bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of platelet activation factor and prostaglandins on cholesterol esterification in human plasma
Authors: Bergelson L.D. , Kulikov V.I. , Muzia G.I.
Published: July 2002

Platelet activating factor
High density lipoprotein
Cholesterol esterification
Succinate thiokinase in pigeon breast muscle mitochondria
Authors: Allen D.A. , Ottaway J.H.
Published: July 2002

Succinate thiokinase has been purified from pigeon breast muscle. It has been confirmed that the enzyme is entirely specific for ATP, and the K
m is very high (0̃.8 mM). Activity in mitochondrial sonicates is low enough for it to be doubtful whether the enzyme can support citric acid cycle flux in the tissue. The enzyme appears to have an M
r of 80000–100000, and to have two unequal subunits. As determined by SDS gel electrophoresis one subunit certainly has an M
r of 40000.
Regulatory subunit of type II cAMP‐dependent protein kinase as substrate and inhibitor of protein phosphatase‐1 and ‐2A
Authors: Vereb György , Erd″odi Ferenc , Tóth Béla , Bot György
Published: July 2002

The dissociated regulatory subunit (RII) of autophosphorylated cAMP‐dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase‐1 and ‐2A (phosphatase‐1c and ‐2Ac) and by a high‐M
r, polycation‐dependent form of phosphatase‐2A (2A0) with K
m values of 5,0.3 and 1 μM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase‐1c, whereas polycations (histone H1 or polybrene) markedly stimulated phosphatase‐2Ac and ‐2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half‐maximum inhibitory concentrations of 0.1–0.36 μM.
A suppressive role of c‐kinase for the stimulation of hepatic ketogenesis by glucagon and epinephrine
Authors: Kojima Hideto , Harano Yutaka , Kosugi Keisuke , Nakano Takamitsu , Shigeta Yukio
Published: July 2002

Long‐chain fatty acid oxidation
Ketogenesis
c‐Kinase
Glucagon
Epinephrine
Carnitine palmitoyltransferase
Sequence‐specific assignments of downfield‐shifted amide proton resonances of calmodulin Use of two‐dimensional NMR analysis of its tryptic fragments
Authors: Ikura Mitsuhiko , Minowa Osamu , Yazawa Michio , Yagi Koichi , Hikichi Kunio
Published: July 2002

Two‐dimensional NMR methods were applied to assign the extremely downfield‐shifted amide‐proton resonances in the 500‐MHz 1H‐NMR spectra of the NH2‐terminal fragment of residues 1–75 of calmodulin. The low‐field resonances of the 1H‐NMR spectra of intact calmodulin were assigned to specific amino acid residues by comparison with spectra of the tryptic fragments of residues 1–75 and 78–148, in both the Ca2+‐free and Ca2+‐bound states. The hydrogen bonding of glycine residues connecting the two amino acid residues at the Z and − Y positions in the octahedral Ca2+ coordination site was investigated. The Gly 134 in site IV showed a different property from the other glycines, 25, 61 and 98, involved in sites I, II and III, respectively.
Activation by calcium of AMP deaminase from the human red cell
Authors: Almaraz Laura , García-Sancho Javier
Published: July 2002

We have investigated the effects of Ca2+ on AMP deaminase from human red cells. At variance with the other known modulators, Ca2+ increased the apparent affinity for AMP without modifying the characteristic positive cooperativity of the enzyme towards the substrate. Ca2+ sensitivity was not modified by dialysis, but dilution of the haemolysate produced an activation of the enzyme similar to that induced by Ca2+. Simultaneously, the Ca2+ dependence was lost. The sensitivity to other modulators, such as ATP, diphosphoglycerate or phosphate, was not modified by dilution. Partial purification of the enzyme produced the same effects as haemolysate dilution. These results may be interpreted to mean that Ca2+ acts by antagonizing an endogenous inhibitor present in red cell lysates.
Calcium‐dependent binding between calmodulin and lysozyme
Authors: Fierro J.F. , Pajares M.A. , Hardisson C.
Published: July 2002

Calmodulin, an acidic protein that binds calcium with high affinity, has multiple roles in the activation of many enzymes involved in cellular regulation of eukaryotes. In this study we show that calmodulin binding to hen egg‐white lysozyme, in a Ca2+‐dependent way, was observed using electroblots incubated with biotinylated calmodulin and detected with avidin‐alkaline phosphatase or for affinity chromatography on a gel calmodulin column. Antimicrobial activity of lysozyme was not modified in the presence of Ca2+‐calmodulin.
Expression of catalytically active rat S‐adenosylmethionine decarboxylase in Escherichia coli
Authors: Salmikangas Paula , Keranen Marja-Riitta , Pajunen Antti
Published: July 2002

The cDNA coding for rat S‐adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50) has been cloned into a plasmid expression vector, pKK‐223‐3, and expressed in E. coli. The authenticity of the expressed protein has been demonstrated by reactivity with antibodies specific for rat Ado‐MetDC, by size analysis on SDS gels visualized with immunotransblots, and, finally, by catalytic activity. The expression of the enzyme results in a decrease in the activity of the bacterial enzyme suggesting the replacement of the bacterial enzyme by the rat AdoMetDC. Similarly, the addition of exogenous spermidine to the growth medium reduces bacterial enzyme activity affecting only marginally the expression of the recombinant protein.
Monoclinic crystals of lignin peroxidase
Authors: Glumoff Tuomo , Winterhalter Kaspar H. , Smit Jan Derk G.
Published: July 2002

