bluuebunny/crossref_metadata_2025 huggingface dataset:
The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus
Authors: FAURE Grazyna , CHOUMET Valérie , BOUCHIER Christiane , CAMOIN Luc , GUILLAUME Jean‐Luc , MONEGIER Bertrand , VUILHORGNE Marc , BON Cassian
Published: March 2005

Crotoxin, the main toxin from the venom of the South American rattlesnake Crotalus durissus terrificus, is a β‐neurotoxin which consists of the non‐covalent association of two subunits: a phospholipase A2 subunit B (CB), and a non‐enzymic subunit A (CA). We have previously purified and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Furthermore, three cDNAs encoding two CB isoforms and the precursor, pro‐CA, of subunit A have been isolated from a cDNA library prepared from a single venom gland of Crotalus durissus terrificus.

The aim of this study is to analyse an individual snake venom from an animal that has been used to construct a cDNA library. Several isoforms of subunit A and two isoforms of subunit B were isolated and compared to purified and characterized subunit isoforms from pooled venom. The result of this study showed that the multiplicity and the diversity of crotoxin isoforms result from post‐translational modifications occurring on a precursor and from the expression of different messenger RNAs present in an individual snake. It allowed for the identification of the two CB isoforms encoding cDNAs expressed in the individual venom with two isoforms from pooled venom, CBc and probably CBa2, that belong to two classes of crotoxin complexes which can be distinguished biochemically and pharmacologically.
Expression of functional proliferating‐cell nuclear antigen from rice (Oryza sativa) in Escherichia coli
Authors: MATSUMOTO Takashi , HATA Shingo , SUZUKA Iwao , HASHIMOTO Junji
Published: March 2005

Proliferating‐cell nuclear antigen (PCNA), the auxiliary protein for DNA polymerase δ, is one of the key factors for both PCNA‐dependent DNA synthesis and cell‐cycle progression. Plant PCNA genes have previously been cloned from rice, carrot, tobacco, and soybean cells by screening the cDNA libraries using similarity to the human or rat PCNA genes. We subcloned the relevant gene from the rice PCNA cDNA into an Escherichia coli expression vector pMAL, and the PCNA protein was expressed in the bacteria in the form of a fusion protein (70 kDa) with maltose‐binding protein (MBP). Monoclonal antibody against human PCNA reacted with both purified fusion protein and a 32‐kDa fragment, resulting from restriction protease (factor Xa) digestion of the fusion protein. The N‐terminal amino acid sequence of the 32‐kDa fragment was identical to that of rice PCNA sequence. Rice PCNA fusion protein was found to stimulate DNA synthesis catalyzed by DNA polymerase δ from human cells (although much less effectively), while having no effect on DNA polymerase α activity. The results indicate that plant PCNA functions as one of the cofactors of DNA synthesis as is the case with other eukaryotes.
Kinetic measurement of the interaction between an oligosaccharide and lectins by a biosensor based on surface plasmon resonance
Authors: SHINOHARA Yasuro , KIM Fimi , SHIMIZU Mizuho , GOTO Masanori , TOSU Mariko , HASEGAWA Yukio
Published: March 2005

Kinetic measurements of the interaction between an oligosaccharide and various lectins were performed using a biosensor based on surface plasmon resonance (SPR). A glycopeptide, prepared from asialofetuin and having a nearly homogeneous N‐linked sugar chain, was immobilized on the surface of a sensor chip via the amino groups of its peptide moiety. The interactions of this bound glycopeptide with six lectins [Sambucus sieboldiana lectin, Maackia amurensis lectin, Aleuria aurantia lectin, Ricinus communis agglutinin‐120 (RCA120), Datura stramonium lectin (DSA) and Phaseolus vulgaris leukoagglutinating lectin] were monitored in real‐time with the change in the SPR response. Of these lectins, only RCA120 and DSA showed an increase in the SPR response, indicating that these two lectins bound specifically to the immobilized glycopeptide. The other lectins did not show any significant changes in the SPR response. These results are in good agreement with the binding specificity previously demonstrated with affinity chromatography. The association‐rate constant (kass) and the dissociation‐rate constant (kdiss) for the glycopeptide‐RCA120 interaction were 3.4 × 105 M−1 s−1 and 2.1 × 10−3 s−1, respectively. The kass and kdiss determined for DSA were 5.7 × 105 M−1 s−1 and 1.3 × 10−3 s−1, respectively. Furthermore, the relative binding molar ratio to the glycopeptide was three times higher for RCA120 than for DSA, suggesting that this sugar chain possesses three binding sites for RCA120 and one for DSA. These parameters are expected to provide useful information for defining the interaction between oligosaccharides and lectins.
Protein kinase C V3 domain mutants with differential sensitivities to m‐calpain are not resistant to phorbol‐ester‐induced down‐regulation
Authors: JUNCO Maria , WEBSTER Catherine , CRAWFORD Catherine , BOSCA Lisardo , PARKER Peter J.
Published: March 2005

Distinct linker sequences were introduced into the protease‐sensitive V3 domain of protein kinase C‐α and the mutant proteins were expressed in COS‐1 cells. Partially purified preparations of these mutants were functionally similar to wild‐type protein kinase C‐α, however their susceptibility to m‐calpain was quite distinct, with one mutant being insensitive to cleavage. The three mutants, after expression in COS‐1 cells, were found to behave in a manner indistinguishable from wild‐type protein kinase C‐α with respect to subcellular distribution, acute responses to 12‐O‐tetradecanoyl‐phorbol 13‐acetate and 12‐O‐tetradecanoyl‐phorbol‐13‐acetate‐induced down‐regulation. The data imply that down‐regulation of protein kinase C‐α is likely to involve a general degradative process rather than cleavage by a site‐specific protease.
Purification of the AMP‐activated protein kinase on ATP‐γ‐Sepharose and analysis of its subunit structure
Authors: DAVIES Stephen P. , HAWLEY Simon A. , WOODS Angela , CARLING David , HAYSTEAD Timothy A. J. , HARDIE D. Grahame
Published: March 2005

