bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and structural characterization of the human endothelial nitric‐oxide‐synthase gene
Authors: MIYAHARA Kaoru , KAWAMOTO Takeshi , SASE Kazuhiro , YUI Yoshiki , TODA Katsumi , YANG Li‐Xia , HATTORI Ryuichi , AOYAMA Takeshi , YAMAMOTO Yasutake , DOI Yoshinori , OGOSHI Shohei , HASHIMOTO Kozo , KAWAI Chuichi , SASAYAMA Shigetake , SHIZUTA Yutaka
Published: March 2005

Nitric oxide accounts for the activity of endothelium‐derived relaxing factor, which seems to have an important role in vasodilation and inhibition of platelet aggregation. In endothelial cells, one isoform of nitric‐oxide synthase is constitutively expressed. Analysis of the cDNA encoding the human endothelial nitric‐oxide synthase revealed that the mRNA is 4.1 kb in size and that the translated protein consists of 1203 amino acids. We have cloned a genomic DNA encoding the human endothelial nitric‐oxide synthase and analyzed the entire nucleotide sequence of the gene. The gene consists of 26 exons with a total size of 21 kb. The 5′ flanking region of the gene lacks TATA boxes, but it contains putative Sp1‐binding sites in (G+C)‐rich regions. Of particular interest is the fact that a shear‐stress‐responsive element is located at position –985, which probably regulates the nitric‐oxide‐synthase gene in response to fluid mechanical forces at the transcriptional level in the vascular endothelium. Two minisatellite sequences are detectable in introns 2 and 8; a 32‐bp consensus sequence repeats 38 times and a 57‐bp consensus sequence repeats ten times. We found polymorphisms of the BamHI fragment containing the former minisatellite sequence in genomic DNA from pedigree family members. Furthermore, five tandem repeats of a 27‐bp core consensus sequence and 35 repeats of a dinucleotide (CA) are located in introns 4 and 13, respectively. These repeat sequences will probably provide genetic markers for gene mapping and linkage analysis of inherited diseases including cardiovascular diseases.
Induction of cytosolic phospholipase A2 in human leukocytes by lipopolysaccharide
Authors: RODEWALD Eva , TIBES Ulrich , MAASS Gerhard , SCHEUER Werner
Published: March 2005

Stimulation of peripheral blood leukocytes with lipopolysaccharide results in the synthesis of inflammatory cytokines including interleukin‐1β, interleukin‐6, tumor necrosis factor‐α and prostaglandin E2 correlating with an increase in phospholipase A2 activity. Mammalian cells contain several phospholipase A2 isoforms including the 14‐kDa secretory isoform and the more recently described high‐molecular‐mass cytosolic isoform. It is commonly believed that during inflammatory responses secretory phospholipase A2 becomes activated. However, we could not detect secretory phospholipase A2 nor its corresponding mRNA after lipopolysaccharide‐induced activation. By contrast, we found increased mRNA levels for cytosolic phospholipase A2 following activation of peripheral blood leukocytes when levels were compared to non‐stimulated controls. Our results demonstrate that cytosolic phospholipase A2, rather than the secretory isoform may be the mediator of the lipopolysaccharide‐induced inflammatory cascade in human peripheral blood leukocytes.
An unusual conformation of the methionine haem ligand in cytochrome cL established by two‐dimensional 1H‐NMR
Authors: COSTA Helena S. , SANTOS Helena , TURNER David L.
Published: March 2005

A complete relaxation‐matrix analysis of NOESY cross‐peak intensities was used to determine the conformation of the methionine ligand to the haem group in two ferrocytochromes cL from Methylophilus methylotrophus and Methylobacterium extorquens, including the configuration at the sulphur. The conformation of the axial methionine is of a type reported only for the cytochromes c5 from Pseudomonas mendocina and Azotobacter vinelandii. Although the conformation of the methionine is unusual, the paramagetic shifts of the haem methyl proton resonances in the oxidized proteins indicate that the electronic structure of the haem groups is similar to that found in the mitochondrial type of cytochrome c.
Regulation of lipogenic enzyme and phosphoenolpyruvate carboxykinase gene expression in cultured white adipose tissue
Authors: FOUFELLE Fabienne , GOUHOT Betty , PERDEREAU Dominique , GIRARD Jean , FERRE Pascal
Published: March 2005

In cultured adipose tissue of suckling rats, glucose alone is able to induce the appearance of fatty‐acid synthase and acetyl‐CoA carboxylase mRNA by a mechanism involving glucose‐6‐phosphate accumulation; insulin alone has no effect but potentiates the effect of glucose. In the present study, we have analysed in cultured adipose tissue the effects of other hormones on the expression of these enzymes as well as on phosphoenolpyruvate carboxykinase. Triiodothyronine has only a marginal effect on fatty‐acid synthase expression, in the absence or presence of glucose and insulin. A synthetic glucocorticoid, dexamethasone, opposes the inductive effect of glucose and insulin on fatty‐acid synthase expression but increases the expression of phosphoenolpyruvate carboxykinase. A β‐agonist, isoproterenol totally inhibits the inductive effect of glucose and insulin on acetyl‐CoA carboxylase and fatty‐acid synthase expression whereas it increases the expression of phosphoenolpyruvate carboxykinase. Similarly, glucagon and cAMP have antagonistic effects on glucose and insulin‐induced fatty‐acid synthase expression. These inhibitory effects cannot be explained only by a reduction in glucose‐6‐phosphate concentration. We conclude that, in adipose tissue, dexamethasone and cAMP‐generating hormones are negative regulators of lipogenic enzyme expression. Finally, the regulation of phosphoenolpyruvate carboxykinase expression in adipose tissue is similar to that found in the liver, i.e. inhibition by insulin and glucose and activation by glucocorticoids and cAMP.
Effect of pH and MgCl2 on the binding of purine nucleotides to the uncoupling protein in membrane particles from brown fat mitochondria
Authors: RAFAEL Johannes , PAMPEL Ingelore , WANG Xuemin
Published: March 2005

