNAP‐catalyzed abortive transcription.
Lipopolysaccharide‐caused fragmentation of individual microtubules in vitro observed by video‐enhanced differential interference contrast microscopy
Authors: Böhm Konrad J , Vater Wolfram , Russwurm Stefan , Reinhart Konrad , Unger Eberhard
Published: July 2002

Microtubule disassembly is commonly believed to be a process of endwise tubulin dimer release. The present study demonstrates by video interference contrast microscopy that Escherichia coli lipopolysaccharide (LPS) caused microtubule disassembly in vitro by both endwise shortening and fragmentation. In contrast, the microtubules were only shortened from their ends in the presence of DNA, used as another example of a macromolecular microtubule effector. LPS‐caused microtubule fragmentation was confirmed by transmission electron microscopy. Because of its ability to induce both fragmentation and endwise shortening, LPS, which is involved in sepsis pathogenesis, has to be regarded as a highly active microtubule‐destabilizing agent.
Rapid activation of the novel serine/threonine protein kinase, protein kinase D by phorbol esters, angiotensin II and PDGF‐BB in vascular smooth muscle cells
Authors: Abedi Husna , Rozengurt Enrique , Zachary Ian
Published: July 2002

Protein kinase D (PKD) is a novel serine/threonine kinase structurally distinct from all protein kinase C (PKC) isoforms but which like classic and novel PKCs is activated by phorbol esters and diacylglycerol. This study investigated the regulation of PKD in vascular smooth muscle cells (VSMC) by physiological regulators of VSMC function and growth factors. Treatment of rabbit aortic VSMC with phorbol ester, angiotensin II and PDGF‐BB all stimulated PKD activity in a time‐ and concentration‐dependent manner in VSMC. The effect of angiotensin II was particularly rapid and potent (maximum stimulation within 1 min and at 0.5 nM). In contrast, the maximum effect of PDGF‐BB was obtained after 5 min. Other factors, including basic FGF, IGF‐I, IGF‐II, endothelin‐1 and endothelin‐2, had no effect on PKD activity in VSMC. These results show for the first time that PKD activity is regulated in VSMC, and is activated by the vasoconstrictor angiotensin II. PKD may be an important mediator for the biological function(s) of one or more PKC isoforms in VSMC and/or may represent a component of a novel PKC‐independent signalling pathway in VSMC.
Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae
Authors: Paulsen Ian T , Sliwinski Marek K , Nelissen Bart , Goffeau André , Saier Milton H
Published: July 2002

We present the complete inventory of currently recognized and putative transporters encoded within the genome of Saccharomyces cerevisiae. These 258 transporters are classified into 42 families according to phylogenetic and substrate specificity criteria. Twelve of these yeast families are found only in eukaryotic organisms, and four are so far unique to yeast. Putative yeast‐specific families transport heavy metals, arsenite and calcium. The phylogenetic analyses reported allow classification of 139 functionally uncharacterized yeast transporters into families of known functions. The relative proportions of yeast transporters specific for different classes of substrates differ only slightly from those reported for Escherichia coli. However, the ratio of secondary transporters (uniporters, cation symporters and antiporters) to primary ATP‐driven transporters is much higher for yeast than for bacteria.
14‐3‐3 proteins activate a plant calcium‐dependent protein kinase (CDPK)
Authors: Camoni Lorenzo , Harper Jeffrey F , Palmgren Michael G
Published: July 2002

Plants and protozoa contain a unique family of calcium‐dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl‐terminal calmodulin‐like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14‐3‐3 proteins. Isoform CPK‐1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium‐dependent activity of this recombinant CPK‐1 was shown to be stimulated almost twofold by three different 14‐3‐3 isoforms with 50% activation around 200 nM. 14‐3‐3 proteins bound to the purified CPK‐1, as shown by binding assays in which either the 14‐3‐3 or CPK‐1 were immobilized on a matrix. Both the 14‐3‐3 binding and activation of CPK‐1 were specifically disrupted by a known 14‐3‐3 binding peptide LSQRQRSTpSTPNVHMV (IC50=30 μM). These results raise the question of whether 14‐3‐3 can modulate the activity of CDPK signal transduction pathways in plants.
Three‐dimensional structure of binase in solution
Authors: Reibarkh M.Ya , Nolde D.E , Vasilieva L.I , Bocharov E.V , Shulga A.A , Kirpichnikov M.P , Arseniev A.S
Published: July 2002