The AMP‐activated protein kinase has been purified by affinity chromatography on ATP‐γ‐Sepharose. A proportion of the activity can be eluted using AMP, while the remainder is eluted using ATP. The AMP eluate contains three polypeptides of 63, 38 and 35 kDa (p63, p38 and p35) in a molar ratio (by Coomassie blue binding) close to 1:1:1. p63 was previously identified as the AMP‐binding catalytic subunit [Carling, D., Clarke, P. R., Zammit, V. A. & Hardie, D. G. (1989) Eur. J. Biochem. 186, 129–136]. All three polypeptides exactly comigrate both on native gel electrophoresis and on gel filtration, suggesting that p38 and p35 are additional subunits. Estimation of Stokes radius (5.4–5.8 nm) by gel filtration, and sedimentation coefficient (7.9–8.4 S) by glycerol gradient centrifugation, suggest that the kinase has an asymmetric structure with a native molecular mass for the complex of 190 ± 10 kDa. Thus the native enzyme appears to be a heterotrimer with a p63/p38/p35 (1:1:1) structure. Despite the fact that the ATP eluate has a higher specific activity than the AMP eluate (3.5 ± 0.2 vs 2.3 ± 0.2 μmol · min−1· mg−1), it appears to be less pure, containing p63, p38 and p35 plus other polypeptides. Experiments examining the effects of protein phosphatase‐2A and kinase kinase, and analysis by Western blotting with anti‐p63 antibody, suggests that the AMP eluate is entirely in the low‐activity dephosphorylated form, while the ATP eluate is a mixture of that form and the high‐activity phosphorylated form. As well as establishing the subunit structure of the AMP‐activated protein kinase, these results suggest that the kinase can bind to ATP‐γ‐Sepharose through either the allosteric (AMP/ATP) site or the catalytic (ATP) site, and that phosphorylation by the kinase kinase increases the affinity for the latter site.
Initial expression of glucokinase gene in cultured hepatocytes from suckling rats is linked to the synthesis of an insulin‐dependent protein
Authors: BOSSARD Pascale , DECAUX Jean‐François , JUANES Maria , GIRARD Jean
Published: March 2005

The initial accumulation of glucokinase mRNA in response to insulin in cultured hepatocytes from 10‐day‐old suckling rats was characterized by a delay of 18–24 h with a maximal level reached after 48 h. This delay is not observed in cultured adult rat hepatocytes. When hepatocytes from 10‐day‐old suckling rats were cultured for 48 h in the presence of insulin (to obtain a maximal accumulation of glucokinase mRNA) and then deprived of insulin for 18 h, glucokinase mRNA returned to very low levels. Reexposure of these cultured hepatocytes to insulin allowed a rapid accumulation of glucokinase mRNA, with a maximal level reached after 8 h, as in adult rat hepatocytes. The aim of the present study was to investigate the factors responsible for the delay in insulin action during first exposure to insulin. The difference in the kinetics of glucokinase mRNA accumulation after the first and secondary exposure to insulin was due to differences in the rate of transcriptional activity of the glucokinase gene, as shown by a run‐on assay on isolated nuclei. The half‐life of glucokinase mRNA was similar after the first and second exposure to insulin. The delay in the initial accumulation of glucokinase mRNA in response to the first exposure to insulin was not due to elevated levels of cAMP (a potent inhibitor of glucokinase gene expression) or to a defect in insulin signalling (insulin inhibited without delay phosphoenolpyruvate carboxykinase gene expression). In contrast, it was markedly dependent upon whether glucokinase has been already expressed in vivo. Hepatocytes from rats that had already expressed glucokinase in vivo (suckling rats force‐fed with glucose or rats weaned to a high‐carbohydrate diet) showed no delay in their response to insulin in culture, whereas hepatocytes from rats that have never expressed glucokinase in vivo (suckling rats or rats weaned to a high‐fat diet) showed a delay of 24 h. Two different inhibitors of protein synthesis (cycloheximide and puromycin) prevented the initial accumulation of glucokinase mRNA in response to the first exposure to insulin but not to the secondary accumulation of glucokinase mRNA in response to reexposure to insulin. This suggests that the synthesis of one or several insulin‐dependent proteins is necessary for the first activation of glucokinase gene transcription in response to the first exposure to insulin.
A peptide C‐terminal to the second Zn finger of human vitamin D receptor is able to specify nuclear localization
Authors: LUO Zhongjun , ROUVINEN Juha , MÄENPÄÄ Pekka H.
Published: March 2005

A peptide of 27 amino acids, VDR(102–76), representing residues 76–102 immediately C‐terminal to the second Zn finger of human vitamin D receptor (hVDR) was conjugated to fluorescein‐labelled IgG using a bifunctional coupling reagent, m‐maleimidobenzoyl n‐hydroxysuccinimide. Upon microinjection into the cytoplasm of human osteosarcoma MG‐63 cells, the chimeras accumulated in the nuclei. This transport was arrested by chilling or energy depletion. Two other peptides, VDR(80–67), spanning the N‐terminal part of VDR(102–76), and VDR(108–97), spanning the C‐terminal part of VDR(102–76), were not able to target the linked proteins to the nuclei. SV40(135–112), a peptide containing a well‐characterized nuclear localization sequence (amino acids 112–135) of simian virus 40 (SV40) large T‐antigen, caused complete nuclear accumulation under the same conditions. Wheat germ agglutinin, which inhibits SV40(135–112) transport, also inhibited the nuclear accumulation of VDR(102–76) as did energy depletion.
Purification and cDNA cloning of a four‐domain Kazal proteinase inhibitor from crayfish blood cells
Authors: JOHANSSON Mats W. , KEYSER Pia , SÖDERHÄLL Kenneth
Published: March 2005