Lignin peroxidase; Protein crystallization; Semi‐continuous cultivation; (Phanerochaete chrysosporium)
Identification of a reversible structural transition in the metal‐depleted glycerol dehydrogenase from Bacillus stearothermophilus
Authors: Spencer P. , Paine L.J. , Scawen M.D. , Atkinson T. , Gore M.G.
Published: July 2002

Evidence is presented to demonstrate that the Zn2+‐depleted, inactive form of the glycerol dehydrogenase from Bacillus stearothermophilus exists in one of two possible conformations in equilibrium, the position of which is temperature sensitive. The conformation of the metal‐depleted enzyme favoured by higher temperatures (20–40°C) is able to bind Zn2+ and regain catalytic activity, whereas that favoured at lower temperatures (0–10°C) is unable to bind metal ions and is thus inactive. This equilibrium is also pH dependent with a pK of 6.6. At pH 6.0, the equilibrium lies in favour of the form of the enzyme able to bind metal ions and exhibit activity.
Proteolysis of Bacillus stearothermophilus IF2 and specific protection by GTP
Authors: Severini M. , Choli T. , La Teana A. , Gualerzi C.O.
Published: July 2002

Translation initiation factor IF2 from Bacillus stearothermophilus (741 amino acids, M
r = 82 043) was subjected to trypsinolysis alone or in the presence of GTP. Following electroblotting and automated amino acid sequencing of the resulting peptides, the location and the sequential order of the main cleavage sites were identified. Trypsinolysis of IF2 ultimately generates two compact domains: a 24.5 kDa C‐terminal fragment and a 40 kDa G‐fragment which is obtained only in the presence of GTP which strongly protects a cleavage site within the GTP binding domain.
Insulin‐secreting β‐cells possess specific receptors for interleukin‐1β
Authors: Hammonds Peter , Beggs Mark , Beresford Guy , Espinal Joseph , Clarke Jennifer , Mertz Robert J.
Published: July 2002

The effect of the cytokine interleukin‐1β on the insulin secretory responsiveness of single β‐cells (HIT‐T15) was investigated. In the short‐term, IL‐1β induced a dosage‐dependent stimulation of insulin release. In contrast, in the long‐term, IL‐1β, inhibited both basal and secretagogue‐stimulated insulin secretion. We also demonstrate the simultaneous presence of specific high and low affinity binding sites for IL‐1β on β‐cells. IL‐1β, which has been implicated in the pathogenesis of insulin‐dependent diabetes, may therefore mediate its opposing effects on β‐cells through a specific plasma membrane receptor.
A small G‐protein involved in phosphatidylinositol‐4‐phosphate kinase activation
Authors: Urumow T. , Wieland O.H.
Published: July 2002

A guanine nucleotide binding protein tentatively designated Gs,(PIPK) which activates purified phosphatidylinositol‐4‐phosphate kinase in vitro, has been partially purified from rat liver membranes and identified as a small G‐protein with a molecular mass between 20 and 25 kDa.
Primary structure of NADP‐dependent malic enzyme in the dicotyledonous C4 plant Flaveria trinervia
Authors: Börsch Dagmar , Westhoff Peter
Published: July 2002

The primary structure of NADP‐dependent malic enzyme (NADP‐ME) of the dicotyledonous C4 plant Flaveria trinervia was determined from sequence analysis of a cDNA clone containing the complete coding region. Comparison of the mature F. trinervia NADP‐ME with the maize enzyme reveals extensive sequence similarity. In contrast, no significant similarity can be detected between the putative transit peptides of the two enzymes. This suggests that the corresponding parts of the genes arose independently from each other during evolution of mono‐ and dicotyledonous C4 plants.
Heme‐CO as a probe of the conformational state of calmodulin
Authors: Marden M.C. , Leclerc L. , Poyart C.
Published: July 2002

The interaction of heme‐CO with calmodulin, in the presence of calcium, leads to a complex of four heme‐CO molecules per protein. No interaction was observed in the absence of calcium. The binding of heme‐CO to calmodulin was monitored by the shift in the Soret absorption band from 407 to 420 nm (bound form); the four sites are not spectrally identical. The ligand CO can be photodissociated from the calmodulin‐heme‐CO complex and the bimolecular recombination kinetics also indicate a heterogeneous mixture. The complex does not bind oxygen reversibly. As calmodulin has only one histidine, the hemes are apparently not bound by the iron atom as in hemoglobin, but are probably loosely associated (K
d=0.5 μM) in hydrophobic pockets which apparently open when the protein is activated by calcium.
The processed pseudogene of mouse thymidine kinase is active after transfection
Authors: Seiser Christian , Beck Gerhard , Wintersberger Erhard
Published: July 2002

Aside of the gene coding for cytoplasmic thymidine kinase, the genome of mouse cells carries two pseudogenes. Both are inactive in situ. One of the pseudogenes is a processed pseudogene in which a two base pair deletion caused a shift of the reading frame and a shortening of the gene product from the 233 amino acids of thymidine kinase to 177 amino acids in the pseudogene product. We report here that introduction of this pseudogene into LTK− cells gave rise to cells with a thymidine kinase positive phenotype. The transformed cells carried multiple copies of the pseudogene the upstream region of which exhibited low but measurable promoter activity. Replacement of the upstream region of the pseudogene by a promoter of Simian virus 40 or of the mammary tumor virus resulted in high transfection efficiencies and in cell lines exhibiting high thymidine kinase activities.
Recognition of target colloid species by conjugates of a linear polyelectrolyte with a vector protein
Authors: Kabanov V.A. , Yaroslavov A.A. , Sukhishvili S.A. , Polynsky A.S. , Chechik O.S.
Published: July 2002

