bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of the lymphoid cell kinase p56lck is stimulated by micromolar concentrations of Zn2+
Authors: Pernelle Jean-Jacques , Creuzet Claudine , Loeb Jacques , Gacon Gérard
Published: July 2002

In particulate fraction from LSTRA lymphoma cells, tyrosine phosphorylation of the lymphoid specific tyrosine kinase p56lck is elicited by Zn2+ in the absence of other divalent cations. Zn2+ alone also induces autophosphorylation of immunoprecipitated p56lck. The effect of Zn2+ is dose dependent; it is detected at concentrations of Zn2+ as low as 5 μM and reaches a maximum at 100 μM Zn2+. Among other divalent cations tested. Mn2+, and Co2+ to a lesser extent, were also effective. Zn2+ also stimulated p56lck phosphorylation in the presence of Mg2+ ions at physiological concentration, whereas orthovanadate had no effect. These results suggest that Zn2+ activates the autophosphorylation of p56lck; this fact could be related with the stimulating effect of Zn2+ in the activation of T lymphocytes.
Identification and partial characterisation of a low Mr collagen synthesised by bovine retinal pericytes Apparent relationship to type X collagen
Authors: Canfield A.E. , Schor A.M.
Published: July 2002

Bovine retinal pericytes (BRP) in culture synthesise a low M
r collagenous polypeptide which appears similar, but not identical, to bovine type X collagen and which we have called ‘BRP collagen’. This polypeptide displays the following characteristics: (i) it is sensitive to digestion by bacterial collagenase and is resistant to pepsin digestion; (ii) it has an apparent M
r of 45 kDa (pepsinised form); (iii) it is recognised by specific antibodies to type X collagen using immunoblotting; (iv) it is present in the cell layer/matrix but not in the medium of pericyte cultures; and (v) it is not disulphide‐bonded into higher M
r multimers. The latter two properties distinguish BRP collagen from bovine type X collagen. We have recently shown that pericytes calcify in vitro. We now report that this calcification is associated with an increased synthesis of BRP collagen.
The promoter of Saccharomyces cerevisiae FBA1 gene contains a single positive upstream regulatory element
Authors: Compagno Concetta , Ranzi Bianca Maria , Martegani Enzo
Published: July 2002

The glycolytic enzyme fructose 1,6‐bisphosphate aldolase is encoded by the FBA1 gene of Saccharomyces cerevisiae. Transcription of aldolase gene is not regulated by glucose and high levels of expression have been observed also during growth on nonfermentable carbon source. A FBA1::lacZ gene fusion was constructed and a deletion analysis demonstrated the presence of a unique cis‐acting positive upstream element (UAS) required for high levels of FBA1 expression. This element is located between positions −550 and −440 upstream of the aldolase open reading frame and it contains sequences known to constitute the binding sites for the multifunctional proteins RAP1 and ABF1 and two TTCC motifs.
Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi
Authors: Barras F. , Bortoli-German I. , Bauzan M. , Rouvier J. , Gey C. , Heyraud A. , Henrissat B.
Published: July 2002

Endoglucanase Z from the phytopathogenic bacterium Erwinia chrysanthemi (strain 3937) was purified by affinity chromatography on microcrystalline cellulose Avicel PH101. A kinetic characterization using p‐nitrophenyl β‐d‐cellobioside and p‐nitrophenyl β‐d‐lactoside as substrates was conducted: endoglucanase Z exhibited K
m values of 3 mM and 7.5 mM and V
m values of 129 and 40 nmol·min−1·mg−1 towards p‐nitrophenyl β‐d‐cellobioside (k
cat=0.1 s−1) and p‐nitrophenyl β‐d‐lactoside (k
cat=0.03 s−1), respectively). The hydrolysis of cellotetraitol by endoglucanase Z was followed by HPLC and 1H NMR. Results show that cellobiitol and β‐cellobiose are initially formed, demonstrating that the enzyme is acting by a molecular mechanism retaining the anomeric configuration. This suggests the involvement of a glycosyl‐enzyme intermediate.
Function of the lipopolysaccharide‐binding protein of Periplaneta americana as an opsonin
Authors: Jomori Takahito , Natori Shunji
Published: July 2002

Previously, we reported the purification of an LPS‐binding protein from the hemolymph of the American cockroach that was specific for E. coli LPS. In this study we found that this protein participated in the clearance of E. coil cells injected into the abdominal cavity of the cockroach, and that hemocytes ingested E. coli cells treated with this LPS‐binding protein in vitro. These findings suggest that this LPS‐binding protein acts as an opsonin.
Glucose has to be phosphorylated to activate glycogen synthase, but not to inactivate glycogen phosphorylase in hepatocytes
Authors: Carabaza Assumpta , Ciudad Carlos J. , Baqué Susanna , Guinovart Joan J.
Published: July 2002

2‐Deoxyglucose and 5‐thioglucose, in the same fashion as glucose, cause the inactivation of the rat hepatocyte glycogen phosphorylase and the activation of glycogen synthase. However, 6‐deoxyglucose and 1,5‐anhydroglucitol inactivate phosphorylase without increasing the activation state of glycogen synthase. With 3‐O‐methylglucose no changes in the activity or these enzymes occurred. These results prove that while glucose is the molecule that triggers the inactivation of phosphorylase, glucose 6‐phosphate is the signal for glucose synthase activation and that a metabolite control of the activation state of glycogen synthase is operative in hepatocytes.
Isolation and characterization of tissue inhibitors of metalloproteinases (TIMP‐1 and TIMP‐2) from human rheumatoid synovial fluid
Authors: Osthues Anja , Knäuper Vera , Oberhoff Renate , Reinke Heinz , Tschesche Harald
Published: July 2002