A cDNA with an open reading frame of 684 base pairs was isolated from a library from blood cells of the crayfish Pacifastacus leniusculus. It codes for a signal sequence and a mature protein of 209 amino acids with a predicted molecular mass of 22.7 kDa. The amino acid sequence consists of four repeated stretches (45–73% identical to each other), indicating that the protein has four domains. The domains have significant sequence similarity to serine proteinase inhibitors of the Kazal family. The three first domains have a leucine residue in the putative reactive site, suggesting that the protein is a chymotrypsin inhibitor.

A monomeric 23‐kDa proteinase inhibitor, which by amino terminal sequencing of the mature protein was confirmed to be the cloned Kazal inhibitor, was purified from crayfish blood cells. It inhibited chymotrypsin or subtilisin, but not trypsin, elastase or thrombin. The inhibitor seemed to form a 1 : 1 complex with chymotrypsin or subtilisin.

This protein seems to be the first described Kazal inhibitor from blood cells of any animal and the first one with four domains.
The calpain cleavage sites in the epidermal growth factor receptor kinase domain
Authors: GREGORIOU Mary , WILLIS Anthony C. , PEARSON Mark A. , CRAWFORD Catherine
Published: March 2005

The proteolysis of the human epidermal growth factor receptor cytoplasmic domain by calpain has been studied in vitro using purified recombinant cytoplasmic domain expressed in insect cells. Limited proteolysis produced kinase that was truncated at either N‐ or C‐termini, as well as in the hinge region. We identified seven sites of calpain proteolysis by N‐terminal sequencing of purified fragments. Calpain cleaved between the catalytic and autophosphorylation domains at two sites in the sequence Gln996–Asp1059, in the hinge region. Three new sites were also found in the autophosphorylation domain, preceding each of the major autophosphorylation sites. A fourth new site was located in the juxtamembrane domain, C‐terminal to the regulatory Thr654. We purified an active 42‐kDa fragment generated by calpain proteolysis between Leu659–Gln660 in the juxtamembrane domain, and in the hinge region.

A fifth new site of calpain cleavage was found between the nucleotide binding motif Gly‐Xaa‐Gly‐Xaa‐Xaa‐Gly and the essential Lys721 in the catalytic core of the kinase. Since both of these features are required for catalysis, calpain cleavage at this site may potentially provide a mechanism for down‐regulation of kinase activity in vivo, under conditions of calpain activation. Thus the distribution of calpain cleavage sites along the kinase domain is consistent with a role for calpain both as a processing and as a degradative protease in epidermal growth factor receptor signalling.
Amino acid sequence of spinach ferredoxin:thioredoxin reductase variable subunit
Authors: IWADATE Hiromoto , YANO Keiichi , KAMO Masaharu , GARDET‐SALVI Laura , SCHÜRMANN Peter , TSUGITA Akira
Published: March 2005

Ferredoxin:thioredoxin reductase (FTR) is an iron‐sulfur protein, which, in the presence of ferredoxin and thioredoxin, catalyses the light‐dependent activation of several photosynthetic enzymes. Spinach FTR consists of two dissimilar polypeptide chains, A and B, present in equal amounts. Whereas subunit B seems to be responsible for the catalytic activity, subunit A has no known catalytic function. We found earlier that the N‐terminus of subunit A, also called the variable subunit, shows terminal redundancy and that 2–3 of its serine residues are phosphorylated [Tsugita, A. Yano, K., Gardet‐Salvi, L. & Schürmann, P. (1991) Protein Sequence Data Anal. 4, 9–13]. We now report the complete amino acid sequence of subunit A, determined by conventional protein sequencing methods. The polypeptide chain with a calculated molecular mass of 12 669 Da consists of 112 amino acids and has a calculated isoelectric point of 5.4. The analysis of the sequence supports the idea that this subunit has no catalytic function. The comparison with a known cyano‐bacterial FTR reveals about 58% similarity and the striking presence of a N‐terminal extension in the spinach protein. This extension may be responsible for the reported size variability of this subunit.
A hydrogen peroxide assay based on the peroxidase‐oxidase reaction
Authors: BOUTELET Isabelle , ALEXANDRE Stéphane , VINCENT Jean‐Claude
Published: March 2005

In order to explain an assay method for H2O2, based on the horseradish peroxidase/NADH/H2O2 system, we performed a numerical simulation of this complex reaction mechanism. For this purpose, we applied a simplified version of a model designed by Yokota and Yamazaki to our experimental conditions [Yokota, K. & Yamazaki, I. (1977) Biochemistry 16, 1913–1918]. This modification leads to a kinetic behaviour of the system very similar to our experimental results when the presence of superoxide dismutase is simulated. These simulations confirm that hydrogen peroxide concentrations are proportional to the total amount of NADH oxidized. We also showed that superoxide dismutase activity can be determined by this assay method. Moreover, in samples containing superoxide dismutase and hydrogen peroxide, the simulations showed that both superoxide dismutase and hydrogen peroxide can be determined using our assay method.
The effects of methylprednisolone on oxidative phosphorylation in Concanavalin‐A‐stimulated thymocytes
Authors: BUTTGEREIT Frank , GRANT Andrea , MÜLLER Marianne , BRAND Martin D.
Published: March 2005