Redistribution reaction of quatermized poly‐4‐vinylpyridine polycations and their conjugates with x‐chymotrypsin by oppositely charged latex particles is disclosed. The polycatrons are strongly absorbed on the latex surface. Nevertheless, they are able to migrate between the latex species via occasional interparticle contracts. In the case of a homogeneous latex such interchange results in uniform distribution of polycations by latex particles. The distribution drastically changes, when x‐chymotrypsin‐polycation conjugates interact with a mixture of two latexes; one chemically modified by bovine serum albumin and the other one by specific protein inhibitor of x‐chymotrypsin. In this case the interchanging polycations are finally fixed on the latex particles carrying the centres of specific binding of the enzyme vector, i.e. recognize them in the latex mixture. The obtained results are considered to minus physico‐chemical interaction and recognition of target supermolecular bio‐objects by large macromolecules carrying relatively small molecular vectors.
Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein
Authors: Aoki Ikuo , Shindoh Yutaka , Nishida Tsutomu , Nakai Satoru , Hong Yeong-Man , Mio Mitsunobu , Saito Tadayuki , Tasaka Kenji
Published: July 2002

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 μg/ml both in the presence and in the absence of extracellular Ca2+. After reverse‐phase high‐performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pl values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPx was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 × 103 clones, a positive clone, which encoded a full length of pre‐proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre‐proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.
Short‐term energy deficit causes net accumulation of linoleoyl‐enriched triacylglycerols in rat liver
Authors: Chen Zhen-Yu , Cunnane Stephen C.
Published: July 2002

Energy depletion by reduced food intake over 4 days resulted in a 73% reduction in total rat liver triacylglycerols (TG). In liver TG of energy‐depleted rats, dilinoleoyl oleoyl glycerol (OLL) and trilinoleoyl glycerol (LLL)) were quantitatively increased by 85% and 147%, respectively. The net increase in linoleoyl‐enriched species could be quantitatively accounted for by the release of linoleate from monolinoleoyl species and its subsequent reacylation into dilinoleoyl species and trilinolein during energy depletion. Hence while palmitate, oleate and some linoleate are being hydrolyzed, presumably for oxidation some linoleate is retained and contributes to the remodelling of hepatic triacylglycerols during energy deficit.
Subcellular distribution of enzymes involved in the biosynthesis of cyanelle murein in the protist Cyanophora paradoxa
Authors: Plaimauer Barbara , Pfanzagl Beatrix , Berenguer José , de Pedro Miguel A. , Löffelhardt Wolfgang
Published: July 2002

Cyanelle containing organisms, notably Cyanophora paradoxa, best studied among them, are unique with respect to the occurrence of peptidoglycan (murein) within an eukaryotic cell. Enzyme activities involved in the biosynthesis of UDP‐N‐acetyl‐muramylpentapeptide could be localized within the cyanelle compartment. Some of the enzymes performing later steps of murcin biosynthesis were detected in the postcyanelle supernatant rather than in the cyanelle lysate. This is taken to reflect a ‘periplasmic’ location of these enzymes that are partially liberated upon rupture of the cyanelle outer membrane.
A rat gene encoding heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase
Authors: Darville Martine I. , Chikri Mohamed , Lebeau Etienne , Hue Louis , Rousseau Guy G.
Published: July 2002

There are at least 3 isozymes of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase, a bifunctional enzyme which catalyzes the synthesis and degradation of fructose 2,6‐bisphosphate. A 22‐kb rat gene that encodes the heart isozyme has been identified and compared with the 55‐kb rat gene encoding the liver and muscle isozymes which had been described earlier. Although these 2 genes include 12 successive similar exons, they contain dissimilar exons at both ends, consistent with the occurrence of different regulatory domains at the N‐ and C‐termini in the 3 isozymes.
The induction of lamellar stacking by cholesterol in lecithin‐bile salt model systems and human bile studied by synchrotron X‐radiation
Authors: Sömjen G.J. , Coleman R. , Koch M.H.J. , Wachtel E. , Billington D. , Towns-Andrews E. , Gilat T.
Published: July 2002

Small angle X‐ray scattering (SAXS) with synchroton radiation was used to investigate interactions among lipid particles in lecithin‐bile salt model systems and in native gallbladder biles. In model systems in the absence of cholesterol. isotropic, continuous spectra were found, indicating the absence of periodic structures. In the presence of excess cholesterol, interaction in the form of lamellar stacking was detected by the appearance of discrete diffraction peaks. In the supersaturated cholesterol region of the commonly accepted phase diagram [1]. where cholesterol crystals were expected. we found lamellar stacking. The high proportion of cholesterol to bile salts seems to be the common denominator of these models. The lamellar stacking was also found in native unprocessed bile. This effect of cholesterol on lipid structure has not been previously described. Lamellar stacking may contribute to cholesterol solubilization. Its influence on the kinetics of cholesterol crystallization is presently unknown.
P26 — calcium binding protein from bovine retinal photoreceptor cells
Authors: Kutuzov M.A. , Shmukler B.E. , Suslov O.N. , Dergachev A.E. , Zargarov A.A. , Abdulaev N.G.
Published: July 2002

The primary structure of bovine retinal calcium binding protein P26 has been determined by parallel analysis of protein and corresponding cDNA, This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. Preliminary data are presented on expression of P26 as a fusion protein in E. coli.
Single channel recording in the chloroplast envelope
Authors: Pottosin Igor I.
Published: July 2002