The tissue inhibitors of metalloproteinases TIMP‐1 and TIMP‐2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non‐covalent gelatinase/TIMP complexes. TIMP‐1 migrated as a single polypeptide with M
r 28 500 on SDS‐PAGE, while the M
r of TIMP‐2 was 21 000. The inhibitory activity was stable under heat and acid pH. N‐terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP‐1 and TIMP‐2. This is the first demonstration that TIMP‐1 and TIMP‐2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting.
Expression and purification of mouse TIMP‐1 from E. coli
Authors: Cocuzzi Enzo T. , Walther Susan E. , Rajan Shyamala , Denhardt David T.
Published: July 2002

Tissue inhibitors of metalloproteinases (TIMPs) constitute a family of secreted glycoproteins involved in regulating extracellular matrix degradation in both normal and malignant tissues. We have expressed a cDNA clone of mouse TIMP‐1 as a 22‐kDa protein with 12 cysteine residues in E. coli and purified protein that shows inhibitory activity against collagenase following renaturation by chemical means. The low specific activity and circular dichroism measurements suggest, however, that the renaturation of the mouse recombinant (non‐glycosylated) protein is not efficient under the conditions we have used, indicative of either thermodynamic instability or the transition to kinetic intermediates which have very low in vitro refolding rates.
The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin
Authors: Calvete Juan J. , Wang Yuqin , Mann Karlheinz , Schäfer Wolfram , Niewiarowski Stefan , Stewart Gwendolyn J.
Published: July 2002

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin β1 and β3 inhibitors of low molecular weight RGD‐containing, cysteine‐rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4‐Cys10 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys13‐Cys14 and Cys13‐Cys14. In echistatin, links between Cys14‐Cys37 and Cys20‐Cys34 were identified by direct chemical analysis.
Solid phase synthesis of the proteinase of bovine leukemia virus Comparison of its specificity to that of HIV‐2 proteinase
Authors: Bláha Ivo , Tözsér József , Kim Young , Copeland Terry D. , Oroszlan Stephen
Published: July 2002

The 126‐residue proteinase (PR) of bovine leukemia virus (BLV) was synthesized by solid‐phase peptide synthesis and its activity was shown using various oligopeptide substrates representing cleavage sites in BLV, human T‐cell leukemia virus type 1 (HTLV‐1), murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV‐1). The specificity of the BLV PR was also compared to that of chemically synthesized human immunodeficiency virus type 2 (HIV‐2) PR. Many of the peptides were cleaved at the expected site, however, 6 out of 15 were hydrolyzed only by one of the PRs. Furthermore, one BLV peptide was processed differently by the two enzymes. These results, together with the relative activities and the lack of inhibition of BLV PR by two HIV‐1 PR inhibitors, suggest that the BLV PR specificity is substantially different from that of HIV PRs.
Na+‐dependent high‐affinity uptake of l‐glutamate in cultured fibroblasts
Authors: Balcar Vladimir J.
Published: July 2002

Uptake of 1 μM[3H]l‐glutamate by cultured 3T3 fibroblasts was strongly dependent on extracellular Na+; it was reduced by elevated concentrations of K+ (60 mM) but it was not influenced by variations in the concentration of Ca2+ (0–9.6 mM). d‐ and l‐Asparate, d‐ and l‐threo‐3‐hydroxyaspartate dl‐threo‐3‐methylaspartate and a few other glutamate derivatives and analogues inhibited the uptake but several close analogues of l‐glutamate (including d‐glutamate) had no effect, implying that the uptake system is highly structurally selective. The recently identified inhibitor of glutamate uptake in synaptosomal preparations, l‐trans‐pyrrolidine‐2,4‐dicarboxylate, was also among the inhibitors. Apparent K
m of the uptake was found to be < 10 μM. The present observations indicate that Na+‐dependent ‘high‐affinity’ uptake of l‐glutamate may appear in structures which are apparently unrelated to glutamatergic synaptic transmission in the CNS.
A mechanism for NADPH inhibition of catalase compound II formation
Authors: Hillar A. , Nicholls P.
Published: July 2002

Catalase‐bound NADPH both prevents and reverses the accumulation of inactive bovine liver catalase peroxide compound II generated by ‘endogenous’ donors under conditions of steady H2O2 formation without reacting rapidly with either compound I or compound II. It thus differs both from classical 2‐electron donors of the ethanol type, and from 1‐electron donors of the ferrocyanide/phenol type. NADPH also inhibits compound II formation induced by the exogenous one‐electron donor ferrocyanide. A catalase reaction scheme is proposed in which the initial formation of compound II from compound I involves production of a neighbouring radical species. NADPH blocks the final formation of stable compound II by reacting as a 2‐electron donor to compound II and to this free radical. The proposed behaviour resembles that of labile free radicals formed in cytochrome c peroxidase and myoglobin. Such radical migration patterns within haem enzymes are increasingly common motifs.
Three‐dimensional structure of apotransketolase flexible loops at the active site enable cofactor binding
Authors: Sundström Michael , Lindqvist Yiva , Schneider Gunter
Published: July 2002