The glucocorticoid methylprednisolone has clinically important anti‐inflammatory effects at high concentrations through unknown mechanisms. Methylprednisolone at 0.2 mg/107 cells inhibits respiration in Concanavalin‐A(ConA)‐stimulated thymocytes from rats by about 20%. We have used top‐down elasticity analysis to identify the blocks of reactions within oxidative phosphorylation in thymocytes whose kinetics are significantly affected by treatment with methylprednisolone. At this concentration methylprednisolone greatly inhibited the reactions of substrate oxidation and increased mitochondrial proton leak but did not significantly affect the synthesis and turnover of ATP by the phosphorylating system. Metabolic control analysis showed that oxygen consumption by ConA‐treated thymocytes was controlled largely (0.51) by the phosphorylating system but also by proton leak (0.32) and substrate oxidation (0.17); this is similar to the distribution of control in hepatocytes, suggesting that this pattern may be general in cells. Methylprednisolone lowered control by the phosphorylating system to 0.26 and raised control by substrate oxidation to 0.37. From these results we conclude that the inhibition of respiration in ConA‐stimulated thymocytes by methylprednisolone at this concentration results from an inhibition of substrate oxidation and a smaller stimulation of mitochondrial proton leak, with only a minor contribution of any effects within the phosphorylating system.
Indirect regulation of Ca2+ entry by cAMP‐dependent and cGMP‐dependent protein kinases and phospholipase C in rat platelets
Authors: HEEMSKERK Johan W. M. , FEIJGE Marion A. H. , SAGE Stewart O. , WALTER Ulrich
Published: March 2005

The Ca2+ responses of rat platelets are dominated by the influx of extracellular Ca2+ across the plasma membrane [Heemskerk, J. W. M., Feijge, M. A. H., Rietman, E. & Hornstra, G. (1991) FEBS Lett. 284, 223], which allows the study of Ca2+ entry into these cells by measuring increases in cytosolic Ca2+ concentration, [Ca2+]i. Several pieces of evidence indicated that, as in human platelets [Sage, S. O., Reast, R., & Rink, T. J. (1990) Biochem. J. 265, 675–680; Alonso, M., Alvarez, J., Montero, M., Sanchez, A. & García‐Sancho, J. (1991) Biochem. J. 280, 783–789], agonist‐stimulated Ca2+ entry was linked to the mobilisation of Ca2+ from intracellular stores: there was good correlation between the potency of receptor agonists in elevating [Ca2+]i in the presence or absence of external CaCl2; agonist‐induced Ca2+ entry was inhibited to a similar degree as internal mobilisation by activators of cAMP‐dependent or cGMP‐dependent protein kinase or by the phospholipase C inhibitor, U73122; thapsigargin (an inhibitor of endomembrane Ca2+ ‐ATPases) evoked store depletion and Ca2+ entry, which were both reduced by prior activation of cAMP‐dependent or cGMP‐dependent protein kinase but were not affected by U73122. In platelets with depleted Ca2+ stores, the addition of CaCl2 resulted in a considerable entry of Ca2+ which was insensitive to cAMP‐dependent and cGMP‐dependent protein kinase activation. In control platelets with full Ca2+ stores, CaCl2 potentiated the thrombin‐induced generation of myo‐inositol phosphates, suggesting that Ca2+ entry potentiated phospholipase C activity. Taken together, these results indicate that Ca2+ entry in rat platelets, (a) is mostly secondary to store depletion, (b) is not directly downregulated by cAMP‐dependent and cGMP‐dependent protein kinase, but indirectly by inhibition of store depletion, (c) can proceed in the absence of phospholipase C activation, but is stimulated by this activity probably by increased mobilisation of Ca2+ from the stores. These results lead to the concept that a major part of receptor‐mediated Ca2+ entry in rat platelets is regulated in an indirect way by factors that stimulate or inhibit the degree of Ca2+ mobilisation from the internal stores.
A β‐glucosidase gene (bgl3) from Streptomyces sp. strain QM‐B814
Authors: PEREZ‐PONS Josep A. , CAYETANO Antonio , REBORDOSA Xavier , LLOBERAS Jorge , GUASCH Alicia , QUEROL Enrique
Published: March 2005

A β‐glucosidase gene (bgl3) from Streptomyces sp. QM‐B814 (American Type Culture Collection 11238) has been cloned by functional complementation of a β‐glucosidase‐negative mutant of Streptomyces lividans. An open‐reading frame of 1440 nucleotides encoding a polypeptide of 479 amino acids was found by sequencing. The encoded protein (Bgl3) shows extensive similarity (over 45% identity) with β‐glycosidases from family‐1 glycosyl hydrolases. The cloned enzyme, purified following ammonium sulphate precipitation and two chromatographic steps, is monomeric with molecular mass 52.6 kDa, as determined by mass spectrometry, and an isoelectric point of pI 4.4. The enzyme appears to be a β‐glucosidase with broad substrate specificity, is active on cellooligomers, and performs transglycosylation reactions. The estimated apparent Km values for p‐nitrophenyl‐β‐d‐glucopyranoside and cellobiose are 0.27 mM and 7.9 mM, respectively. The Ki values for glucose and δ‐gluconolactone, using p‐nitrophenyl‐β‐d‐glucopyranoside as a substrate, are 65 mM and 0.08 mM, respectively. The purified enzyme has a pH optimum of pH 6.5 and the temperature optimum for activity is 50°C.
A possible regulation of negative factor (Nef) activity of human immunodeficiency virus type 1 by the viral protease
Authors: FREUND Jens , KELLNER Roland , KONVALINKA Jan , WOLBER Vera , KRÄUSSLICH Hans‐Georg , KALBITZER Hans R.
Published: March 2005