The patch‐clamp technique was applied to study ion channels in the intact chloroplast envelope. Three channel types were characterized: two cation‐selective, with a conductance (in 100 mM KCl) of 517 and 1016 pS, respectively, and one anion‐selective with a conductance of 159 pS. All three channels showed voltage‐dependent closures at both positive and negative membrane potentials.
Interleukin‐1β induces the expression of an isoform of nitric oxide synthase in insulin‐producing cells, which is similar to that observed in activated macrophages
Authors: Eizirik Décio L. , Cagliero Enrico , Björklund Andres , Welsh Nils
Published: July 2002

The suppressive and cytotoxic effects of interleukin‐1β(IL‐β) on rodent insulin‐producing cells observed in vitro are probably mediated through formation or nitric oxide (NO). In this study we demonstrate that IL‐1‐induced NO formation in isolated rat islets and insulin‐producing HIT cells is more sensitive to inhibition by N
G‐monomethyl‐l‐arginine than to inhibition by N
G‐nitro‐l‐arginine, thus suggesting that IL‐1‐exposed insulin producing cells express an isoform of nitric oxide synthase similar to that present in activated macrophages, Furthermore, IL‐1β markedly increased the mRNA levels or the inducible microphage form of nitric oxide synthase in HIT cells.
Reversible inactivation and binding to mitochondria of nitrate reductase by heat shock in the yeast Hansenula anomala
Authors: Siverio José M. , González Celedonio , Mendoza-Riquel Alvaro , Pérez Ma Dolores , González Gregorio
Published: July 2002

Heat shock from 25°C to 40°C of Hansenula anomalacells resulted in a rapid and reversible inactivation of the NADPH‐nitrate reductase (NR) activity. The inactive enzyme retained partial activity with the non‐physiological co‐substrates, reduced methyl viologen and reduced flavin mononucleotide. The inactive NR pelleted after centrifugation at 12,000 × g for 30 min and was associated with mitochondria. In untreated cells around 10% of the total NR is inactive and associated with mitochondria, while the active enzyme is soluble. In vitro, inactive NR could be partially dissociated from the mitochondria by incubating them at pH 11.5 or in the presence of 15 mM CHAPS.
Inhibition of neutral cholesteryl ester hydrolase by a naturally occurring cytosolic protein in macrophages
Authors: Shand John H. , Crilly Peter J. , West David W.
Published: July 2002

The soluble fraction from a number of macrophage cell lines has been shown to contain a protein(s) that inhibits neutral cholesteryl ester hydrolase. The inhibition is dependent on the concentration of soluble protein used, and the efficiency of the inhibitor can be influenced by the inclusion of oleate and an inhibitor of ACAT. It is suggested that the presence of this material indicates that the macrophage contains a means of negatively controlling the activity of the hydrolytic phase of the cholesterol/cholesteryl ester cycle.
pH dependence of the formation of an M‐type intermediate in the photocycle of 13‐cis‐bacteriorhodopsin
Authors: Drachev L.A. , Dracheva S.V. , Kaulen A.D.
Published: July 2002

An M‐type intermediate is formed in the 13‐cis‐bR photocycle in purple membranes at high pH. This is presumably due to deprotonation of the same group whose deprotonation causes a large increase in rate of M formation in the trans‐bR photocycle (the ‘alkaline transition’). For Triton X‐100‐solubilized bR, the alkaline transition is shifted to a lower pH value by more than 2 pH units. The alkaline transition in Triton‐solubilized preparations changes the efficiency of the M intermediate formation in the 13‐cis‐sbR photocycle. The M intermediate formation in 13‐cis‐sbR, as in the case of trans‐sbR, is completely inhibited when the blue ‘acidic’ bR is formed at low pH. The protonation state of the group affecting formation of the M intermediate in 13‐cis‐bR at high pH and the group which is responsible for the transition to the blue acidic form influence in a similar way the equilibrium between bR isomers in the dark‐adapted form as well as the rate of dark adaptation.
The human sperm protein PH‐20 has hyaluronidase activity
Authors: Gmachl Michael , Sagan Sandrine , Ketter Sigrid , Kreil Günther
Published: July 2002

The PH‐20 protein present on the membrane of guinea pig sperm was characterized using a monoclonal antibody [(1991) J. Cell Biol. 111, 2939‐2949]. We have isolated the cDNA encoding the human PH‐20 protein from a testis library. This cDNA was expressed in RK 13 cells using a vaccinia virus expression system. Cells expressing the human PH‐20 protein possess hyaluronidase activity. Treatment with PI‐PLC releases the hyaluronidase into the the medium with a concomitant large increase in enzymatic activity. These results demonstrate that the human PH‐20 protein has hyaluronidase activity.
Diacylglycerol pyrophosphate, a novel phospholipid compound
Authors: Wissing Josef B. , Behrbohm Heike
Published: July 2002

In studies on lipid kinase activities of microsomal membranes from cultured plant cells a new, hitherto unknown, lipid kinase product was detected. The new phospholipid, labeled by [γ‐32P]ATP, could be separated from known phospholipid species by thin layer chromatography using different solvent mixtures. After partial purification of the related enzyme activity, the substrate of the unknown lipid kinase was elucidated as phosphatidic acid. With authentic phosphatidic acid and partially purified enzyme, the lipid kinase product was prepared in mg quantities and its structure was determined by mass spectrometry and NMR analyses as diacylglycerol pyrophosphate, a hitherto unknown phospholipid. The possible physiological role of this novel phospholipid metabolite is discussed.
Effect of divalent cations on Na+,K+‐ATPase obtained from human placenta
Authors: Zolese G. , Staffolani R. , Mazzanti L. , Gratton E.
Published: July 2002