The structure determination of apotransketolase and the comparison of its three‐dimensional structure with that of the holoenzyme has revealed that no large conformational changes are associated with cofactor binding. Two loops at the active site are flexible in the apoenzyme which enables ThDP to reach its binding site. Binding of the cofactor induces defined conformations for these two loops at the active site. One of these loops is directly involving in binding of the cofactors, Ca2+ and ThDP. This loop acts like a flap which closes off the diphosphate binding site. After binding of the cofactor, residues of this loop form interactions to residues of loop 383–398 from the second subunit. These interactions stabilize the conformation of the two loops from a flexible to a ‘closed’ conformation.
Human surfactant polypeptide SP‐B Disulfide bridges, C‐terminal end, and peptide analysis of the airway form
Authors: Johansson Jan , Jörnvall Hans , Curstedt Tore
Published: July 2002

Human hydrophobic surfactant polypeptide, SP‐B, purified from lung tissue by exclusion chromatography in organic solvents, has been characterized. The polypeptide is 79 residues long, has a C‐terminal methionine, and contains seven Cys residues. Native human SP‐B lacks free thiol groups. Three intrachain disulfide bridges were defined, linking CysK to Cys77, Cys11 to Cys71 and Cys35 to Cys46. The remaining Cys48 is concluded to link the protein chains into homodimers via an interchain disulfide to its counterpart in a second SP‐B polypeptide. These SS bridges are identical to those in the porcine form and confirm a consistant and unique disulfide pattern for SP‐B polypeptides in general.
The second cholera toxin, Zot, and its plasmid‐encoded and phage‐encoded homologues constitute a group of putative ATPases with an altered purine NTP‐binding motif
Authors: Koonin Eugene V.
Published: July 2002

It is shown that the second cholera toxin, Zot, ORF3 product of Pseudomonas plasmid pKB740, and ORF424 product of bacteriophage Pfl are a group of closely related proteins containing a modified version of the purine NTP‐binding motif, with a drastic substitution of tyrosine for a conserved glycine. They are distantly but reliably related to the product of gene I of filamentous bacteriophages which is a putative ATPase containing the classical NTP‐binding motif and is involved in bacteriophage assembly and exit from the bacterial cell. Hydropathy analysis suggests that the Zot and gene I product may have a similar transmembrane topology. It is hypothesized that Zot may possess ATPase activity and modify the membrane structure of its target cells in an ATP‐dependent fashion. Genes for Zot and the related protein of pKB740 are likely to have evolved from gene I of a Pf1‐like bacteriophage.
SC‐0051, a 2‐benzoyl‐cyclohexane‐1,3‐dione bleaching herbicide, is a potent inhibitor of the enzyme p‐hydroxyphenylpyruvate dioxygenase
Authors: Schulz Arno , Ort Oswald , Beyer Peter , Kleinig Hans
Published: July 2002

Growth inhibition of Lemna gibba plantlets by the bleaching herbicide, SC‐0051 (2‐(2‐chloro‐4‐methanesulfonylbenzoyl)‐1,3‐cyclohexanedione)) was alleviated by the addition of homogentisic acid to the growth medium. Homogentisic acid is a key intermediate in the biosynthesis of tyrosine‐derived plant quinones as well as in tyrosine metabolism. The herbicide prevented the incorporation of radioactivity from [14C]tyrosine into lipophilic plant metabolites and, in rat liver extracts, the herbicide inhibited the conversion of tyrosine to homogentisic acid. The enzyme p‐hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) from both Zea mays seedlings and liver tissues, was found to be subject to strong inhibition by SC‐0051. Inhibition of plant quinone biosynthesis is a new mode of herbicidal action. One of the consequences of quinone depletion in plants in vivo is apparently an indirect inhibition of phytoene desaturation. The enzyme phytoene desaturase itself, however, is not afflicted by the herbicide.
Identification of a cross‐linked double‐peptide from the catalytic site of the Ca2+‐ATPase of sarcoplasmic reticulum formed by the Ca2+‐ and pH‐dependent reaction with ATP γP‐imidazolidate
Authors: Gutowski-Eckel Zeynep , Mann Karlheinz , Bäumert Hans G.
Published: July 2002

The Ca2+‐ATPase from sarcoplasmic reticulum can be inhibited by the Ca2+‐ and pH‐dependent reaction with ATP γP‐imidazolidate. The chemically and monofunctionally activated inhibitor introduces an intramolecular cross‐link between two neighbouring peptides of the active site. This can be followed by the reduced mobility of the ATPase upon SDS‐PAGE analysis which becomes even more pronounced after limited trypsinolysis. After cleavage of the cross‐linked ATPase molecule by cyanogen bromide and separation of the peptides a double‐peptide can be detected which upon sequencing can be identified as part of the phosphorylation and the nucleotide binding site, respectively.
Characterization of the 97 and 103 kDa forms of starch branching enzyme from potato tubers
Authors: Khoshnoodi Jamshid , Ek Bo , Rask Lars , Larsson Håkan
Published: July 2002

N‐Terminal analysis, peptide mapping and partial peptide sequencing of the 97 and 103 kDa forms of starch branching enzyme from potato tubers showed that the two forms are highly related. A comparison with sequence data in the literature showed that these forms belong to the starch branching enzyme isoform I family. An internal cDNA fragment was obtained using PCR technology on potato tuber RNA with two oligonucleotide primers constructed from the peptide sequence data. Southern blot analysis using the PCR fragment as probe showed that there is only one gene locus encoding this isoform of the enzyme in Solanum tuberosum as well as in Solanum commersonii.
The rat β3‐adrenergic receptor gene contains an intron
Authors: Bensaid M. , Kaghad M. , Rodriguez M. , Le Fur G. , Caput D.
Published: July 2002