Negative factor (Nef) protein from human immunodeficiency virus type 1 (HIV‐1) is cleaved into two well‐defined domains by the HIV‐1‐encoded protease. The cleavage site is located between Trp57 and Leu58 and is well conserved. The two domains are stable in the presence of protease for more than 48 h. The C‐terminal core domain contains a well‐conserved well‐folded region. The cleavage releases the core domain from the myristoylated membrane anchor domain. As is the case for other HIV proteins, cleavage of Nef could be crucial for correct biological function.
Production of active, insect‐specific scorpion neurotoxin in yeast
Authors: MARTIN‐EAUCLAIRE Marie‐France , SØGAARD Morten , RAMOS Cayo , CESTÈLE Sandrine , BOUGIS Pierre E. , SVENSSON Birte
Published: March 2005

A cDNA encoding the Androctonus australis Hector insect toxin 1 (AaH IT1) was expressed in yeast leading to secretion of fully biologically active protein. Three different multicopy plasmids were constructed using PCR. Expression was directed by the strong PGK1 promoter of the yeast vector pMA 91. Plasmid pMA 91‐AaH IT1 encodes AaH IT1 and its own signal peptide. In the two other constructions, the cDNA encoding the mature part of AaH IT1 is fused to the prepro‐signal sequence of the yeast α‐mating‐factor precursor; the pBAL 7‐α‐KREAEA‐AaH IT1 includes the cDNA sequence encoding the KR(EAEA) processing sequence of the α‐mating‐factor, and pBAL 7‐α‐KR‐AaH IT1 encodes the KR fused directly to the AaH IT1 gene. The yeast α‐mating‐factor signal peptide launched the pro‐α‐mating‐factor−AaH IT1 fusion protein into the secretory pathway. The fusion proteins are expected to be cleaved in the Golgi by the KEX2 endopeptidase and the STE13 dipeptidyl aminopeptidase, leading to release of mature AaH IT1. Pulse/chase labelling of transformed yeast protoplasts, followed by SDS/PAGE analysis of proteins immunoprecipitated from either the lysate or the extracellular fluid, showed that AaH IT1 was produced. The highest concentration of recombinant AaH IT1 in the culture medium, as determined using a 125I‐AaH IT1 specific radioimmunoassay, was 4 μg/l (0.5 nM). The recombinant toxin was fully biologically active against cockroaches as assessed by injection and comparison to native AaH IT1. Moreover, it competed with radiolabelled native toxin for its receptor on the voltage‐sensitive Na+ channel with a dissociation constant of 0.5 nM.
Two promoters control the transcription of the human α2(VI) collagen gene
Authors: SAITTA Biagio , CHU Mon‐Li
Published: March 2005

Our previous studies have demonstrated that the human α2(VI) collagen gene produces four mRNA species with different 5′‐untranslated regions [Saitta, B., Timpl, R. & Chu, M.‐L. (1992) J. Biol. Chem. 267, 6188–6196]. The major mRNA species initiates from exon 1, located at the most 5′ end, whereas three minor mRNAs start from an alternative exon, 1A, located 657 bp downstream of exon 1. In this study, we have investigated whether or not these different mRNAs are transcribed from two separate promoters. DNA fragments preceding exons 1 and 1A were fused with a reporter gene for chloramphenicol acetyl transferase (CAT) and transfected into human dermal fibroblasts and fibrosarcoma HT1080 cells. Strong CAT activity in both cell types was observed using a construct containing DNA from nucleotide −502 to +115 preceding exon 1. The CAT activity of a construct containing nucleotide +514 to +894 preceding exon 1A was almost as high as that of the former construct, indicating the presence of two promoters, P1 and P2, preceding exons 1 and 1A, respectively. Transient transfection assays also identified positive and negative regulatory regions for the P1 promoter, located from nucleotide −2152 to −1384 and from nucleotide −1383 to −503, respectively. A negative regulatory region located at nucleotide +116 to +513 was found for the P2 promoter. This region strongly inhibits the P2 promoter in dermal fibroblasts, and thus may be responsible for the low expression of the endogenous exon‐1A‐containing mRNAs in these cells. Footprinting analysis of the two promoters with purified Sp1 protein and AP2 protein extract showed several sites of DNA‐protein interaction. The specificity of these sites was confirmed by competition experiments using consensus Sp1 and AP2 oligonucleotides. The results thus demonstrate that the human α2(VI) collagen gene contains two promoters, which are regulated by positive and negative cis‐acting DNA elements and trans‐acting factors.
Subtractive hybridization cloning of novel genes differentially expressed during intestinal development
Authors: BARILÀ Daniela , MURGIA Chiara , NOBILI Fabio , GAETANI Sancia , PEROZZI Giuditta
Published: March 2005