We present the spatial structure of binase, a small extracellular ribonuclease, derived from 1H‐NMR* data in aqueous solution. The total of 20 structures were obtained via torsion angle dynamics using DYANA program with experimental NOE and hydrogen bond distance constraints and φ and χ1 dihedral angle constraints. The final structures were energy minimised with ECEPP/2 potential in FANTOM program. Binase consists of three α‐helices in N‐terminal part (residues 6–16, 26–32 and 41–44), five‐stranded antiparallel β‐sheet in C‐terminal part (residues 51–55, 70–75, 86–90, 94–99 and 104–108) and two‐stranded parallel β‐sheet (residues 22–24 and 49–51). Three loops (residues 36–39, 56–67 and 76–83), which play significant role in biological functioning of binase, are flexible in solution. The differences between binase and barnase spatial structures in solution explain the differences in thermostability of binase, barnase and their hybrids.
Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N‐1H spin‐spin coupling of the Arg guanidinium moiety in the triple‐helix structure
Authors: Consonni Roberto , Santomo Laura , Tenni Ruggero , Longhi Renato , Zetta Lucia
Published: July 2002

CB2, a CNBr peptide of 36 residues from type I collagen α1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg9 showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons (1
J
NH=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (Δδ/ΔT of −3.6 ppb/°C) suggest that the H atoms of the protonated guanidinium moiety of Arg9 in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354–7359), similar to CB2, our data could indicate that the guanidinium group of Arg9 form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the Nϵ hydrogen, leaving the four Nη hydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric α1(I) CB2 triple helix. The behaviour of Arg9 has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17°C.
Constitutive association of JAK1 and STAT5 in pro‐B cells is dissolved by interleukin‐4‐induced tyrosine phosphorylation of both proteins
Authors: Erhardt Ingrid , Lischke Antje , Sebald Walter , Friedrich Karlheinz
Published: July 2002

The bipartite human interleukin‐4 (IL‐4) receptor was functionally expressed in murine pro‐B cells and activated by human IL‐4 to evoke intracellular signaling. Mutual association of signal transducing proteins within the receptor complex was then studied in dependence of ligand stimulation. Besides ligand‐induced receptor heterodimerization and contacts of the two IL‐4 receptor subunits α and γ with Janus kinases JAK1 and JAK3 a prominent constitutive binding between JAK1 and signal transducer and activator of transcription STAT5 was detected. Since both these proteins become phosphorylated in response to IL‐4 receptor stimulation, the influence of tyrosine phosphorylation on their mutual contact was analyzed. Association of JAK1 and STAT5 was found to occur exclusively between unphosphorylated proteins.
Influence of endogenous GM1 ganglioside on TrkB activity, in cultured neurons
Authors: Pitto Marina , Mutoh Tatsuro , Kuriyama Masaru , Ferraretto Anita , Palestini Paola , Masserini Massimo
Published: July 2002

We verified the hypothesis that changes in the endogenous GM1 ganglioside density in the environment of TrkB, receptor of brain‐derived neurotrophic factor, can affect receptor activity, and focused on rat cerebellar granule cells expressing both GM1 and TrkB. Changes of the amount of GM1 associated to immunoprecipitated TrkB and of receptor tyrosine phosphorylation were evaluated after treatment with phorbol‐12‐myristate‐13‐acetate (1 μM, 7 min), reported to affect the plasma membrane distribution of endogenous gangliosides in the same cells. After treatment, the amount of GM1 associated to receptor and TrkB phosphorylation decreased by about 40%. The amount of associated GM1 decreased by about 33% also after concomitant treatment with phorbol ester and brain‐derived neurotrophic factor, but in this case the neurotrophin was unable to enhance receptor tyrosine phosphorylation. These results for the first time suggest that changes in the amount of endogenous GM1 in the environment of TrkB can modulate receptor activity, and offer new clues for a better understanding of physiological and pathological events of the nervous system.
Cloning and expression of a unique inorganic pyrophosphatase fromBacillus subtilis: evidence for a new family of enzymes
Authors: Shintani Toshio , Uchiumi Toshio , Yonezawa Tomoki , Salminen Anu , Baykov Alexander A. , Lahti Reijo , Hachimori Akira
Published: July 2002