We report that the rat β3‐adrenergic receptor (β3‐AR) gene has an intron. The intron starts with an in‐frame stop codon with the result that unspliced transcripts will encode a C‐terminal truncated protein. The reported protein sequences of mouse and human β3‐AR were both deduced from genomic DNA sequences. Given the heterogeneity at the C‐termini of the otherwise highly similar rat, mouse and human sequences, we discuss the intriguing possibility that the β3‐AR gene of the latter two species also contain an intron near the extremity of the open reading frame. A β‐adrenergic receptor (β‐AR) cDNA we have cloned from rat colonic tissue which has a sequence essentially identical to that previously reported for the rat adipose β3‐AR cDNA [(1991) J. Chem. 266, 24053], encodes the spliced version of the β3‐AR.
Down‐modulation of CD4 antigen during programmed cell death in U937 cells
Authors: Malorni Walter , Rivabene Roberto , Teresa Santini Maria , Paradisi Silvia , Iosi Francesca , Donelli Gianfranco
Published: July 2002

It has been hypothesized that programmed cell death (PCD), an active cell suicide process occurring in place of necrosis, can be associated with the pathogenesis of acquired immunodeficiency syndrome (AIDS). The entry of human immunodeficiency virus (HIV) into competent cells is mediated by the CD4 molecule present on the surface of certain lymphocyte subpopulations as well as on some cultured cell lines, e.g. U937 myelomonocytic cells. The present paper focuses on some specific aspects of PCD induced by the cytokine tumor necrosis factor (TNF). The results obtained indicate that the exposure of U937 cells to cycloheximide facilitates TNF‐mediated PCD via a short term cell death program and modifies the expression of CD4 surface molecules. This change in surface antigen expression, manifested by internalization of the CD4 molecule, occurs in cells in which apoptosis has been triggered, but not in cells undergoing necrosis. These results indicate that the progression of cell death could be associated with specific alterations of certain surface molecules and could have a role in the entry of HIV into cells.
Organisation of the chicken annexin V gene and its correlation with the tertiary structure of the protein
Authors: Pfannmüller Eva , Turnay Javier , Bertling Wolf , von der Mark Klaus
Published: July 2002

Chicken annexin V (anchorin CII) is a collagen binding, membrane‐associated molecule with Ca2+ channel activity. Here we report on the coding sequences, promoter region, size and distribution of exons, and exon‐intron junctions of the chicken annexin V gene. It is about 25 kb long and codes for 13 short exons between 50 and 581 bp length. Exon sizes and locations of splice sites are almost completely homologous to those of the human and mouse annexin II or pigeon annexin I genes, although there is only 50–60% homology in the sequence of the corresponding proteins. The four repeat structure and symmetry of the annexin V as evident from sequence and X‐ray analysis studies is only partially reflected in this highly conserved exon distribution. In the first two repeats of chicken annexin V the exons correlate with protein domains containing one, two, or three α‐helices, while in the repeats 3 and 4 exon junctions and α‐helical domains do not correlate. The analysis of the promotor structure revealed the absence of a typical TATA‐box, but a GC‐rich region which may possibly promote transcription from several start sites.
Rapid and transient thrombin stimulation of phosphatidylinositol 4,5‐bisphosphate synthesis but not of phosphatidylinositol 3,4‐bisphosphate independent of phospholipase C activation in platelets
Authors: Racaud-Sultan Claire , Mauco Gérard , Guinebault Christine , Plantavid Monique , Payrastre Bernard , Breton Monique , Chap Hugues
Published: July 2002

When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5‐bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin‐2‐tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5‐bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4‐bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5‐bisphosphate and phosphatidylinositol 3,4‐bisphosphate.
Strontium binding to sarcoplasmic reticulum Ca2+‐ATPase
Authors: Orlowski Stéphane , Champeil Philippe
Published: July 2002

We investigated the consequences of Sr2+ binding to the transport sites of sarcoplasmic reticulum (SR) Ca2+‐ATPase for two fluorescent conformational probes located in different regions of the ATPase. Using SR vesicles in which Lys‐515 in the ATPase had been previously labeled with fluorescein 5'‐isothiocyanate (FITC), we found that the Sr2+‐induced a drop in the fluorescein fluorescence of this FITC‐labeled ATPase shifted toward lower Sr2+ concentrations than the Sr2+‐induced rise in Trp fluorescence for the same FITC‐labeled ATPase. The curve describing the Sr2+‐dependent rise in Trp fluorescence had a characteristic asymmetric shape, and the changes in Trp fluorescence occurred in parallel with the activation by Sr2+ of pNPP hydrolysis by the ATPase. Analysis of these results in terms of the simplest scheme describing the sequential binding of the two Sr2+ ions suggests that under the conditions of these experiments, i.e. at neutral pH in the presence of potassium, the Sr2+‐induced rise in the Trp fluorescence mainly reflected the formation of ATPase with two ions bound to the transport sites, whereas the binding of a single Sr2+ ion was virtually sufficient to reduce the fluorescence of bound FITC to its minimal level.
Proper processing of a G protein γ subunit depends on complex formation with a β subunit
Authors: Pronin Alexey N. , Gautam Narasimhan
Published: July 2002

G protein β and γ subunits function as a tightly associated complex. We show that complex formation with the β subunit is a critical step for post‐translational processing of a γ subunit. When expressed alone in a cell line, the γ3 subunit type is isoprenylated but degraded; co‐expression with theβ1 subunit type stabilizes the γ3 protein. Furthermore, our experiments with partial cell fractionation indicate that the γ3 protein is localized differently in the cell depending on whether or not it is bound to the β subunit. Binding of the γ subunit to the β subunit is thus one of the prerequisites for the appropriate intracellular localization of the βγ complex and potentially, for normal G‐protein function.
High resolution proton NMR investigations of rat blood plasma Assignment of resonances for the molecularly mobile carbohydrate side‐chains of ‘acute‐phase’ glycoproteins
Authors: Grootveld Martin , Claxson Andrew W.D. , Chander Chaman L. , Haycock Peter , Blake David R. , Hawkes Geoffrey E.
Published: July 2002