Intestinal genes whose expression is regulated during development and differentiation were identified and cloned from a rat villi cDNA library using a subtracted cDNA probe. The isolated clones are transcribed in the fully differentiated intestinal epithelium 21 days after birth and absent or poorly expressed in the fetal gut at 15 days of gestation. Two of the DRI (differentially‐expressed in rat intestine) genes are novel, while the others encode the microvillar protein ezrin and intracellular carrier proteins for retinol and fatty acids. Expression of the newly isolated DRI27 and DRI42 clones parallels epithelial differentiation during development and it is more pronounced in the distal portions of the small intestine. In situ hybridization experiments indicate that the DRI mRNAs are expressed in the differentiated cell types of the gut epithelium. Moreover, the expression of DRI27 and DRI42 is strongly related to the stage of epithelial differentiation during gut development. This relationship holds true also for the expression of DRI42 in other tissues. These clones will be a valuable tool to identify regulatory sequences and factors responsible for confining gene expression to the differentiated epithelial cell types in mammalian small intestine.
Cloning and expression of the gene for hydroxypyruvate reductase (d‐glycerate dehydrogenase from an obligate methylotroph Hyphomicrobium methylovorum GM2
Authors: YOSHIDA Toyokazu , YAMAGUCHI Kenji , HAGISHITA Tairo , MITSUNAGA Toshio , MIYATA Atsuro , TANABE Tadashi , TOH Hiroyuki , OHSHIRO Takashi , SHIMAO Masayuki , IZUMI Yoshikazu
Published: March 2005

The gene encoding hydroxypyruvate reductase, catalyzing the asymmetric reduction of hydroxypyruvate to d‐glycerate, and its flanking regions were isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2. Nucleotide sequencing of the recombinant plasmids revealed that the hydroxypyruvate‐reductase gene codes for the 322‐amino‐acid protein with calculated molecular mass 35726 Da. The sequence was confirmed by sequencing the intact enzyme and peptides obtained by digestion of the enzyme with Achromobacter proteinase I. The amino acid sequence of the enzyme showed similarity to members of the d‐isomer‐specific 2‐hydroxyacid dehydrogenase family. The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223‐3, was introduced into Escherichia coli HB101. The recombinant enzyme purified from the transformed E. coli cells was indistinguishable from the enzyme isolated from H. methylovorum GM2 by immunological and enzymological analyses.
Site‐specific oxidation of histidine residues in glycated insulin mediated by Cu2+
Authors: CHENG Rong‐zhu , KAWAKISHI Shunro
Published: March 2005

The site‐specific oxidation of histidine residues in glycated insulin mediated by copper ions and the relationship between the oxidation sites and the steric conformation of insulin are discussed in this study. Glycated insulin was prepared by incubating native insulin with glucose in 67 mM sodium phosphate, pH 7.5, at 37°C for 30 h. In the presence of micromolar concentrations of Cu2+, glycated insulin was oxidized and its fragmentation or aggregation was detected. Accompanying the fragmentation, new N‐termini were generated. The residues in these N‐termini were identified as alanine, proline, valine, leucine and isoleucine by comparing dansyl derivatives with standard dansyl‐amino acid products. Furthermore, several oxidized products of glycated insulin were isolated using reverse‐phase HPLC (P1–P3). From amino acid composition and sequence analyses, it was determined that His10 on the insulin B‐chain was modified in each of these peptides, while His5 was also modified in P3. The difference in susceptibility of His10 and His5 to oxidative modification is considered to be due to easier coordination of Cu2+ with His10, which further forms a complex with the Amadori compound at B‐chain Phe1 that is vicinal to His10 in the steric conformation of insulin. This complex may generate an active oxygen species, which induces the degradation of the imidazole ring at His10, leading to aggregation or fragmentation of insulin.
Purification and characterization of an alkaline elastase from Myxococcus xanthus
Authors: DUMONT Laurence , VERNEUIL Bernard , WALLACH Jean , JULIEN Raymond
Published: March 2005

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAP1), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion‐exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,10‐phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin‐orcein and succinyl‐Ala3p‐nitroanilide as substrates. Despite its low pI (5.2), MAP1 was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAP1 and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30‐min prior incubation of the enzyme at 40°C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50°C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAP1 readily hydrolyzes the Gly23‐Phe24 bond in the oxidized insulin B chain; the peptide bonds Ala14‐Leu15, Leu15‐Tyr16, Phe24‐Phe25, Phe25‐Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C‐terminus from the cleavage site (P′1 position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157–162]. This hypothesis is consistent with the fact that Ala2‐Phe‐Ala and Ala3‐Phe‐Ala are hydrolyzed even though tri‐alanine to hexa‐alanine oligomers are not.

The evidence of an elastase with the same molecular mass and pI as MAP1 is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A‐signal [Kuspa, A., Plamann, L. & Kaiser, D. (1992) J. Bacteriol. 174, 3319–3326] is consistent with the hypothesis that, regarding its specificity, MAP1 is likely to play a role in development of myxobacteria.
Modulation of thermoprotection and translational thermotolerance induced by Semliki Forest virus capsid protein
Authors: MICHEL Marcel R. , FAVRE Daniel , STUDER Erwin , ARRIGO André‐Patrick , KEMPF Christoph
Published: March 2005

Low amounts of Semliki Forest virus capsid protein transferred into target cells by electroporation‐mediated delivery (103–104 molecules incorporated/cell) confer thermal resistance resulting in enhanced survival. Furthermore, when exposed to 43°C, these cells display an enhanced expression of heat‐shock protein‐70 and a translational thermotolerance. Similarly, low amounts of capsid protein transferred into cells in which transcription is blocked by actinomycin D, also protect the translation machinery at 43°C. In a cell‐free translation system, added capsid protein appears to modulate translational efficiency of endogenous mRNSs. At approximately 1 molecule/ribosome, capsid protein is able to enhance translation at 30°C and at 43°C. In contrast, high concentrations of capsid protein are responsible for a marked inhibition of protein synthesis at 30°C, but only hamper translational thermotolerance at 43°C. Our results favor the hypothesis that small amounts of capsid protein trigger a chaperone‐like activity that is able to protect the translational machinery from thermal damage.
Binding of human serum amyloid P component (hSAP) to human neutrophils
Authors: LANDSMANN Pavel , ROSEN Oren , PONTET Michel , PRAS Mordechai , LEVARTOWSKY David , SHEPHARD Enid G. , FRIDKIN Mati
Published: March 2005