An open reading frame located in the COTF‐TETB intergenic region ofBacillus subtiliswas cloned and expressed inEscherichia coliand shown to encode inorganic pyrophosphatase (PPase). The isolated enzyme is Mn2+‐activated, like the authentic PPase isolated fromB. subtilis. Although 13 functionally important active site residues are conserved in all 31 soluble PPase sequences so far identified, only two of them are conserved inB. subtilisPPase. This suggests thatB. subtilisPPase represents a new family of soluble PPases (a Bs family), putative members of which were found inArchaeoglobus fulgidus,Methanococcus jannaschii,Streptococcus mutansandStreptococcus gordonii.
EVH1/WH1 domains of VASP and WASP proteins belong to a large family including Ran‐binding domains of the RanBP1 family
Authors: Callebaut Isabelle , Cossart Pascale , Dehoux Pierre
Published: July 2002

The two cytoskeletal proteins VASP and WASP and the protein Homer share a conserved domain, currently designated the WH1 domain (ASP omology domain ) or EVH1 domain (NA/ASP omology domain ), which could play an important role in various cellular events such as transport, folding of proteins, and signal transduction. We report here additional occurrences of this domain in Ran‐binding proteins of the RanBP1 family and various others proteins, or putative proteins of eukaryotic organisms, suggesting that the EVH1/WH1 domain may be more widely used than originally thought.
Bacterial proteins carrying twin‐R signal peptides are specifically targeted by the ΔpH‐dependent transport machinery of the thylakoid membrane system
Authors: Halbig Dirk , Hou Bo , Freudl Roland , Sprenger Georg A. , Klösgen Ralf Bernd
Published: July 2002

Glucose‐fructose oxidoreductase (GFOR), a periplasmic protein of Zymomonas mobilis, is synthesized as a precursor polypeptide with a twin‐R signal peptide for Sec‐independent protein export in bacteria. In higher plant chloroplasts, twin‐R signal peptides are specific targeting signals for the Sec‐independent ΔpH pathway of the thylakoid membrane system. In agreement with the assumed common phylogenetic origin of the two protein transport mechanisms, GFOR can be efficiently translocated by the ΔpH‐dependent pathway when analyzed with isolated thylakoid membranes. Transport is sensitive to the ionophore nigericin and competes with specific substrates for the ΔpH‐dependent transport route. In contrast, neither sodium azide nor enzymatic destruction of the nucleoside triphosphates in the assays affects thylakoid transport of GFOR indicating that the Sec apparatus is not involved in this process. Mutagenesis of the twin‐R motif in the GFOR signal peptide prevents membrane translocation of the protein emphasizing the importance of these residues for the transport process.
Regulation of p53 mediated transactivation by the β‐subunit of protein kinase CK2
Authors: Schuster Norbert , Prowald Alexandra , Schneider Eberhard , Scheidtmann Karl-Heinz , Montenarh Mathias
Published: July 2002