Binding of purine nucleotides to the uncoupling protein (UCP) was investigated in membrane particles prepared from brown fat mitochondria of cold‐acclimated rats. Mitochondrial membranes were separated from soluble protein with Lubrol WX and treated with 3 M urea at basic pH. The resulting membrane vesicles were permeable to GDP and contained up to 3 nmol UCP/mg protein with unchanged nucleotide binding, as compared to the mitochondria (GDP/UCP ratio = 1.0; pKd GDP = 6.0 at pH 7.0). UCP bound nucleotides to one type of specific binding sites, located exclusively on the cytosolic side of the mitochondrial membrane. The binding affinity of guanine nucleotides was 3–18‐times higher than that of the corresponding adenine nucleotides, when measured in membrane particles from cold‐acclimated rats, hamsters, and guinea pigs. The pH‐dependent binding affinities of GDP and ADP attained a maximum at pH 5.0–6.0 (pKd GDP = 6.8, pKd ADP = 5.8) and were decreased by a factor of 102 at pH 4.0 and pH 8.0, respectively, whereas the binding affinity of ATP was maximal at pH 4.0 (pKd= 7.0) and was decreased by a factor of 103 at pH 7.5. Participation of the protein binding center in nucleotide interaction with UCP in the membrane was highly pH‐dependent. Mg2+ modified the number of binding sites engaged at a given nucleotide concentration by complex binding of nucleotides; the Kd for Mg ° GTP2– and Mg ° GDP− was 20–50‐times lower than that of the free nucleotides.
Functional characterization of carrier‐mediated transport of uridine diphosphate N‐acetylglucosamine across the endoplasmic reticulum membrane
Authors: BOSSUYT Xavier , BLANCKAERT Norbert
Published: March 2005

Uptake and metabolism of uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc) by rough‐endoplasmic‐reticulum (rER)‐derived vesicles was studied. Analysis of the molecular species, double‐label experiments, cis‐inhibition and trans‐stimulation experiments revealed that uptake represented entry of intact UDP‐GlcNAc into the microsomal lumen. The amount of vesicle‐associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space, and permeabilized microsomes were unable to retain UDP‐GlcNAc. These findings indicate that uptake constituted effective translocation from the medium into the lumen of the vesicles. The microsomal uptake of UDP‐GlcNAc met the criteria of bidirectional, carrier‐mediated translocation. Transport was time and temperature dependent, saturable, selective, capable of trans‐stimulation and operational against a concentration gradient. Uptake studies performed in membrane preparations that were highly enriched in either rER, smooth ER, or Golgi revealed that UDP‐GlcNAc was taken up by the ER and by the Golgi apparatus.
Involvement of Ets‐related proteins in hormone‐independent mammary cell‐specific gene expression
Authors: WELTE Thomas , GARIMORTH Katja , PHILIPP Sonja , JENNEWEIN Patricia , HUCK Carmen , CATO Andrew C. B. , DOPPLER Wolfgang
Published: March 2005

Regulatory regions have been located in the 5′ flanking sequence of the mouse whey acidic protein gene which contribute to its tissue‐ and stage‐specific expression in the mammary gland. They can be functionally separated into elements which mediate the action of lactogenic hormones prolactin and glucocoticoids and elements which control mammary cell‐specific transcription in the absence of hormones. By mutational analysis, we have located a site in the whey acidic protein promoter between – 120 and – 100 which is important for hormone‐independent promoter function. In stably transfected HC11 mammary epithelial cells, the hormone‐independent activity of the mutated promoter was reduced 40‐fold, whereas the capability to respond to lactogenic hormones was retained. The site was specifically recognised by two nuclear factors contained in extracts of cultivated mammary epithelial cells or mammary glands. Electrophoretic mobility shift assay, DNase I footprinting and methylation interference experiments indicated a relation of both factors to the Ets family of DNA‐binding proteins. One of these factors also recognised a functionally important site in the mammary cell‐specific enhancer of the mouse mammary tumor virus long terminal repeat. The results suggest that factors related to the Ets family are important determinants in mammary cell‐specific gene expression.
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis
Authors: LADENSTEIN Rudolf , RITSERT Karl , HUBER Robert , RICHTER Gerald , BACHER Adelbert
Published: March 2005

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 β subunits enclosing a triplet of α subunits. An X‐ray structure of 0.32 nm resolution has been obtained for the icosahedral capsid of the native α3β60 complex [Ladenstein, R., Schneider, M., Huber, R., Bartunik, H. D., Wilson, K., Schott, K. & Bacher, A. (1988) J. Mol. Biol. 203, 1045–1070].

β subunits were isolated after denaturation of the α3β60 complex and were subsequently reconstituted in a ligand‐driven reaction yielding artifactual, hollow β60 capsids with icosahedral symmetry. Hexagonal crystals (space group P6322) of the reconstituted capsids diffracted X‐rays to a resolution of 0.32 nm. Crystallographic intensity data were obtained using synchrotron radiation. Freeze‐etched electron‐microscopic images and rotation function calculations showed that the hexagonal crystal. forms of the artifactual β60 capsids and the native α3β60 complex are isomorphous. Orientation and translation parameters of the β‐subunit model were refined by XPLOR rigid‐body refinement. The electron‐density map was improved by cyclic icosahedral averaging and phase extension from 0.5–0.32 nm resolution. The β‐subunit structure was partially refined by energy minimization and crystallographic refinement (XPLOR) assuming strict icosahedral symmetry (final R factor 30.9% for data at 0.8–0.32 nm resolution).