Human serum amyloid P component (hSAP) and human C‐reactive protein (hCRP) are normal serum constituents related to the pentraxin family of plasma proteins. hSAP has morphological and immunochemical identity and extensive sequence similarity to the amyloid P (AP) component found in normal tissues and particularly in amyloid deposits. hCRP and its proteolytic products have been previously shown to bind and to interact with various types of human leukocytes. Binding‐displacement experiments with 125I‐labeled hSAP and hCRP show that both proteins have specific high‐affinity binding sites on normal human polymorphonuclear leukocytes (PMN) and each can compete efficiently with the binding of the other. Scatchard analysis of hSAP‐displacement curves reveals a heterogeneous population of hSAP‐binding sites existing on the PMN cells, among them about 300000 low‐affinity binding sites with Kd≤ 5X10−6 M and about 30000 high‐affinity binding sites with Kd≤ 5X10−8 M. hAP was found to be degraded by enzymes from human neutrophils to yield a mixture of low‐molecular‐mass peptides, similarly to the case of CRP reported previously. The binding of hSAP can be efficiently inhibited by this peptide mixture. The results suggest that both hCRP and hSAP, together with related peptides, may participate in vivo in an unknown mechanism of regulation of human neutrophils.
Iron metabolism in Rhodobacter capsulatus
Authors: RINGELING Patricia L. , DAVY Sharon L. , MONKARA Fayez A. , HUNT Colette , DICKSON Dominic P. E. , McEWAN Alastair G. , MOORE Geoffrey R.
Published: March 2005

The water‐soluble cytochrome b557 from the photosynthetic bacterium Rhodobacter capsulatus was purified and shown to have the properties of the iron‐storage protein bacterioferritin. The molecular mass of R. capsulatus bacterioferritin is 428 kDa and it is composed of a single type of 18‐kDa subunit. The N‐terminal amino acid sequence of the bacterioferritin subunit shows 70% identity to the sequence of bacterioferritin subunits from Escherichia coli, Nitrobacter winogradskyi, Azotobacter vinelandii and Synechocystis PCC 6803. The absorbance spectrum of reduced bacterioferritin shows absorbance maxima at 557 nm (α band), 526 nm (β band) and 417 nm (Soret band) from the six haem groups/molecule. Antibody assays reveal that bacterioferritin is located in the cytoplasm of R. capsulatus, and its levels stay relatively constant during batch growth under aerobic conditions when the iron concentration in the medium is kept constant. Iron deficiency leads to a decrease in bacterioferritin and iron overload leads to an increase. Bacterioferritin from R. capsulatus has an amorphous iron‐oxide core with a high phosphate content (900–1000 Fe atoms and approximately 600 phosphates/bacterioferritin molecule). Mössbauer spectroscopy indicates that in both aerobically and anaerobically (phototrophically) grown cells bacterioferritin with an Fe3+ core is formed, suggesting that iron‐core formation in vivo may not always require molecular oxygen.
A new function of calphobindin I (annexin V)
Authors: NAKAO Hiroshi , WATANABE Masanao , MAKI Masahiro
Published: March 2005

Calphobindin I (CPB‐I, annexin V) is an anticoagulant protein purified from human placenta; it is a member of the annexin family that binds phospholipids in a calcium‐dependent manner. In this study, we discovered and examined a new function of CPB‐I: promotion of both the migration and the urokinase‐type plasminogen activator (uPA) activity of normal human keratinocytes (NHK). While the treatment of NHK with a 10‐μg/ml concentration of CPB‐I for 24 h or 48 h caused an approximate 30% increase in the migration of NHK (compared with the no treatment), migration was inhibited when anti‐CPB‐I monoclonal antibodies (i.e. A46 and A180) were added along with the CPB‐I. Moreover, while the treatment of NHK with a CPB‐I concentration greater than 10 μg/ml caused a significant increase in the activity of secreted uPA, reflected in an approximately 40% increase in cell migration, uPA activity was inhibited both by cycloheximide and by monoclonal antibodies. This significant increase of secreted uPA was seen 8 h after the addition of CPB‐I. Specific binding of CPB‐I to NHK had a Kd value of 95.2 nM (equivalent to a CPB‐I concentration of 3.2 μg/ml), and was also inhibited by monoclonal antibodies. However, CPB‐I had no effect on NHK proliferation. Furthermore, CPB‐I enhanced reepithelialization when it was applied locally twice a day to full‐thickness cutaneous wounds made in male rats. Our results show that, during an injury, CPB‐I helps reepithelialization through the promotion of both uPA synthesis and migration of keratinocytes without stimulating their proliferation.
Differentiation‐induced increase in Clostridium botulinum C3 exoenzyme‐catalyzed ADP‐ribosylation of the small GTP‐binding protein Rho
Authors: FRITZ Gerhard , JUST Ingo , WOLLENBERG Peter , AKTORIES Klaus
Published: March 2005