The growth suppressor protein p53 plays a main part in cellular growth control. Two of its key functions are sequence specific DNA binding and transactivation. Functions of p53 in growth control are regulated at least in part by its interaction with protein kinases. p53 binds to protein kinase CK2, formerly known as casein kinase 2, and it is phosphorylated by this enzyme. CK2 is composed of two regulating β‐subunits and two catalytic α‐ or α′‐subunits and the interaction with p53 is mediated by the regulatory β‐subunit of CK2. Recently we showed that the β‐subunit could inhibit the sequence specific DNA binding activity of p53 in vitro. Based on this finding, we asked if a coexpression of the β‐subunit of CK2 with p53 in mammalian cells could inhibit the DNA binding activity of p53 in a physiological context. We found that the coexpression of the β‐subunit showed the same inhibitory effect as in the previous assays with purified proteins. Then, we investigated the effects of the coexpression of the β‐subunit of CK2 on the transactivation and transrepression activity of p53. We found that transactivation of the mdm2, p21WAF1/CIP1 and cyclin G promoter was inhibited in three different cell lines whereas transactivation of the bax promoter was not affected in COS1 cells but down‐regulated in MCO1 and SaosS138V21 cells. p53 mediated transrepression of the fos promoter was not influenced by coexpression of the CK2 β‐subunit. Taken together we propose a cell type dependent fine regulation of the p53 transactivation function by the CK2 β‐subunit in vivo, which does not affect p53 mediated transrepression.
Energy‐dependent transformation of the catalytic activities of the mitochondrial F0·F1‐ATP synthase
Authors: Galkin M.A. , Vinogradov A.D.
Published: July 2002

The ADP(Mg2+)‐deactivated, azide‐trapped F0·F1‐ATPase of coupled submitochondrial particles is capable of ATP synthesis being incapable of ATP hydrolysis and ATP‐dependent generation [FEBS Lett. (1995) 366, 29–32]. This puzzling phenomenon was studied further. No ATPase activity of the submitochondrial particles catalyzing succinate‐supported oxidative phosphorylation in the presence of azide was observed when ATP was added to the assay mixture after an uncoupler. Rapid ATP hydrolysis was detected in the same system when ATP followed by an uncoupler was added. Less than 5% of the original ATPase activity was seen when the reaction (assayed with ATP‐regenerating system) was initiated by the addition of ATP to the azide‐trapped coupled particles oxidizing succinate either in the presence or in the absence of the uncoupler. High ATP hydrolytic activity was revealed when the reaction was started by the simultaneous addition of the ATP plus uncoupler to the particles generating . The energy‐dependent conversion of the enzyme into latent uncoupler‐activated ATPase was prevented by free ADP (K
i≈20 μM) and was greatly enhanced after multiple turnovers in oxidative phosphorylation. The results suggest that the catalytic properties of F0·F1 are ‐dependent which is in accord with our hypothesis on different conformational states of the enzyme participating in ATP synthesis or hydrolysis.
The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells
Authors: Kamitani Shigeki , Asakawa Makoto , Shimekake Yoshiyuki , Kuwasako Kenji , Nakahara Koichiro , Sakata Tsuneaki
Published: July 2002

Adrenomedullin, a potently hypotensive peptide isolated from human pheochromocytoma, is known to elicit a rise in cAMP levels within mammalian endothelial and smooth muscle cells. Until now, however, little has been known about the adrenomedullin receptor. Recently, a group called receptor activity‐modifying proteins that complex with the calcitonin receptor‐like receptor, and thereby regulate its transport and ligand specificity, were identified. Here we show that mRNA for both the calcitonin receptor‐like receptor and the receptor activity‐modifying protein 2, but not the receptor activity‐modifying protein 1 or receptor activity‐modifying protein 3, are expressed in human endothelial and vascular smooth muscle cells. We also found that adrenomedullin increased cAMP levels in HeLa EBNA and 293 EBNA cells, expressing both the receptor activity‐modifying protein 2 and calcitonin receptor‐like receptor proteins. Thus, the receptor activity‐modifying protein 2/calcitonin receptor‐like receptor complex apparently serves as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.
Characterization of a soluble class I α‐mannosidase in human serum
Authors: Porwoll S , Fuchs H , Tauber R
Published: July 2002

Class I α‐mannosidases are thought to exist exclusively as integral membrane proteins that play intracellulary an essential role in the N‐glycan biosynthesis. Using [3H]Man9GlcNAc2 as a substrate, we were able to identify a soluble α‐mannosidase in human serum that trims the substrate Man9GlcNAc2 to Man5‐8GlcNAc2 with Man6GlcNAc2 being the major product. This serum mannosidase is Ca2+‐dependent, sensitive to 1‐deoxymannojirimycin but insensitive to the class II inhibitor swainsonine and, hence, belongs to class I mannosidases. The enzymatic properties of the serum class I mannosidase are similar to that of the membrane bound class I mannosidases Golgi‐mannosidase IA and IB and Man9‐mannosidase.
Enhanced anti‐HIV‐1 activity of CC‐chemokine LD78β, a non‐allelic variant of MIP‐1α/LD78α
Authors: Xin Xiaomi , Shioda Tatsuo , Kato Atsushi , Liu Huanliang , Sakai Yuko , Nagai Yoshiyuki
Published: July 2002