Circular dichroism (CD) and acrylamide quenching studies of Na+,K+‐ATPase from human placenta showed that its incorporation into phosphatidylcholine vesicles increased the enzymic activity by 55%. Moreover, both with the purified and the vesicle‐reconstituted protein, Ca2+ and Mg2+ increased the activity, the effect being more pronounced after preincubation of the protein with Mg2+. CD data suggest that this activity increase may be linked to a change in the secondary structure of the ATPase, in particular β‐turn, β‐sheet and random coil. Acrylamide quenching studies suggest that ions could primarily interact with phospholipid head groups, but not directly with the protein.
Serum alleviates the requirement of the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐induced Ras activation for proliferation of BaF3 cells
Authors: Sakamakia Kazuhiro , Yonehara Shin
Published: July 2002

Deletion analysis of the β subunit of the human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptor previously defined two cytoplasmic regions required for distinct signaling. The membrane‐proximal region is responsible for induction of c‐myc and pim‐1, and is indispensable for GM‐CSF‐dependent proliferation of mouse BaF3 transfectants. The distal region is required for activation of Ras, Raf‐1, MAP kinase and p70 S6 kinase as well as induction of c‐fos and c‐jun, but is dispensable for GM‐CSF‐dependent proliferation of transfectants under normal culture conditions containing serum. Here we show that signals induced by the distal region of the β subunit are also required for proliferation. GM‐CSF supported proliferation of BaF3 transfectants expressing the normal β subunit, even in serum‐free medium. However, in the absence of serum, GM‐CSF did not support proliferation of BaF3 transfectants that have the β deletion mutants lacking the distal region. Serum‐induced activation of Ras, phosphorylation of MAP kinase and expression of c‐fos in parental BaF3 cells and antisense oligonucleotide against c‐raf blocked DNA synthesis of BaF3 cells. These results indicate that proliferation of BaF3 cells requires signals induced by the proximal as well as the distal region of the β subunit of the GM‐CSF receptor, and that serum alleviates the requirement of signals induced by the distal region.
Expression and stability of recombinant RQ‐mRNAs in cell‐free translation systems
Authors: Ugarov Victor I. , Morozov Igor Yu. , Jung Gyoo Y. , Chetverin Alexander B. , Spirin Alexander S.
Published: July 2002

Expression of dihydrofolate reductase (DHFR) and chloramphenicol acetyltransferase (CAT) mRNAs in cell‐free Escherichia coli translation systems is greatly enhanced as a result of their insertion into RQ135 RNA, a naturally occurring satellite of phage Qβ. The enhancement is due to protection of the recombinant mRNAs against endogenous ribonucleases and to an increased initial rate of translation in the case of the RQ‐CAT mRNA.
Cardiac L‐type Ca2+ channel triggers transmitter release in PC 12 cells
Authors: Avidor Boaz , Avidor Tomer , Schwartz Lydia , De Jongh Karen S. , Atlas Daphne
Published: July 2002

Among the various voltage‐sensitive Ca2+ channels present in PC 12 cells are the dihydropyridine (DHP)‐sensitive L‐channel, the ω‐conotoxin (ω‐CgTx)‐sensitive N‐channel, and an atypical ω‐CgTx/DHP‐insensitive Ca2+ channel. Depolarization‐evoked Ca2+ entry and [3H]dopamine release is mediated by L‐type Ca2+ channels determined by the use of Ca2+ channel antagonists, and a single protein of 250 kDa is recognized by L‐type‐specific antibodies. Screening of a PC12 cDNA library revealed two types of Ca2+channels which were identified by partial sequencing. A pc12‐L clone displayed virtually identical sequence homology to the cardiac L‐type channel. The identical sequence homology of the single alternative splicing region confirmed clone pc12‐L as the rbC‐I transcript, a cardiac‐neuronal α1 subunit expressed in rat brain. Clone pc12‐N displayed identical sequence homology to rbB‐I, a neuronal al subunit of the N‐type Ca2+ channel expressed in rat brain; Northern blot analysis identified RNA of a size similar to that previously described for rat brain.
Evidence for the existence of a single ubiquitin gene in Giardia lamblia
Authors: Krebber Heike , Wöstmann Claudia , Bakker-Grunwald Tilly
Published: July 2002

All eukaryotes investigated so far contain multiple copies of ubiquitin genes, most of which are arranged in fusions coding for either polyubiquitin or ubiquitin‐ribosomal protein constructs; the former are normally under the control of a heat shock promoter. Giardia lamblia, an intestinal parasite, is the most primitive eukaryote known to date. We have investigated the arrangement and expression of ubiquitin genes in this organism by Southern and Northern blotting. Our data strongly suggest that G. lamblia contains just one ubiquitin gene, which consists of a single copy of the coding sequence and the expression of which is not enhanced by heat shock. By pulsed‐field gel electrophoresis we localized this gene on the largest of the five giardial chromosomes. These data imply that the ubiquitin system in Giardia has probably been trapped at an original stage.
Effect of 6‐ketocholestanol on FCCP‐ and DNP‐induced uncoupling in plant mitochondria
Authors: Vianello A. , Macrì F. , Braidot E. , Mokhova E.N.
Published: July 2002