An intense broad resonance at 2.14 ppm present in high field (400, 500 and 600 MHz) Hahn spin‐echo 1H‐NMR spectra of rat blood plasma, but absent from those of human blood plasma is attributable to the presence of terminal O‐acetylsialate sugars in the molecularly mobile carbohydrate side‐chains of ‘acute‐phase’ glycoproteins (predominantly α1‐acid glycoprotein). The presence of such alternative acetylsugars in the carbohydrate side‐chains of rat plasma glycoproteins are of much physiological and experimental significance in view of the regular use of these animals in model systems of human inflammatory conditions.
Calcium homeostasis in yeast cells exposed to high concentrations of calcium Roles of vacuolar H+‐ATPase and cellular ATP
Authors: Halachmi D. , Eilam Y.
Published: July 2002

Cytosolic Ca2+ concentrations ([Ca2+]i) were determined in haploid and diploid cells of Saccharomyces cerevisiae, loaded with indo‐1 and exposed to media containing a range of Ca2+ concentrations. [Ca2+]i homeostasis was maintained at the 100–150 nM level in cells which were pre‐incubated with glucose and exposed to O.1 μM–1O mM Ca2+ in the medium. Slightly higher levels of [Ca2+]i were determined in cells exposed to 50 mM Ca2+. Pre‐incubation with metabolic inhibitors instead of glucose caused a reduction in cellular ATP levels and an impaired [Ca2+]i homeostasis; [Ca2+]i reached 800 nM in cells exposed to 10 mM CaCl2. Cells of the Δvma4 mutant strain, with no functional vacuolar H1‐ATPase, had elevated levels of [Ca2+]i, reaching 1.8 μM when pre‐incubated with glucose and exposed to 10 mM CaCl2. Higher levels of [Ca2+]i were measured in the mutant cells which were pre‐incubated with metabolic inhibitors. This result indicates the central role of the vacuoles in maintaining [Ca2+]i‐homeostasis and suggests the presence of an additional non‐vacuolar ATP‐requiring mechanism which contributes to keeping [Ca2+]i at low levels.
Retention of native‐like structure in an acyclic counterpart of a β‐sheet antibiotic
Authors: Maplestone Rachael A. , Cox Jonathan P.L. , Williams Dudley H.
Published: July 2002

An acyclic derivative of the cyclic peptide antibiotic, ramoplanin, has been prepared. In aqueous solution, two‐dimensional NMR spectroscopy indicates that the acyclic form adopts a threshold population of conformers in which at least part of the β‐sheet characteristic of the intact ramoplanin persists. Thus, despite losing the entropic benefit which the macrocycle must lend to β‐sheet formation, the polypeptide chain of the acyclic ramoplanin appears to display an innate tendency to adopt a native‐like conformation.
Surface engineered nanospheres with enhanced drainage into lymphatics and uptake by macrophages of the regional lymph nodes
Authors: Moghimi S.M. , Hawley A.E. , Christy N.M. , Gray T. , Illum L. , Davis S.S.
Published: July 2002

The concept of steric stabilization as used in colloid science is applied to carefully manipulate the drainage and lymphatic distribution of subcutaneously administered model polystyrene nanospheres. A wide range of synthetic polyoxyethylene (POE)/polyoxypropylene (POP) block co‐polymers of poloxamine and poloxamer series have been used to produce sterically stabilized nanospheres. We have found a correlation between the length of the stabilizing POE chains of the block copolymers and nanosphere drainage and passageway across tissue lymph interface in dermal lymphatic capillaries in the rat footpads; the longer the POE chains, the faster the particle drainage. Nanospheres conditioned with block co‐polymers of POE chains of 5–15 ethylene oxide units are effectively opsonized in lymphatics; a process which dramatically enhances sequestration (up to 40% of the administered dose) by macrophages of the regional lymph nodes. If the dimensions of the stabilizing POE chains of the poloxamines and poloxamers exceed the range of the Van der Waals force of attraction, opsonization fails to occur and rapidly drained engineered vehicles escape clearance by macrophages of the regional nodes, reach the systemic circulation and remain in the blood for prolonged periods. These observations suggest that a lymphatic delivery composition based on polymer‐coated particles will be advantageous for many applications in clinical and experimental medicine.
Calcium mobilisation controls tyrosine protein phosphorylation independently of the activation of protein kinase C in human platelets
Authors: Falet Hervé , Rendu Francine
Published: July 2002

We have investigated the regulation of tyrosine proteins phosphorylation by intracellular Ca2+ level ([Ca2+]i) and protein kinase C (PKC) during platelet stimulation. We found that chelation of extracellular calcium completely prevented phosphorylation of tyrosine proteins induced by thapsigargin and phorbol 12‐myristate 13‐acetate (PMA), whereas, when induced by thrombin, it prevented a subset of tyrosine proteins. The selective inhibition of PKC by OF 109203X did not abolish tyrosine protein phosphorylation when induced by thrombin and thapsigargin. The results suggest that in human platelets tyrosine protein phosphorylation is dependent on [Ca2+]i, although direct PKC activation can also induce phosphorylation of tyrosine proteins.
Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C‐terminal sequence
Authors: Sommer Jürg M. , Nguyen Tien T. , Wang C.C.
Published: July 2002