We compared the anti‐HIV‐1 activity of CC‐chemokine LD78β with that of MIP‐1α, another CC‐chemokine which shows 94% sequence homology with LD78β. Despite its close similarity to MIP‐1α, the anti‐HIV‐1 activity of LD78β appeared to be nearly 10 times higher than that of MIP‐1α. Mutagenesis of MIP‐1α showed that the N‐terminal additional tetrapeptide, which was present in LD78β and absent in MIP‐1α, is responsible for enhanced anti‐HIV‐1 activity. The N‐terminal structure‐function relationship of LD78β described here will be of value in understanding the chemokine‐receptor interactions and designing anti‐HIV‐1 compounds based on LD78β.
Kluyveromyces lactis HIS4 transcriptional regulation: similarities and differences to Saccharomyces cerevisiae HIS4 gene
Authors: Lamas-Maceiras Mónica , Cerdán M.Esperanza , Freire-Picos M.Angeles
Published: July 2002

Sequence analysis of the Kluyveromyces lactis HIS4 (KlHIS4) gene promoter reveals relevant differences in comparison to the Saccharomyces cerevisiae HIS4 homologous gene. Among them are the absence of a Rap1 binding site and the presence of only three putative Gcn4 binding consensus sites instead of the five described in the S. cerevisiae promoter. Since these factors are implicated in the general control, we investigated the transcriptional regulation of the KlHIS4 gene under conditions of amino acid starvation and discovered that the mechanisms previously described for S. cerevisiae HIS4 regulation and related to general control are not functional in K. lactis. The expression analysis of the KlHIS4 gene under phosphate starvation or high adenine supply shows that factors, such as Bas1 or Bas2, involved in the basal control may also operate in a different way in K. lactis. Interestingly, and also in contrast to the HIS4 regulation in S. cerevisiae, we found domains for Nit2‐like and yeast‐Ap1‐like binding sequences. Northern analyses showed transcriptional activation under ammonia starvation and oxidative stress. The EMBL accession number for the KlHIS4 promoter is AJ238494.
Reduced extractability of the XPA DNA repair protein in ultraviolet light‐irradiated mammalian cells
Authors: Svetlova Maria , Nikiforov Andrey , Solovjeva Liudmila , Pleskach Nadezhda , Tomilin Nikolai , Hanawalt Philip C.
Published: July 2002

The XPA protein is essential for both of the known modes of nucleotide excision repair (NER) in human cells: transcription‐coupled repair (TCR) and global genome repair (GGR). In TCR, this protein is thought to be recruited to lesion sites in DNA at which RNA polymerase II is blocked and in GGR, by direct recognition of damages by repair protein complex containing XPC/HR23B or DNA damage‐binding protein. However, details of the recruitment of the XPA protein in vivo are unknown. It was shown earlier that a portion of another NER protein, PCNA, which is completely extractable from non‐S‐phase mammalian nuclei, becomes insoluble after ultraviolet (UV) light irradiation and cannot be extracted by methanol or buffer containing Triton X‐100. In the present study, we have found that UV light irradiation of human or Chinese hamster cells leads to decrease of extractability of the XPA protein by Triton X‐100. Maximal insolubilization of the XPA protein is observed 1–4 h after irradiation but it is not detectable by 22 h. This effect is dose‐dependent for UV light from 2.5 to 15 J/m2 and is unaffected by the pre‐treatment of cells with sodium butyrate, an inhibitor of histone deacetylation. The UV light‐induced insolubilization of the XPA protein was also observed in two lines of Cockayne syndrome complementation group A cells, indicating that the effect is not dependent upon TCR. The results are discussed in relation to possible mechanisms of NER.