Effect of 6‐ketocholestanol on FCCP‐induced and DNP‐induced uncoupling in beef liver and pea stem mitochondria was studied, under experimental conditions at which this steroid abolished the effect of low concentrations of FCCP and other most potent uncouplers in rat mitochondria [Starkov et al. (1994) FEBS Lett., 355, 305–308]. It is shown that, in both types of mitochondria, 6‐ketocholestanol prevents or reverses the uncoupling induced by low concentrations of FCCP, but not that caused by high concentrations of FCCP or by any concentration of DNP. Progesterone and male sex hormones, showing recoupling capability in animal mitochondria, appear to be ineffective in the plant system. Cholesterol does not recouple in both animal and plant mitochondria. Plant steroids, such as β‐sitosterol and stigmasterol, are also without effect.
Identification and characterization of a low temperature regulated gene encoding an actin‐binding protein from wheat
Authors: Danyluk Jean , Carpentier Eric , Sarhan Fathey
Published: July 2002

A cDNA corresponding to a putative actin‐binding protein was cloned from a cold‐acclimated wheat cDNA library. The cDNA, designated Wcor719, encodes a polypeptide of 142 amino acids with a calculated molecular mass of 15.8 kDa and a pI of 4.27. The protein has the two conserved domains identified as actin and phosphatidylinositol 4,5‐biphosphate (PIP2) binding sites found in members of the cofilin family. Northern analyses revealed that Wcor719 transcript accumulation is rapid and strongly up‐regulated by low temperature. This accumulation was greater in the tolerant winter wheat and rye species compared to the less tolerant ones. The rapidity of transcript induction and the significant homology with actin‐binding proteins (ABP) from different organisms suggest that the product of this gene might be involved in the dynamic reorganization of the cytoskeleton during low temperature acclimation. It may also serve as a key factor in the signal transduction pathway during cold acclimation.
Transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H+ antiporter of Staphylococcus aureus
Authors: Fujihira Erika , Kimura Tomomi , Shiina Yasuko , Yamaguchi Akihito
Published: July 2002

Three transmembrane aspartyl residues play essential roles in the transposon Tn10‐encoded metal‐tetracycline/H+ antiporter (Tet(B)) [Yamaguchi, A. et al. (1992) J. Biol. Chem. 267, 7490–7498]. The tetK gene‐encoding tetracycline resistance protein (Tet(K)) of Staphylococcus aureus mediates metaltetracycline/H+ antiport similarly to Tet(B); however, it has no transmembrane aspartyl residue. On the other hand, Tet(K) has three glutamyl residues, Glu‐30, Glu‐152 and Glu‐397, in the putative transmembrane regions. In the present work, tet(K) gene was expressed in Escherichia coli and the transport activity was measured in everted membrane vesicles. When these glutamyl residues were replaced with Gln, the tetracycline transport activity was almost completely lost, indicating the important roles of these residues in Tet(K). In the case of Glu‐397, even the charge‐conserved mutation to Asp caused complete loss of the activity. On the other hand, the mutation of Glu‐30 and Glu‐152 to Asp resulted in significant retention of transport activity. These results are similar to those on the mutation of the three transmembrane aspartyl residues in Tet(B), indicating that the transmembrane glutamyl residues in Tet(K) play roles similar to those of the transmembrane aspartyl residues in Tet(B).
Multiple-outcome criteria in schizophrenia: an overview of outcome with clozapine
Authors: Meltzer HY
Published: July 2002

The outcome of treatment in schizophrenia is best considered as a multidimensional construct. This report lists thirteen categories of outcome measures that have been studied in schizophrenia research, among the most important of which are psychopathology, cognitive function, interpersonal social function, quality of life, extrapyramidal function, the suicide tendeccy and the need for hospitalisation. Clozapine is effective in achieving improved outcome in all these measures in a high percentage of neuroleptic-resistant patients. Ninety-two of 180 (51.1%) clozapine-treated neuroleptic-resistant patients achieved a decrease of 20% in Brief Psychiatric Rating Scale total scores at six weeks using the 0–6 scaling system. Cognitive function contributes importantly to overall function for schizophrenia. Clozapine has been shown to improve some types of cognition in schizophrenia, especially semantic memory and some tests of executive function. Clozapine has also been shown to decrease the incidence of suicide and hospitalisation. The task of deciding which therapies to use in schizophrenia should encompass the spectrum of benefits of each therapy, their risks and costs, in relation to all alternatives.
Epidemiology of schizophrenia. The disease model of schizophrenia in the light of current epidemiological knowledge
Authors: Häfner H
Published: July 2002

Attempting an update of the epidemiology of schizophrenia, it is pointed out that schizophrenia seems to occur with the same core symptoms and almost at the same frequency in all countries and cultures studied. Methodologically sound studies have failed to produce evidence for a secular trend of the morbid risk. The genotype of schizophrenia is expressed as psychosis, personality disorders and non-specific disorders or it goes without manifest psychopathology. Minor brain anomalies are present in most cases. The British Child Development Study showed that behavioural, cognitive, emotional and neuromotor antecedents occur in 50% of cases, thus pointing to disordered brain development, very likely not specific to schizophrenia, since found in many other mental disorders as well. A look into the hidden early course of schizophrenia revealed a significant sex difference in age of onset and a prodromal phase of some 3 to 4 years throughout the cases. A case-control study showed that it is mainly during this early course before first admission that social disadvantage in schizophrenia arises. In the prephase a disease-related lack of social ascent plays a greater role than steps of social decline. The early social course differs between the sexes mainly due to an earlier onset of the disorder in males. The actual disease variables, that is, core symptoms and type of course, do not show any essential differences between males and females. These results indicate that schizophrenia is one of the rare uniform patterns of response of the brain, capable of being triggered by a large number of causes or favoured by non-specific risk factors. In this context the protective effect of estrogens will be discussed.
Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL‐12 toward the novel substrates, partially N‐deacetylated 4‐methylumbelliferyl chitobiosides
Authors: Honda Yuji , Tanimori Shinji , Kirihata Mitsunori , Kaneko Satoshi , Tokuyasu Ken , Hashimoto Masayuki , Watanabe Takeshi , Fukamizo Tamo
Published: July 2002