The topology and chain folding of the β subunits in the artifactual β60 capsid are similar to the native α3β60 enzyme. Structural features of the substrate‐binding site and the binding of the substrate‐analogous ligand 5‐nitro‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione are discussed. Ligand binding occurs at the pentamer interfaces and includes van der Waals' interactions and hydrogen bonding. The binding pocket shows a hydrophobic region which accomodates the pyrimidinedione ring and a hydrophilic region to which the ribityl side chain binds. Most amino acid residues involved in the active site are conserved as shown by sequence comparisons with the putative lumazine‐synthase genes of Escherichia coli and Photobacterium leiognathi. In the final electron‐density map, a residual density feature was tentatively assigned to a bound phosphate ion which mimics the binding of the second substrate, 3,4‐dihydroxy‐2‐butanone 4‐phosphate. This putative phosphate‐binding site involves a highly conserved amino acid sequence containing three basic residues.
Conformational changes of the recombinant extracellular domain of E‐cadherin upon calcium binding
Authors: POKUTTA Sabine , HERRENKNECHT Kurt , KEMLER Rolf , ENGEL Jürgen
Published: March 2005

The cell‐adhesion protein E‐cadherin/uvomorulin exhibits a calcium‐dependent homoassociation. The effect of Ca2+ on the extracellular fragment of E‐cadherin was studied using the recombinant protein expressed in the baculovirus expression system. The recombinant and native fragment of E‐cadherin were found to be similar by many biochemical criteria [Herrenknecht, K. & Kemler, R. (1993) J. Cell Sci. 17, 147–154]. A large and reversible conformational transition was observed upon Ca2+ depletion. A change from a rod‐like structure, 22 nm in length, to a more globular assembly of the five subdomains became evident by electron‐microscopical analysis. In the presence of Ca2+, the circular dichroic spectra indicated predominantly β‐structure but a more negative ellipticity was observed in the absence of Ca2+. The intrinsic tryptophan fluorescence decreased by 12% upon Ca2+ depletion. Both effects were used for calcium titrations which indicated calcium binding to several sites with average Kd values of 45–150 μM. Cleavage of the protein fragment by trypsin occurred only at low Ca2+ concentrations and from the calcium‐dependence of cleavage rates, a Kd value of 24 μM was derived. The major site of cleavage was identified by partial sequencing to be located between the two putative calcium‐binding sites in the third subdomain from the N‐terminus. In agreement with earlier results with the native fragment, the recombinant protein did not associate in the presence or absence of Ca2+. We suggest the calcium‐dependent homoassociation therefore depends on additional effects connected with the cell surface association of E‐cadherin.
Identification of the dihydrolipoamide acetyltransferase subunit of the human pyruvate dehydrogenase complex as an autoantigen in halothane hepatitis
Authors: CHRISTEN Urs , QUINN Janet , YEAMAN Stephen J. , KENNA J. Gerald , CLARKE Janet B. , GANDOLFI A. Jay , GUT Josef
Published: March 2005

Trifluoroacetylated (CF3CO‐) proteins, elicited upon exposure of animals or humans to halothane, were recognized by anti‐CF3CO antibody, monospecific for the hapten derivative N6‐trifluoroacetyl‐l‐lysine. Anti‐CF3CO antibodies cross‐reacted with the dihydrolipoamide acetyltransferase (E2 subunit) of pyruvate dehydrogenase, indicating that epitopes on the E2 subunit of pyruvate dehydrogenase molecularly mimic those on CF3CO‐proteins. Lipoic acid, the prosthetic group of the E2 subunit of pyruvate dehydrogenase was essential in this process, in that only the lipoylated form of the recombinantly expressed inner lipoyl domain of the human E2 subunit of pyruvate dehydrogenase, but not the unlipoylated form, was recognized by anti‐CF3CO antibody. Furthermore, based on a high degree of structural relatedness, both CF3CO‐Lys and (6RS)‐lipoic acid, as well as the lipoylated peptide ETDK(lipoyl)ATIG specifically inhibited the recognition by anti‐CF3CO antibody of the E2 subunit of pyruvate dehydrogenase, of trifluoroacetylated rabbit serum albumin and of human liver CF3CO‐proteins. In sera of patients was halothane hepatitis, autoantibodies with properties identical to those of anti‐CF3CO antibody were identified which could not discriminate between CF3CO‐proteins and the E2 subunit of pyruvate dehydrogenase. These data suggest that the E2 subunit pyruvate of dehydrogenase is an autoantigen in halothane hepatitis and that molecular mimicry of CF3CO‐proteins by the E2 subunit of pyruvate dehydrogenase is due to the similar structures of CF3CO‐Lys and lipoic acid.
RNAs and ribonucleoproteins in recognition and catalysis
Authors: WITTOP KONING Tom H. , SCHÜMPERLI Daniel
Published: March 2005

CONTENTS. 1. Did life begin with catalytic RNA?–2. Self‐splicing and self‐cleaving RNAs–2.1 Self‐splicing of group I introns – 2.2 Self‐splicing of group II introns – 2.3 Self‐cleaving RNAs–3. Splicing mediated by trans‐acting factors–3.1 Group III introns – 3.2 Splicing of nuclear pre‐mRNAs – 3.3 Trans‐splicing – 3.4 Is nuclear pre‐mRNA splicing evolutionarily related to group I and group II self‐splicing?– 3.5 Non‐RNA mediated splicing of tRNAs–4. Processing of ribosomal precursor RNAs–5. Processing of pre‐mRNA 3′ ends–5.1 Polyadenylation – 5.2 Histone pre‐mRNA 3′ processing–6. Other RNPs involved in metabolic mechanisms–6.1 5′ end processing of pre‐tRNAs by RNase P – 6.2 The signal recognition particle – 6.3 Telomerase – 6.4 RNA editing in trypanosomatid mitochondria–7. Why RNA?
Biosynthesis of carnosine and related peptides by skeletal muscle cells in primary culture
Authors: BAUER Karl , SCHULZ Michael
Published: March 2005