Import of proteins into the glycosomes of T. brucei resembles the peroxisomal protein import in that C‐terminal SKL‐like tripeptide sequences can function as targeting signals. Many of the glycosomal proteins do not, however, possess such C‐terminal tripeptide signals. Among these, phosphoenolpyruvate carboxykinase (PEPCK (ATP)) was thought to be targeted to the glycosomes by an N‐terminal or an internal targeting signal. A limited similarity to the N‐terminal targeting signal of rat peroxisomal thiolase exists at the N‐terminus of T. brucei PEPCK. However, we found that this peroxisomal targeting signal does not function for glycosomal protein import in T. brucei. Further studies of the PEPCK gene revealed that the C‐terminus of the predicted protein does not correspond to the previously deduced protein sequence of 472 amino acids due to a −1 frame shift error in the original DNA sequence. Readjusting the reading frame of the sequence results in a predicted protein of 525 amino acids in length ending in a tripeptide serine‐arginine‐leucine (SRL), which is a potential targeting signal for import into the glycosomes. A fusion protein of firefly luciferase, without its own C‐terminal SKL targeting signal, and T. brucei PEPCK is efficiently imported into the glycosomes when expressed in procyclic trypanosomes. Deletion of the C‐terminal SRL tripeptide or the last 29 amino acids of PEPCK reduced the import only by about 50%, while a deletion of the last 47 amino acids completely abolished the import. These results suggest that T. brucei PEPCK may contain a second, internal glycosomal targeting signal upstream of the C‐terminal SRL sequence.
Epidermal growth factor and transforming growth factor‐α mRNA in rat small intestine: In situ hybridization study
Authors: Dvořák Bohuslav , Holubec Hana , LeBouton Albert V. , Wilson Jean M. , Koldovský Otakar
Published: July 2002

The expression of epidermal growth factor (EGF) and transforming growth factor‐α (TGF‐α) mRNA in the small intestine of suckling and adult rats was examined by in situ hybridization. EGF mRNA was found mainly in the intestinal crypts in adult rats. Adult rats also exhibited a considerably stronger signal for EGF mRNA in comparison to suckling rats, where the signal was very low or absent. In contrast to EGF, very strong expression of TGF‐α mRNA was observed in the small intestine of both adult and suckling rats. These data suggest the differences between the expression of EGF and TGF‐α in the developing small intestine.
Refined structure for the complex of d‐gluco‐dihydroacarbose with glucoamylase from Aspergillus awamori var. X100 to 2.2 Å resolution: dual conformations for extended inhibitors bound to the active site of glucoamylase
Authors: Stoffer Bjarne , Aleshin Alexander E. , Firsov Leonid M. , Svensson Birte , Honzatko Richard B.
Published: July 2002

The crystal structure at pH 4 of the complex of glucoamylase II(471) from Aspergillus awamori var. X100 with the pseudotetrasaccharide d‐gluco‐dihydroacarbose has been refined to an R‐factor of 0.125 against data to 2.2 Å resolution. The first two residues of the inhibitor bind at a position nearly identical to those of the closely related inhibitor acarbose in its complex with glucoamylase at pH 6. However, the electron density bifurcates beyond the second residue of the d‐gluco‐dihydroacarbose molecule, placing the third and fourth residues together at two positions in the active site. The position of relatively low density (estimated occupancy of 35%) corresponds to the location of the third and fourth residues of acarbose in its complex with glucoamylase at pH 6. The position of high density (65% occupancy) corresponds to a new binding mode of an extended inhibitor to the active site of glucoamylase. Presented are possible causes for the binding of d‐gluco‐dihydroacarbose in two conformations at the active site of glucoamylase at pH 4.
Structure and mechanism of inositol monophosphatase
Authors: Atack John R. , Broughton Howard B. , Pollack Scott J.
Published: July 2002

Since lithium inhibits IMPase and modulates phosphatidylinositol (PtdIns) cell signalling at therapeutically relevant concentrations (0.5–1.0 mM), IMPase has attracted attention as a putative molecular target for lithium in the treatment of manic depression. IMPase is a homodimer, with each subunit organised in an αβαβα arrangement of α‐helices and β‐sheets, and this type of structure seems crucial to the two‐metal catalysed mechanism in which an activated water molecule serves as a nucleophile. Lithium appears to inhibit the enzyme following substrate hydrolysis by occupying the second metal binding site before the phosphate group can dissociate from its interaction with the site 1 metal. The understanding of IMPase structure and the mechanism of substrate hydrolysis and lithium inhibition should be useful in the development of novel inhibitors which may prove clinically useful in the treatment of manic depression.
Class III alcohol dehydrogenase from Saccharomyces cerevisiae: Structural and enzymatic features differ toward the human/mammalian forms in a manner consistent with functional needs in formaldehyde detoxication
Authors: Rosario Fernández M. , Biosca Josep A. , Norin Annika , Jörnvall Hans , Parés Xavier
Published: July 2002

Alcohol dehydrogenase class III (glutathione‐dependent formaldehyde dehydrogenase) from Saccharomyces cerevisiae was purified and analyzed structurally and enzymatically. The corresponding gene was also analyzed after cloning from a yeast genome library by screening with a probe prepared through PCR amplification. As with class III alcohol dehydrogenase from other sources, the yeast protein was obtained in two active forms, deduced to reflect different adducts/modifications. Protein analysis established N‐terminal and C‐terminal positions, showing different and specific patterns in protein start positions between the human/mammalian, yeast, and prokaryotic forms. K
m values with formaldehyde differ consistently, being about 10‐fold higher in the yeast than the human/mammalian enzymes, but compensated for by similar changes in κ
cat values. This is compatible with the different functional needs, emphasizing low formaldehyde concentration in the animal cells but efficient formaldehyde elimination in the microorganisms. This supports a general role of the enzyme in formaldehyde detoxication rather than in long‐chain alcohol turnover.
Business environment, operations strategy, and performance: An empirical study of Singapore manufacturers
Authors: Ward Peter T. , Duray Rebecca , Keong Leong G. , Sum Chee‐Chuong
Published: July 2002

Consideration of the task environment, those forces which are out of the short‐run control of management, has been relatively neglected in operations strategy research. The neglect of environmental factors in operations strategy research is surprising when one considers that the fit between environment and organizational capabilities and resources is a central tenet of major stretegic management paradigms. We use a path analytic framework to study the effects of environment on operations strategy selection and performance (self‐reported change in profits) for a sample of Singapore manufacturers.