The kinetic behavior of chitinase A1 from Bacillus circulans WL‐12 was investigated using the novel fluorogenic substrates, N‐deacetylated 4‐methylumbelliferyl chitobiosides [GlcN‐GlcNAc‐UMB (2), GlcNAc‐GlcN‐UMB (3), and (GlcN)2‐UMB (4)], and the results were compared with those obtained using 4‐methylumbelliferyl N,N′‐diacetylchitobiose [(GlcNAc)2‐UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. k
cat/K
m values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s−1 M−1 for 3. The lack of an N‐acetyl group at subsite (−1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N‐acetyl group at subsite (−2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a ‘substrate‐assisted’ mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (−2) toward the N‐acetyl group of the substrate sugar residue. The (−2) subsite of chitinase A1 was found to specifically recognize an N‐acetylated sugar residue, but this specificity was not as strict as that found in subsite (−1).
Srb7p is essential for the activation of a subset of genes
Authors: Gromöller Anne , Lehming Norbert
Published: July 2002

The mediator complex in the RNA polymerase II holoenzyme is known to be involved in transcriptional activation. The role of the essential mediator component Srb7p has been difficult to investigate, since no conditional lethal allele has been available to date. While the expression of Srb7p under the control of a repressible promoter is not sufficient to reduce the level of Srb7p beneath the threshold for survival, we have been able to isolate a clone termed ts16 which confers a temperature sensitive phenotype. ts16 contains an insertion mutation that requires translational frameshifting for correct expression of Srb7p, leading to extremely low protein levels. Strains bearing the ts16 construct show mild defects in the transcription of constitutive genes like TDH1 but severely affect activated transcription, e.g. of the GAL1 gene. In contrast, CUP1, which is also independent of other holoenzyme components, is not affected by ts16.
Electrogenic proton transfer in Rhodobacter sphaeroides reaction centers: effect of coenzyme Q10 substitution by decylubiquinone in the QB binding site
Authors: Gopta Oxana A , Semenov Alexey Y , Bloch Dmitry A
Published: July 2002

An electrometric technique was used to investigate the effect of coenzyme Q10 (UQ), substitution by decylubiquinone (dQ) at the QB binding site of reaction centers (UQ‐RC and dQ‐RC, respectively) on the electrogenic proton transfer kinetics upon QB reduction in Rhodobacter sphaeroides chromatophores. Unlike dQ‐RC, the kinetics of the second flash‐induced proton uptake in UQ‐RC clearly deviated from the mono‐exponential one. The activation energy (about 30 kJ/mol) and the pH profile of the kinetics in dQ‐RC were similar to those in UQ‐RC, with the power law approximation used in the latter case. The interpretation of the data presumed the quinone translocation between the two binding positions within the QB site. It is proposed that the native isoprenyl side chain (in contrast to decyl chain) favors the equilibrium binding of neutral quinone at the redox‐active ‘proximal’ position, but causes a higher barrier for the hydroquinone movement from ‘proximal’ to ‘distal’ position.
Identification of the cardiac ryanodine receptor channel in membrane blebs of sarcoplasmic reticulum
Authors: Böhle Thomas , Brandt Mathias C. , Henn Nadine , Schmidt Annette , Bloch Wilhelm , Beuckelmann Dirk J.
Published: July 2002

Blebs of the sarcoplasmic reticulum (SR) membrane of heart muscle cells were generated after saponin perforation of the plasma membrane followed by complete hypercontraction of the cell. Although characteristic proteins of the plasma membrane, namely the β1‐adrenoreceptor and Gαi, were stained by monoclonal antibodies in the hypercontracted cells, these proteins could not be detected in the adjacent blebs. Monoclonal antibodies to the cardiac ryanodine receptor (RyR2), calsequestrin and SERCA2 bound at different amounts to surface components of the blebs and to components of the hypercontracted cells. From the immunofluorescence signals we conclude that the blebs are mainly constituted of corbular and junctional SR membrane, and only to a lesser extent of network SR membrane. Deconvolution microscopy revealed that the membrane location of RyR2, calsequestrin and SERCA2 in the bleb is comparable to native SR membrane. At the bleb membrane giga‐ohm seals could be obtained and patches could be excised in a way that single‐channel currents could be measured, although these are not completely identified.
Post‐transcriptional regulation of plant ferritin accumulation in response to iron as observed in the maize mutant ys1
Authors: Fobis-Loisy Isabelle , Aussel Laurent , Briat Jean-Francois
Published: July 2002

The maize mutant ys1 accumulates iron in leaves to a lower extent than a Fe‐efficient genotype. In this mutant, ferritin mRNA accumulates in response to iron to a similar level as in other genotypes. However, ferritin protein and mRNA abundance does not correlate in ys1 leaves, demonstrating that iron also controls plant ferritin protein accumulation at the posttranscriptional level.
The reaction mechanism of phosphomannomutase in plants
Authors: Oesterhelt Christine , Schnarrenberger Claus , Gross Wolfgang
Published: July 2002