Synthesis of carnosine (β‐alanyl‐l‐histidine) and related dipeptides could be demonstrated in primary muscle cell cultures derived from embryonic chick pectoral muscle. After incubation with radiolabeled β‐alanine or γ‐aminobutyric acid, the radiolabeled dipeptides were isolated from the cell extracts and also in small amounts from the culture medium. The kinetics of dipeptide formation indicated that anserine (β‐alanyl‐1‐methylhistidine) is not formed directly by these cells but as a secondary product via the methylation of carnosine. Coinciding with the morphological differentiation of the mononucleated myoblast to form multi‐nucleated myotubes, a rapid increase in β‐alanine uptake and also in dipeptide synthesis could be observed. These results demonstrate that carnosine and related peptides are not merely deposited in skeletal muscles but that they are actively synthesized by muscle cells in culture.
Heat shock enhances the amount of prenylated Dnaj protein at membranes of glyoxysomes
Authors: PREISIG‐MÜLLER Regina , MUSTER Gerhard , KINDL Helmut
Published: March 2005

Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described in tissues of dark‐grown cucumber seedlings. Using a cucumber Dnaj protein produced by expression in bacteria, we raised polyclonal antibodies against the protein and used them for localization studies. In etiolated cucumber seedlings, both cotyledons and hypocotyledons were found to contain Dnaj proteins. Cell fractionation of etiolated cotyledons showed that Dnaj proteins were detectable mainly in the postnuclear cell fraction after sedimentation at 10000Xg, and in the microsomes. Following subfractionation by sucrose density gradient centrifugation and analysis by immunoblotting, a 53‐kDa protein was attributed to the glyoxysomal fraction and an 80‐kDa protein to the mitochondrial fraction. The glyoxysomal Dnaj protein behaved as a membrane‐bound form. Upon heat shock, a slight increase in the content of the glyoxysomal Dnaj protein was found. When glyoxysomes were treated with protease and subsequently isolated by gradient centrifugation, virtually all immunologically detectable Dnaj protein was removed. Administration of radiolabelled mevalonic acid to cotyledons and isolation of glyoxysomes yielded labelled Dnaj protein which remained membrane bound during the purification of glyoxysomal membranes by floatation in a density gradient.
Undecagold cluster modified tRNAPhe from Escherichia coli and its activity in the protein elongation cycle
Authors: BLECHSCHMIDT Bernd , SHIROKOV Vladimir , SPRINZL Mathias
Published: March 2005

An undecagold cluster (Au11) of molecular mass 6200Da was attached to the 3‐(3‐amino‐3‐carboxypropyl)uridine at position 47 of tRNAPhe from Escherichia coli. This modified tRNA can be enzymically aminoacylated with phenylalanine in the reaction catalyzed by phenylalanyl‐tRNA synthetase. Au11‐labeled Phe‐tRNAPhe forms a ternary complex with the elongation factor Tu · GTP and is active in poly(U)‐dependent poly(phe) synthesis. The Au11 modification does not hinder the specific binding of tRNA to distinct ribosomal binding sites or the precise positioning of the aminoacyl and peptidyl residues in the peptidyltransferase center, and does not impair the translocation. The modified tRNA is suitable for the identification of ribosomal binding sites by scanning transmission electron microscopy and for crystallographic studies of the 70S ribosome at different states of the protein‐elongation cycle.
Genuine and apparent cross‐reaction of polyclonal antibodies to proteins and peptides
Authors: LEDER Lukas , WENDT Hans , SCHWAB Christian , JELESAROV Ilian , BORNHAUSER Susanne , ACKERMANN Friederike , BOSSHARD Hans Rudolf
Published: March 2005

Antiserum to a native protein may cross‐react with the corresponding denatured protein or with peptides. The cross‐reaction is either a genuine property of the antibodies or caused by antibodies produced against some unfolded protein contaminating the native protein used for immunization. Appropriate conformation‐sensitive immunoassays must be employed to distinguish a genuine from an apparent cross‐reaction.

In the present study, we have analyzed critically the cross‐reaction of rabbit antisera against proteins and peptides. We have distinguished between genuine and apparent cross‐reaction with the help of the protein A antibody‐capture ELISA, a new conformation‐sensitive ELISA format. Three systems were analyzed: cross‐reaction of antisera to native yeast and horse cytochrome c with unfolded apo‐cytochrome c; cross‐reaction of antisera to a coiled‐coil leucine‐zipper peptide with a homologous random‐coil peptide obtained by introducing two proline residues into the leucine‐zipper sequence; cross‐reaction of antisera to two peptides that correspond to the N‐terminal and an internal sequence of ferredoxin:NADP+ reductase (FNR), with the native enzyme. The reaction of the anti‐(cytochrome c) sera was clearly due to antibodies produced against unfolded protein, it was an apparent and not a genuine cross‐reaction. Furthermore, the apparently cross‐reactive antibodies to horse cytochrome c did not discriminate against sequence‐related proteins from dog, beef, rabbit and pigeon. In contrast, antibodies to the leucine‐zipper peptide did cross‐react in a genuine way with the homologous random‐coil peptide, that is, the cross‐reactive antibodies do not seem to have been produced against the unfolded form of the leucine‐zipper peptide. Of the two anti‐peptide sera the one against the unstructured and highly accessible N‐terminal segment reacted strongly with the native protein. The second serum against a solvent‐accessible turn‐like sequence of FNR showed apparent cross‐reactivity: antibodies recognizing the native protein were directed against a minor conformational isoform of the free peptide and did not react with the principal form(s) of the free peptide.