We identify strong relationships between environmental factors such as labor availability, competitive hostility, and market dynamism and the operations strategy choices encompassed by competitive priorities. The data also indicate that, when faced with the same environmental stimuli, high performers choose to emphasize different competitive priorities than low performers. In addition to exploring substantive questions about the importance of the environment in explaining operations strategy, this research also demonstrates that environmental variables can provide effective controls for industry effects in multiple industry empirical studies in operations strategy.
Synchronous glucose‐dependent [Ca2+]i oscillations in mouse pancreatic islets of Langerhans recorded in vivo
Authors: Fernandez J , Valdeolmillos M
Published: July 2002

Using microfluorescence in combination with image‐analysis techniques we monitored intracellular calcium ([Ca2+]i) dynamics in mouse islets of Langerhans loaded with fura‐2 and recorded in vivo. [Ca2+]i oscillates in the glycaemias range 5–10 mM, the duration of the oscillations being directly proportional to the blood glucose concentration. The analysis of different areas within the same islet shows that [Ca2+]i oscillations are synchronous throughout the islet. These results show that in vivo, individual islets of Langerhans behave as a functional syncytium and suggest the existence of secretory pulses of insulin.
Role of reactive oxygen species and phosphatidylinositol 3‐kinase in cardiomyocyte differentiation of embryonic stem cells
Authors: Sauer Heinrich , Rahimi Gohar , Hescheler Jürgen , Wartenberg Maria
Published: July 2002

Cardiotypic development in embryonic stem cell‐derived embryoid bodies may be regulated by reactive oxygen species (ROS). ROS were generated by a NADPH oxidase‐like enzyme which was transiently expressed during the time course of embryoid body development. Incubation with either H2O2 or menadione enhanced cardiomyogenesis, whereas the radical scavengers trolox, pyrrolidinedithiocarbamate and N‐acetylcysteine exerted inhibitory effects. The phosphatidylinositol 3‐kinase (PI‐3‐kinase) inhibitors LY294002 and wortmannin abolished cardiac commitment and downregulated ROS in embryoid bodies. Coadministration of LY294002 with prooxidants resumed cardiomyocyte differentiation, indicating a role for PI‐3‐kinase in the regulation of the intracellular redox state.
Evidence for an active role of the DnaK chaperone system in the degradation of σ32
Authors: Tatsuta Takashi , Joo Daniel M. , Calendar Richard , Akiyama Yoshinori , Ogura Teru
Published: July 2002

Under non‐stressed conditions in Escherichia coli, the heat shock transcription factor σ32 is rapidly degraded by the AAA protease FtsH. The DnaK chaperone system is also required for the rapid turnover of σ32 in the cell. It has been hypothesized that the DnaK chaperone system facilitates the degradation of σ32 by sequestering it from RNA polymerase core. This hypothesis predicts that mutant σ32 proteins, which are deficient in binding to RNA polymerase core, will be degraded independently of the DnaK chaperone system. We examined the in vivo stability of such mutant σ32 proteins. Results indicated that the mutant σ32 proteins as similar as authentic σ32 were stabilized in ΔdnaK and ΔdnaJ/ΔcbpA cells. The interaction between σ32 and DnaK/DnaJ/GrpE was not affected by these mutations. These results strongly suggest that the degradation of σ32 requires an unidentified active role of the DnaK chaperone system.
Two nuclear localization signals are required for nuclear translocation of nuclear factor 1‐A
Authors: Imagawa Masayoshi , Sakaue Reiko , Tanabe Atsuhiro , Osada Shigehiro , Nishihara Tsutomu
Published: July 2002

Nuclear factor 1 (NF1) proteins are encoded by at least four genes (NF1‐A, B, C, X). Although DNA‐binding and the transcription regulation domains of these proteins are well characterized, the nuclear localization signals (NLSs) are still unknown in all NF1s. We have identified two NLSs in NF1‐A, and both are required for full translocation to the nucleus, although one of them itself has a partial translocation ability. These two NLSs are conserved in all four NF1s. Interestingly, three isoforms of NF1‐A (NF1‐A1, A2, A4) have two NLSs and translocate completely to the nucleus. In contrast, NF1‐A3 lacks the second NLS and partially stays in the cytoplasm. Since NF1s construct homodimer and heterodimer, these findings indicate the differential regulations of the NF1 translocation.
Inhibition of Mycobacterium smegmatis topoisomerase I by specific oligonucleotides
Authors: Bhaduri Tisha , Basak Shashwati , Sikder Devanjan , Nagaraja Valakunja
Published: July 2002