The enzyme phosphomannomutase catalyzes the interconversion of mannose‐1‐phosphate (Man‐1‐P) and mannose‐6‐phosphate (Man‐6‐P). In mammalian cells the enzyme has to be activated by transfer of a phosphate group from a sugar‐1.6‐P2 (Guha, S.K. and Rose, Z.B. (1985) Arch. Biochem. Biophys. 243, 168). In contrast, in the red alga Galdieria sulphuraria the co‐substrate (Man‐1.6‐P2 or Glc‐1.6‐P2) is converted to the corresponding sugar monophosphate while the substrate is converted to the sugar bisphosphate in each reaction cycle. Evidence is presented that the same reaction mechanism occurs in spinach and yeast.
A fragment of the major histocompatibility complex class II – associated p41 invariant chain inhibits cruzipain, the major cysteine proteinase from Trypanosoma cruzi
Authors: Bevec Tadeja , Stoka Veronika , Pungerčič Galina , Cazzulo Juan José , Turk Vito
Published: July 2002

A peptide fragment derived from the p41 form of the invariant chain (Ii) associated with the major histocompatibility complex (MHC) class II molecule has been shown to inhibit the mammalian lysosomal cysteine proteinase, cathepsin L, and to be a novel cysteine proteinase inhibitor, distinct from cystatins. Here we report that this same fragment also binds to and inhibits cruzipain, the cathepsin L‐like enzyme from the protozoan parasite Trypanosoma cruzi. The binding of the Ii fragment to cruzipain is fast (k
ass=2.4×107 M−1 s−1) and tight (K
i=5.8×10−11 M). The inhibition is competitive. These results suggest the possibility of using the invariant chain as a model for the specific inhibition of cruzipain in vivo, i.e. as a potential drug to combat Chagas' disease.
19F‐NMR studies of retinol transfer between cellular retinol binding proteins and phospholipid vesicles
Authors: Rong Ding , Lin Chan-Lan S , d'Avignon D.Andre , Lovey Allen J , Rosenberger Michael , Li Ellen
Published: July 2002

The cellular retinol binding proteins, CRBP and CRBP II, are implicated in the cellular uptake of retinol and intracellular trafficking of retinol between sites of metabolic processing. 19F‐NMR studies of retinol transfer between CRBP and CRBP II and phospholipid vesicles, using either fluorine‐labeled ligand or protein, demonstrated that there was significantly more transfer of retinol from CRBP II to lipid vesicles than from CRBP. Differences in how readily protein‐bound retinol is released to lipid bilayers may lead to differences in how these two proteins modulate intracellular retinol metabolism.
Rapid method to separate the domains of soybean lipoxygenase‐1: identification of the interdomain interactions
Authors: Sudharshan E , Appu Rao A.G
Published: July 2002

Lipoxygenase‐1 (LOX1) from soybeans was cleaved with chymotrypsin (Ramachandran et al., 31 (1992) 7700–7706). The domains were separated on a Sephadex G‐50 column by minimising domain interactions at pH 4.0. The molecular weight and apparent homogeneity of the domains were established by SDS‐PAGE. The solution conformation of the 60 kDa and 30 kDa fragments was compared with that of native LOX1. 1‐Anilino‐8‐naphthalene sulphonate (ANS) binding measurements confirmed the exposure of large hydrophobic residues on the surface of the 60 kDa due to separation of the domains. The monomeric nature of the 60 kDa fragment was confirmed by HPLC gel filtration. The increased number of binding sites and magnitude of binding constant suggested the involvement of extensive hydrophobic interactions between the two domains. The essential cofactor iron was with the C‐terminal domain. The attempts to resolve and reconstitute the catalytic activity of isolated domains were not successful.
The Aspergillus nidulans transcription factor AlcR forms a stable complex with its half‐site DNA: a NMR study
Authors: Cerdan Rachel , Collin Delphine , Lenouvel François , Felenbok Béatrice , Guittet Eric
Published: July 2002

The Aspergillus nidulans transcription factor AlcR is shown by NMR and gel retardation assay to form a stable complex with oligonucleotide sequences comprising the consensus half‐site 5′‐TGCGG‐3′. Apparent μM dissociation constants are evaluated by both methods. The measured lifetime of the complex is 74±7 ms at 20°C with the following DNA sequence: 5′‐C1G2T3G4C5G6G7A8T9C10‐3′. The major chemical shift variations upon binding involve both the two adjacent GC pairs (G6 and G7) and, clearly, the AT pairs at both ends of the consensus sequence (T3 and A8), suggesting additional contacts of the protein with the DNA. This extensive and strong interaction with the half‐site is another example of the variability in contacts of the fungal DNA‐binding proteins containing Zn2Cys6 domains with their consensus sites. It is the first demonstration that a binuclear cluster protein can bind to DNA as a monomer with strong affinity.
Medium‐reductant directed expression of methyl coenzyme M reductase isoenzymes in Methanobacterium thermoautotrophicum (strain ΔH)
Authors: Pennings Jeroen L.A , de Wijs Jeroen L.J , Keltjens Jan T , van der Drift Chris
Published: July 2002

Methanobacterium thermoautotrophicum was grown in a chemostat under various controlled conditions in the presence of either sodium sulfide or sodium thiosulfate. After establishment of the steady state, cells were taken and examined for expression of the mRNA transcripts coding for the different forms of methyl coenzyme M reductase (MCR) and methylene tetrahydomethanopterin dehydrogenase (MDH). MCR isoenzyme II expression varied most markedly. Expression was found not only to depend on known parameters temperature, pH and gassing rate, but also on the medium composition, especially the reductant present.