The generation of cross‐reactive antibodies depends on the conformational stability and intergrity of the immunogen and on the molecular form of its application, i.e., free, polymerized or carrier‐bound. The results clarify the different nature of cross‐reactivity of antisera to proteins and peptides. This knowledge is crucial if antisera are to be used as conformation‐specific probes.
Molecular characterization of a human tyrosinase‐related‐protein‐2 cDNA
Authors: BOUCHARD Brigitte , DEL MARMOL Véronique , JACKSON Ian J. , CHERIF Dorra , DUBERTRET Louis
Published: March 2005

Pigmentation in mammals is under complex genetic control. Amongst the genes involved in this process, those encoding tyrosinase and the tyrosinase‐related‐proteins 1 and 2 have been well characterized and share a number of features. Recently, the murine tyosinase‐related‐protein‐2 gene was shown to encode dopachrome‐tautomerase activity and was mapped to the slaty locus. Human tyrosinase and tyrosinase‐related‐protein‐1 genes have been isolated and demonstrate a high degree of similarity with their murine counterparts. However, there has been limited data regarding the existence of a human homologue for tyrosinase‐related‐protein‐2 and its relationship to the other tyrosinase‐related proteins. In this study, we report the molecular isolation of a cDNA encoding a human homologue of the murine tyrosinase‐related‐protein‐2/dopachrome tautomerase. We have characterized its expression in human melanocytic cells and have analyzed the relationship between dopachrome tautomerase and tyrosinase activities with the level of visible pigmentation in these cells. TYRP2 has been mapped to the chromosomal region 13q32, thus extending a region of synteny with mouse‐chromosome 14.
Antagonism of the intracellular action of botulinum neurotoxin type A with monoclonal antibodies that map to light‐chain epitopes
Authors: DI BELLO Isabelle CENCI , POULAIN Bernard , SHONE Clifford C. , TAUC Ladislav , DOLLY J. Oliver
Published: March 2005

mAbs were produced in mice against highly purified, renatured light chain (LC) of botulinum neurotoxin A (BoNT A) that was immobilised on nitrocellulose to avoid the undesirable use of toxoids. Subcutaneous implants of relatively high amounts (up to 10 μg each) of LC allowed its slow release into the systemic circulation and, thus, yielded much higher antibody titres against the underivatized antigen than had hitherto been obtained by conventional immunization. Seven stable hybridoma cell lines were established which secrete mAb of IgG1 and IgG2b subclasses reactive specifically with BoNT A and LC, in native and denatured states, without showing any cross‐reactivity with types B, E, F or tetanus toxin. The pronounced reactivities of three mAbs towards refolded LC or intact toxin, observed in immunobinding and precipitation assays, relative to that seen in Western blots imply a preference for conformational epitopes. Though mAbs 4, 5 and 7 failed to neutralize the lethality of BoNT in vivo, administration intraneurally of mAb7 prevented the inhibition of transmitter release normally induced by subsequent extracellular administration of BoNT A. Notably, the latter mAb reacted with a synthetic peptide corresponding to amino acids 28–53 in the N‐terminus of the LC, a highly conserved region in Clostridial neurotoxins reported to be essential for maintaining the tertiary structure of the chain. Most importantly, when mAbs 4 or 7 were microinjected inside ganglionic neurons of Aplysia, each reversed, though transiently, the blockade of acetylcholine release by the toxin; this novel finding is discussed in relation to the nature of the zinc‐dependent protease activity of the toxin.
The role of trehalose synthesis for the acquisition of thermotolerance in yeast
Authors: HOTTIGER Thomas , DE VIRGILIO Claudio , HALL Michael N. , BOLLER Thomas , WIEMKEN Andres
Published: March 2005

In baker's yeast (Saccharomyces cerevisiae), accumulation of the non‐reducing disaccharide, trehalose, is triggered by stimuli that activate the heat‐shock response. Previously, trehalose levels have been shown to be closely correlated with thermotolerance, suggesting a protective function of this substance. Genetic evidence in support of this view is presented in an accompanying paper [De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 179–186]. In this study, we have examined the effect of trehalose on the thermal stability of proteins, a parameter thought to be a major determinant of thermotolerance. Physiological concentrations of trehalose (up to 0.5 M) were found to efficiently protect enzymes of yeast (glucose‐6P‐dehydrogenase, phosphoglucose‐isomerase) as well as enzymes of non‐yeast origin (bovine glutamic dehydrogenase, EcoRI) against heat inactivation in vitro. Trehalose also reduced the heat‐induced formation of protein aggregates. The disaccharide proved to be a compatible solute, as even at very high concentrations (up to 1 M) it did not significantly interfere with the activity of test enzymes. Trehalose was at least as good or better a protein stabilizer than any of a number of other compatible solutes (including sugars, polyalcohols and amino acids), while the structurally related trehalose‐6P was devoid of any protective effect. Thermoprotection of enzymes by trehalose was evident even in solutions containing high concentrations of yeast protein or substrate. The data indicate that trehalose accumulation may increase the thermotolerance of yeast by enhancing protein stability in intact cells.
Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen‐limited conditions
Authors: KALLIO Pauli T. , Jin KIM Dong , TSAI Philip S. , BAILEY James E.
Published: March 2005