The specific [32P]ADP‐ribosylation by Clostridium botulinum exoenzyme C3 was used to study differentiation‐dependent changes in the regulation of the low‐molecular‐mass GTP‐binding protein Rho. Differentiation of F9 teratocarcinoma cells to neuronal‐like cells by treatment with retinoic acid and dibutyryl‐adenosine 3′,5′‐monophosphate [(Bt)2cAMP] increased the C3‐catalyzed ADP‐ribosylation of RhoA proteins in cytosolic and membrane fractions by about threefold and sixfold, respectively. Phenotypical differentiation of F9 cells was not required for increase in ADP‐ribosylation. Increase in ADP‐ribosylation after (Bt)2cAMP and retinoic acid treatments was blocked by cycloheximide, indicating the requirement of protein biosynthesis. As deduced from specific rho mRNA amounts and from Western analysis with a monoclonal RhoA antibody, the stimulation in the [32P]ADP‐ribosylation of Rho was not caused by an increased de‐novo synthesis of Rho proteins. GDP increased the ADP‐ribosylation of membrane‐associated Rho from non‐differentiated, but not from differentiated F9 cells. GTP[S] decreased ADP‐ribosylation of membranous Rho from differentiated and much less from non‐differentiated F9 cells. Differentiation‐dependent increase in ADP‐ribosylation of cytosolic Rho was reversed by protein phosphatase type‐1. Treatment with SDS (0.01%) which releases Rho from complexation with guanine nucleotide dissociation inhibitor, increased ADP‐ribosylation both in differentiated and non‐differentiated cells, indicating no differentiation‐specific change of such complexes. In total, our data indicate that the induction of the differentiation process in F9 cells is accompanied by changes in the regulation of cytosolic and membrane‐associated Rho proteins.
Cloning of a wheat 15‐kDa grain softness protein (GSP)
Authors: RAHMAN Sadequr , JOLLY Christopher J. , SKERRITT John H. , WALLOSHECK Andrea
Published: March 2005

The wheat starch 15‐kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides. An antiserum raised against GSP was used to screen a wheat cDNA library. A cDNA family encoding approximately 15‐kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified. A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti‐GSP serum. A new antiserum raised against the fusion protein produced a weak reaction against a 15‐kDa polypeptide extracted from wheat seeds. The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15‐kDa polypeptides defined as GSP. RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half‐way through grain filling. The encoded proteins are novel members of the 2S superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues. The deduced proteins show the highest similarity to the oat 16‐kDa avenin and to wheat puroindoline (a lipid‐binding 15‐kDa protein from wheat). Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid‐binding properties of GSP polypeptides may influence grain softness.
Structures of heat‐stable and unstable homologues of the sweet protein mabinlin.
Authors: NIRASAWA Satoru , NISHINO Tomoko , KATAHIRA Masato , UESUGI Seiichi , HU Zhong , KURIHARA Yoshie
Published: March 2005

There are several analogues of the sweet protein mabinlin. In previous studies, we purified the heat‐stable analogue, mabinlin II, from the seeds of Capparis masaikai Lévl. and determined its amino acid sequence [Liu, X., Maeda, S., Hu, Z., Aiuchi, T., Nakaya, K. & Kurihara, Y. (1993) Eur. J. Biochem. 211, 281–287] and the disulfide structure [Nirasawa, S., Liu, X., Nishino, T. & Kurihara, Y. (1993) Biochim. Biophys. Acta 1202, 277–280]. We have now purified four additional homologues of mabinlin. The sweet activities of mabinlin III and mabinlin IV were unchanged by incubation for 1 h at 80°C, as was found previously for mabinlin II, while the sweet activity of mabinlin I‐1 was completely abolished by a 1‐h incubation at 80°C. The circular dichroic spectrum showed that α‐helical structures of mabinlins II–IV were unchanged by the 1‐h incubation at 80°C, while the α‐helical structures of mabinlin I‐1 were completely destroyed by the 1‐h incubation in parallel with the decrease of the sweet activity. To compare the structures of the heat‐stable and unstable homologues, we determined their amino acid sequences and the disulfide array. The positions of four disulfide bridges of mabinlin I‐1 were the same as those of mabinlin II, suggesting that the disulfide bridges do not contribute to the difference in the heat stability among the homologues. There was a high similarity among amino acid sequences of the homologues. Only three amino acid residues (A‐chain residues at positions 22 and 32 and B‐chain residue at position 47) were different between mabinlin I‐1 and mabinlin III. A‐chain residue at position 32 was lacking in mabinlin IV and the A‐chain residue at position 22 was identical in both mabinlin I‐1 and mabinlin II. The B‐chain residue at position 47 was the only residue present in all three heat‐stable homologues (mabinlins II–IV) and is not present in the unstable homologue (mabinlin I‐1). This suggests that the difference in the heat stability of mabinlin is due to the difference in a B‐chain residue at position 47; the difference in the heat‐stable homologues is due to the presence of an arginine residue and the difference of the unstable homologue is due to the presence of glutamine. It is possible that a salt bridge, formed between a at B‐chain arginine residue at position 47 and the caroxylic group of an A‐chain or B‐chain C‐terminal residue contributes to the heat stability.
Dissociation and reconstitution of the Thermoplasma proteasome
Authors: GRZIWA Anja , MAACK Stephan , PÜHLER Gabriela , WIEGAND Georg , BAUMEISTER Wolfgang , JAENICKE Rainer
Published: March 2005

The proteasome from the thermoacidophilic archaeon Thermoplasma acidophilum in its native state represents a 20S particle with significant secondary structure (∼ 35%α helix) of its subunits. Electron microscopy, ultracentrifugal and spectral analysis demonstrate that at pH of less than 3 dissociation to partially denatured subunits occurs. Upon dialysis against near neutral pH buffers, at low protein concentration, reconstitution occurs, leading to the restoration of up to 90% of the native fluorescence signal. The recovery of activity depends on several parameters, including the buffer system, the pH used to dissociate the complex, and the duration of exposure to low pH. High concentrations of Ca2+ and Mg2+ cause partial dissociation of the Thermoplasma proteasome, yielding distinct subcomplexes. Neither the completely nor the partially dissociated complexes have proteolytic activity, indicating that function is linked to fully assembled proteasomes.