DNA topoisomerase I from Mycobacterium smegmatis unlike many other type I topoisomerases is a site specific DNA binding protein. We have investigated the sequence specific DNA binding characteristics of the enzyme using specific oligonucleotides of varied length. DNA binding, oligonucleotide competition and covalent complex assays show that the substrate length requirement for interaction is much longer (∼20 nucleotides) in contrast to short length substrates (eight nucleotides) reported for Escherichia coli topoisomerase I and III. P1 nuclease and KMnO4 footprinting experiments indicate a large protected region spanning about 20 nucleotides upstream and 2–3 nucleotides downstream of the cleavage site. Binding characteristics indicate that the enzyme interacts efficiently with both single‐stranded and double‐stranded substrates containing strong topoisomerase I sites (STS), a unique property not shared by any other type I topoisomerase. The oligonucleotides containing STS effectively inhibit the M. smegmatis topoisomerase I DNA relaxation activity.
Genomic Exploration of the Hemiascomycetous Yeasts: 5. Saccharomyces bayanus var. uvarum
Authors: Bon Elisabeth , Neuvéglise Cécile , Casaregola Serge , Artiguenave François , Wincker Patrick , Aigle Michel , Durrens Pascal
Published: July 2002

Saccharomyces bayanus var. uvarum investigated here is the species closest to Saccharomyces cerevisiae. Random sequence tags (RSTs) allowed us to identify homologues to 2789 open reading frames (ORFs) in S. cerevisiae, ORFs duplicated in S. uvarum but not in S. cerevisiae, centromeres, tRNAs, homologues of Ty1/2 and Ty4 retrotransposons, and a complete rDNA repeat. Only 13 RSTs seem to be homologous to sequences in other organisms but not in S. cerevisiae. As the synteny between the two species is very high, cases in which synteny is lost suggest special mechanisms of genome evolution. The corresponding RSTs revealed that S. uvarum can exist without any S. cerevisiae DNA introgression. Accession numbers are from AL397139 to AL402278 in the EMBL databank.
Protein kinase C μ selectively activates the mitogen‐activated protein kinase (MAPK) p42 pathway
Authors: Hausser Angelika , Storz Peter , Hübner Susanne , Braendlin Ilona , Martinez-Moya Marina , Link Gisela , Johannes Franz-Josef
Published: July 2002

Here we show that human protein kinase C μ (PKCμ) activates the mitogen‐activated protein kinase (MAPK). Transient expression of constitutive active PKCμ leads to an activation of Raf‐1 kinase as demonstrated by in vitro phosphorylation of MAPK. PKCμ enhances transcriptional activity of a basal thymidine kinase promotor containing serum response elements (SREs) as shown by luciferase reporter gene assays. SRE driven gene activation by PKCμ is triggered by the Elk‐1 ternary complex factor. PKCμ‐mediated activation of SRE driven transcription can be inhibited by the MEK1 inhibitor PD98059. In contrast to the activation of the p42/ERK1 MAPK cascade, transient expression of constitutive active PKCμ does neither affect c‐jun N‐terminal kinase nor p38 MAPK.
Functional consequences of the autosomal dominant G272A mutation in the human GLUT1 gene
Authors: Klepper Jörg , Monden Ingrid , Guertsen Elena , Voit Thomas , Willemsen Michèl , Keller Konrad
Published: July 2002

The first autosomal dominant missense mutation (G272A) reported within the human GLUT1 gene and shared by three affected family members was investigated in respect to functional consequences. Substitution of glycine‐91 by site‐directed mutagenesis with either aspartate or alanine resulted in a significant decrease in transport activity of GLUT1 expressed in Xenopus oocytes. Expression of mutant transporters was confirmed by immunoblot, 2‐deoxy‐glucose uptake and confocal laser microscopy. The data agree with 3‐O‐methyl‐glucose uptake into patient erythrocytes and indicate that the loss of glycine rather than a hydrophilic side chain (Gly91Asp) defines the functional consequences of this mutation.
Novel tetramer assembly of pyruvate decarboxylase from brewer's yeast observed in a new crystal form
Authors: Lu Guoguang , Dobritzsch Doreen , König Stephan , Schneider Gunter
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Glutamine‐330 is not essential for activity in isopenicillin N synthase from Aspergillus nidulans
Authors: Sami Malkit , Brown Toby J.N , Roach Peter L , Schofield Christosper J , Baldwin Jack E
Published: July 2002

© 1997 Federation of European Biochemical Societies.
What is the diameter of the actin filament?
Authors: Grazi Enrico
Published: July 2002

The limits of the most recent models of the actin filament are discussed. These model are generated without taking into account the effect of protein osmotic pressure and, in general, of the solvent conditions. As a result they do not provide a bona fide representation of the actin filament in vivo. A new ‘fluttering wing’ model is proposed which predicts that orientation of the monomers, intermonomer contacts and diameter of the actin filament are sensitive to protein osmotic pressure and to interaction with regulatory proteins.
Substrate and thiol specificity of a stress‐inducible glutathione transferase from soybean
Authors: Skipsey Mark , Andrews Christopher J , Townson Jane K , Jepson Ian , Edwards Robert
Published: July 2002

An RT‐PCR‐derived clone encoding a stress‐inducible glutathione transferase (GSTGm1) from soybean has been over‐expressed in E. coli. The enzyme was active as the dimer GSTGm1‐1 and showed GST and glutathione peroxidase activity toward diverse xenobiotics, including analogues of natural stress‐metabolites. The selective herbicides, fomesafen and acifluorfen, were conjugated more actively with homoglutathione (hGSH), the major thiol in soybean, than with glutathione (GSH). No thiol preference was shown with the related herbicide, fluorodifen, while GSH was preferred with metolachlor and most non‐herbicide substrates. Similar thiol‐dependent specificities were observed in GST preparations from plants of varying GSH/hGSH content.