An earlier stoichiometric analysis of oxygen‐limited metabolism of Escherichia coli expressing cloned Vitreoscilla hemoglobin (VHb) suggested improved efficiency of ATP production relative to wild‐type controls [Khosla, C., Curtis, J. E., DeModena, J., Rinas, J. & Bailey, J. E. (1990) Bio‐Technol. 8, 849–853]. This hypothesis has been further examined by determining several energetic parameters of different VHb‐expressing E. coli (VHb+) strains relative to controls not expressing VHb (VHb–). The H+/O ratio, the transmembrane ΔpH, and the ATP content of VHb+ constructs are 1.5, 1.6 and 2 times, respectively, corresponding values in VHb– controls. VHb was expressed using a low‐copy‐number vector in E. coli mutant strains lacking cytochrome o, cytochrome d, or both terminal oxidases; significant growth enhancement due to VHb expression was observed only in the strain having functional cytochrome o and lacking cytochrome d. All of these data obtained using different E. coli strains are consistent with a model of VHb action that hypothesizes enhancement by VHb of activity of the lower oxygen‐affinity, higher proton‐pumping‐efficiency cytochrome o terminal oxidase under oxygen‐limited growth conditions.
Correlation of high‐temperature stability of α‐chymotrypsin with ‘salting‐in’ properties of solution
Authors: LEVITSKY Vladislav Yu. , PANOVA Anna A. , MOZHAEV Vadim V.
Published: March 2005

A correlation between the stability of α‐chymotrypsin against irreversible thermal inactivation at high temperatures (long‐term stability) and the coefficient of Setchenov equation as a measure of salting‐in/out efficiency of solutes in the Hofmeister series has been found. An increase in the concentration of salting‐in solutes (KSCN, urea, guanidinium chloride, formamide) leads to a manyfold decrease of the inactivation rate of the enzyme. In contrast, addition of salting‐out solutes has a small effect on the long‐term stability of α‐chymotrypsin at high temperatures. The effects of solutes are additive with respect to their salting‐in/out capacities; the stabilizing action of the solutes is determined by the calculated Setchenov coefficient of solution. The correlation is explained by a solute‐driven shift of the conformational equilibrium between the ‘low‐temperature’ native and the ‘high‐temperature’ denatured forms of the enzyme within the range of the kinetic scheme put forward in the preceding paper in this journal: irreversible inactivation of the high‐temperature form proceeds much more slowly compared with the low‐temperature form.
Alteration of the cleavage mode and of the transglycosylation reactions of the xylanase A of Streptomyces lividans 1326 by site‐directed mutagenesis of the Asn173 residue
Authors: MOREAU Alain , SHARECK François , KLUEPFEL Dieter , MOROSOLI Rolf
Published: March 2005

The amino acid replacement of Asn173 by Asp in the xylanase A (Xln A) of Streptomyces lividans significantly altered its enzymic properties. A time‐course hydrolysis of xylan showed that the altered xylanase ([N173D] Xln A) initially produced larger amounts of xylose (X1), xylobiose (X2) and xylotriose (X3) than Xln A, but less xylotetraose (X4). The bond‐cleavage frequencies were determined for both enzymes using xylopentaose (X5), xylotetraose (X4) and xylotriose (X3) labelled at the reducing end of the molecule. Xln A hydrolysed X5, yielding 56% X2 and 44% X3, while [N173D]Xln A liberated 90% X2 and only 10% X3. Both enzymes hydrolysed X4 into 100% X2 and X3 into 100% X1. Transglycosylation reactions were detected in HPLC hydrolysis patterns using high substrate concentrations, where larger products than the starting substrates were formed. Their subsequent degradation also affected the yield of hydrolysis products. Using X5 as substrate, products from xylohexaose (X6) up to xylooligosides larger than xylooctaose (X8) were synthesized by Xln A, while [N173D]Xln A produced only a small amount of xyloheptaose (X7) and X8. Xln A hydrolysed X5 into an equivalent amount of X4 and X2 and 1.5‐fold more X3. However, [N173D]Xln A yielded the same amount of X3 and X2 but half as much X4. With X4 as substrate, Xln A synthesized twofold more X7 and X6 than [N173D]Xln A. Xln A liberated 1.4‐fold more X3 than X2, while [N173D]Xln A yielded twofold more X2 than X3. Xln A liberated almost fourfold more X2 than X1 from X3, while [N173D]Xln A produced only twofold more X2 than X1. These results indicated that the negative charge introduced by the mutation greatly affected the transglycosylation reactions catalysed by this xylanase.
Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein‐ubiquinone oxidoreductase
Authors: GOODMAN Stephen I. , AXTELL Kathleen M. , BINDOFF Laurence A. , BEARD Shannon E. , GILL Ronald E. , FRERMAN Frank E.
Published: March 2005

Electron‐transfer flavoprotein‐ubiquinone oxidoreductase (ETF‐QO) in the inner mitochondrial membrane accepts electrons from electron‐transfer flavoprotein which is located in the mitochondrial matrix and reduces ubiquinone in the mitochondrial membrane. The two redox centers in the protein, FAD and a [4Fe4S]+2,+1 cluster, are present in a 64‐kDa monomer. We cloned several cDNA sequences encoding the majority of porcine ETF‐QO and used these as probes to clone a full‐length human ETF‐QO cDNA. The deduced human ETF‐QO sequence predicts a protein containing 617 amino acids (67 kDa), two domains associated with the binding of the AMP moiety of the FAD prosthetic group, two membrane helices and a motif containing four cysteine residues that is frequently associated with the liganding of ferredoxin‐like iron‐sulfur clusters. A cleavable 33‐amino‐acid sequence is also predicted at the amino terminus of the 67‐kDa protein which targets the protein to mitochondria. In vitro transcription and translation yielded a 67‐kDa immunoprecipitable product as predicted from the open reading frame of the cDNA. The human cDNA was expressed in Saccharomyces cerevisiae, which does not normally synthesize the protein. The ETF‐QO is synthesized as a 67‐kDa precursor which is targeted to mitochondria and processed in a single step to a 64‐kDa mature form located in the mitochondrial membrane. The detergent‐solubilized protein transfers electrons from ETF to the ubiquinone homolog, Q1, indicating that both the FAD and iron‐sulfur cluster are properly inserted into the heterologously expressed protein.
Photoaffinity labelling of cardiac membrane GTP‐binding proteins in response to insulin
Authors: RUSS Martina , WICHELHAUS Antje , UPHUES Ingo , KOLTER Thomas , ECKEL Jürgen
Published: March 2005

Plasma membranes from rat cardiac ventricular tissue and insulin receptors partially purified by wheat‐germ‐agglutinin chromatography were subjected to direct photoaffinity labelling with [α‐32P]GTP in order to elucidate the presence of insulin‐receptor‐coupled GTP‐binding proteins. In plasma membranes three proteins have been identified that exhibit an enhanced photolabelling with the nucleotide in response to insulin. The apparent molecular masses of these proteins were found to be 56, 60 and 74 kDa. Photolabelling of partially purified insulin receptors showed the copurification of the 60‐kDa species, whereas the 56‐kDa and 74‐kDa proteins could not be detected. Furthermore, the 60‐kDa G‐protein was found to be specifically co‐immunoprecipitated with the insulin receptor. Incubation of insulin receptors with insulin increased the labelling of the 60‐kDa band to 205±27% (n= 5) of control. Immuno‐ and ligand‐blotting experiments revealed the additional presence of a 39‐kDa Go‐like protein and two G‐proteins with molecular masses of 24 and 26 kDa in the receptor preparation. Under basal conditions the insulin receptor and the 60‐kDa G‐protein exhibited an apparent inverse distribution between plasma and microsomal membranes with the G‐protein being extensively labelled in the microsomal fraction. In conclusion, our data show that, in its native environment, the cardiac insulin receptor couples to at least three GTP‐binding proteins. Out of these, a 60‐kDa species of microsomal origin, copurifies with the insulin receptor. It is suggested that this G‐protein is associated with the insulin receptor and may be involved in insulin receptor signalling in target cells.
Characterization of native laminin from bovine kidney and comparison with other laminin variants
Authors: LINDBLOM Anders , MARSH Tracey , FAUSER Charlotte , ENGEL Jürgen , PAULSSON Mats
Published: March 2005

A comprehensive characterization of laminin isoforms requires access to native preparations of laminins of a defined subunit composition. For this purpose an optimized isolation procedure was developed and shown to be broadly applicable to normal mammalian tissues. The protocol does in addition yield side fractions highly enriched in collagens XII and XIV.

The major laminin purified from bovine kidney is indistinguishable from mouse Engelbreth‐Holm‐Swarm (EHS) tumor laminin in electron microscopy, but contains an A chain that migrates in a position intermediate to the Ae and the Am chains on SDS/PAGE. Antisera raised against mouse EHS‐tumor laminin crossreact with B chains, but not with the A chain, of kidney laminin. Further, this A chain is not recognized by antisera raised against the Am chain. Laminins from heart and kidney both contain a significant subpopulation with a 190‐kDa polypeptide identified as the B1s chain. The Am‐containing laminins from heart and placenta differ morphologically from the Ae‐containing EHS laminin in having one short arm that does not have the characteristic globule‐rod‐globule appearance. Further, the Am‐containing laminins show a significantly higher thermal stability of the coiled‐coil α‐helical region in the long arm than does Ae‐containing EHS laminin, indicating that certain combinations of laminin chains interact more strongly than others.
Purification, characterization and inhibition of dihydropyrimidinase from rat liver
Authors: KIKUGAWA Mariko , KANEKO Masae , FUJIMOTO‐SAKATA Shigeko , MAEDA Mitsuko , KAWASAKI Koichi , TAKAGI Tatsuya , TAMAKI Nanaya
Published: March 2005

Dihydropyrimidinase (DHPase) was purified 564‐fold over the initial rat liver extract, using heat, ammonium sulfate fractionation, DEAE‐Sepharose CL‐6B, carboxymethyl‐Sepharose CL‐6B, hydroxyapatite and Sephacryl S‐300 chromatography. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 215 kDa and the subunit mass was 54 kDa. DHPase catalyzed the reversible cyclization of 5,6‐dihydrouracil (H2Ura) to N‐carbamoyl‐β‐alanine or 5,6‐dihydrothymine (H2Thy) to N‐carbamoyl‐β‐aminoisobutyric acid.

Authentic 5‐bromo‐5,6‐dihydrouracil (BrH2Ura) and commercially available H2Thy were racemic. However, these 5‐substituted 5,6‐dihydropyrimidines were hydrolyzed by over 96% and 98%, respectively, by DHPase. These results suggest that dihydropyrimidinase has no stereo specificities for 5‐substituents of H2Ura. The addition of H2Ura and H2Thy competitively inhibited the enzyme activity against BrH2Ura. However, the addition of N‐carbamoyl‐β‐alanine or N‐carbamoyl‐β‐aminoisobutyric acid showed hyperbolic mixed‐type inhibition, when BrH2Ura was used as the substrate. The values of the dissociation constants of BrH2Ura, N‐carbamoyl‐β‐alanine and N‐carbamoyl‐β‐aminoisobutyric acid were 17 μM, 0.38 mM and 0.38 mM, respectively.

DHPase from the rat liver contains 4 mol Zn2+/mol active enzyme, presumably one atom/subunit. Zn2+ also inhibited the hydrolysis of BrH2Ura by the enzyme. The Ki for Zn2+ as an inhibitor of DHPase was 23 μM, and the maximum rate of inactivation was 0.057 min−1 at 37°C. H2Ura and H2Thy protected the enzyme activity from Zn2+ inactivation.
Purification cDNA cloning and heterologous expression of human phosphomannose isomerase
Authors: PROUDFOOT Amanda E. I. , TURCATTI Gerardo , WELLS Timothy N. C. , PAYTON Mark A. , SMITH David J.
Published: March 2005

Phosphomannose isomerase catalyses the interconversion of fructose‐6‐P and mannose‐6‐P and has a critical role in the supply of d‐mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose‐isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose‐isomerase cDNA. A full‐length cDNA was isolated from a human testes λgt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose‐isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose‐isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.
Discrimination against misacylated tRNA by chloroplast elongation factor Tu
Authors: STANZEL Manfred , SCHÖN Astrid , SPRINZL Mathias
Published: March 2005

Chloroplast elongation factor Tu was purified from Pisum sativum and the binding properties of glutamylated chloroplast tRNAs were studied by gel‐permeation chromatography. Whereas chloroplast Glu‐tRNAGlu is efficiently bound by this factor, the misacylated Glu‐tRNAGin does not interact with chloroplast elongation factor Tu · GTP and is thus efficiently excluded from protein synthesis. Comparison with the behaviour of Escherichia coli elongation factor Tu · GTP shows that this factor, which is not confronted with the in vivo misacylation phenomenon of organelles, binds both Glu‐tRNAGlu and Glu‐tRNAGln from chloroplasts with approximately equal efficiency.
Isolation and complete sequence of CBR, a gene encoding a putative cytochrome b reductase in Saccharomyces cerevisiae
Authors: CSUKAI Michael , MURRAY Michael , ORR Elisha
Published: March 2005

We have isolated and characterised a novel yeast gene, CBR (cytochrome b reductase), encoding a 35‐kDa yeast novobiocin‐binding protein. The predicted protein sequence of CBR displays considerable similarity to both plant nitrate reductases and mammalian cytochrome b5 reductases indicating that it is a putative member of the flavoprotein pyridine‐nucleotide‐cytochrome‐reductase family. Disruption of CBR is not lethal under various growth conditions, suggesting the presence of some functional overlap with other reductases, possibly with the cytochrome P‐450 reductase.
Nitric oxide reductase from Pseudomonas stutzeri
Authors: ZUMFT Walter G. , BRAUN Cornelia , CUYPERS Heinrich
Published: March 2005

Nitric oxide (NO) reductase is an integral membrane component of the anaerobic respiratory chain of Pseudomonas stutzeri that transforms nitrate to dinitrogen (denitrification). The enzyme catalyzes the reduction of NO to nitrous oxide. The structural genes for the NO reductase complex, norC and norB, were sequenced and their organization established by primer extension and Northern blot analysis. The norCB genes encoding the cytochrome c and cytochrome b subunits of the enzyme are contiguous and transcribed as a single 2.0‐kb transcript. The promoter region has a canonical recognition motif for the transcriptional activator protein Fnr, centered at –40.5 nucleotides from the initiation site of transcription. No similarity of the derived gene products to known cytochromes of b‐ or c‐type was found in a data bank search.

Post‐translational processing of the two subunits was limited to the removal of the terminal methionine to leave an N‐terminal serine in either subunit. The mature cytochrome c subunit (16508Da, 145 residues) is predicted to be a bitopic protein with a single membrane anchor. The mature cytochrome b subunit (53006Da, 473 residues) is a putatively polytopic, strongly hydrophobic membrane‐bound protein with 12 potential transmembrane segments. Several histidine and proline residues were identified with potentially structural and/or functional importance.

Mutational inactivation of NO reductase by deletion of norB or the norCB genes affected strongly the in vivo activity of respiratory nitrite reductase (cytochrome cd1), but to a much lesser extent the expression level of this enzyme. In turn, mutational inactivation of the structural gene for cytochrome cd1, nirS, or loss of in vivo nitrite reduction by mutation of the nirT gene, encoding a presumed tetraheme cytochrome, lowered the expression level of NO reductase to 5–20%, but hardly its catalytic activity. The cellular concentration of NO reductase increased again on restoration of nitrite reduction in the nirS::Tn5 mutant MK202 by complementation with nirS or with the heterologous nirK gene, encoding the Cu‐containing nitrite reductase from Pseudomonas aureofaciens. Thus, NO may be required as an inducer for its own reductase. Our results show that the nitrite‐reducing system and the NO‐reducing system are not operating independently from each other but are interlaced by activity modulation and regulation of enzyme synthesis.
A 40‐kDa myelin basic protein kinase, distinct from erk1 and erk2, is activated in mitotic HeLa cells
Authors: HEIDER Harald , HUG Corinne , LUCOCQ John M.
Published: March 2005

Mitotic HeLa cells showed an increased phosphorylation activity towards myelin basic protein compared to cells in G1 or S phases. Further investigation using renaturation gels revealed that, in mitotic cell lysates, a protein with an apparent molecular mass of around 40 kDa phosphorylates myelin basic protein. This kinase is active early in mitosis, but is then dowregulated concomitantly with p34cdc2 kinase as mitosis proceeds, its activity decreasing to basal levels by early G1. The molecular mass of the kinase suggested that it might be one of the human homologues of rat erk1 or erk2. However, antibodies raised against C‐terminal sequences of erk1 and erk2 failed to immunoprecipitate renaturable kinase activity from mitotic lysates. In addition, in immunoblots erk1 and erk2 failed to show the well established changes in electrophoretic migration that are consequences of their activation. These data indicate that these two mitogen‐activated protein (MAP) kinases are not stimulated during HeLa cell mitosis and indicate that the 40‐kDa kinase is either a new member of the MAP kinase family or it is a novel mitotic kinase that has not yet been described.