bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions of lipoyl domains with the E1p subunits of the pyruvate dehydrogenase multienzyme complex from Escherichiacoli
Authors: Graham Lloyd D. , Perham Richard N.
Published: July 2002

Equilibrium binding experiments were carried out with lipoyl domains and the pyruvate decarboxylase [pyruvate dehydrogenase (lipoamide), Elp, EC 1.2.4.1)] component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The dissociation constant (K
s) was estimated to be not less than 0.3 mM, exceeding the K
m value (33 μM) for reductive acetylation of the domains by an order of magnitude. Thus, the lipoyl domain, which is required to promote reductive acetylation of the lipoyl group, does not appear to do this simply by enhancing the binding to Elp. The difference between K
s and K
m suggests that the formation and release of reductively acetylated lipoyl domains from the enzyme may be a relatively rapid step in the mechanism.
Cyclic diguanylic acid behaves as a host molecule for planar intercalators
Authors: Liaw Yen-Chywan , Gao Yi-Gui , Robinson Howard , Sheldrick George M. , Sliedregt L.A.J.M. , van der Marel Gijs A. , van Boom Jacques H. , Wang Andrew H.-J.
Published: July 2002

Cyclic ribodiguanylic acid, c‐(GpGp), is the endogenous effector regulator of cellulose synthase. Its three‐dimensional structure from two different crystal forms (tetragonal and trigonal) has been determined by X‐ray diffraction analysis at 1 Å resolution. In both crystal forms, two independent c‐(GpGp) molecules associate with each other to form a self‐intercalated dimer. A hydrated cobalt ion is found to coordinate to two N7 atoms of adjacent guanines, forcing these two guanines to destack with a large dihedral angle (32°), in the dimer of the tetragonal form. This metal coordination mechanism may be relevant to that of the anticancer drug cisplatin. Moreover, c‐(GpGp) exhibits unusual spectral properties not seen in any other cyclic dinucleotide. It interacts with planar organic intercalator molecules in ways similar to double helical DNA. We propose a cage‐like model consisting of a tetrameric c‐(GpGp) aggregate in which a large cavity (‘host’) is generated to afford a binding site for certain planar intercalators (‘guests’).
Evidence for communication between nerve growth factor and protein tyrosine phosphorylation
Authors: Gómez Néstor , Tonks Nicholas K. , Morrison Christine , Harmar Tony , Cohen Philip
Published: July 2002

Nerve growth factor (NGF) stimulation of PC12 cells activated two myelin basic protein (MBP) kinase activities > 10‐fold within 5 min, which were resolved by chromatography on Mono Q. Each enzyme phosphorylated MBP on threonine and was inactivated by incubation with either CD45, a protein tyrosine phosphatase, or protein phosphatase 2A (PP2A), a serine/threonine phosphatase. The effects of CD45 and PP2A were prevented by vanadate and okadaic acid, respectively. Activation of the MBP‐kinases provides a mechanism for communication between NGF and intracellular protein tyrosine phosphorylation.
Cytotoxic activity of tumor necrosis factor is inhibited by amiloride derivatives without involvement of the Na+/H+ antiporter
Authors: Vanhaesebroeck B. , Cragoe E.J. , Pouysségur J. , Beyaert R. , Van Roy F. , Fiers W.
Published: July 2002

Cytotoxicity of tumor necrosis factor (TNF) on L929s cells was efficiently blocked by several amiloride analogs but not by amiloride itself. This protection did not require RNA or protein synthesis. Na+/H+ antiporter‐negative L‐M(TK−) cells (LAP) could be killed by TNF, showing that the Na+/H+ exchanger is not required for TNF‐cytotoxicity. Similar protection against TNF‐mediated cell lysis by amiloride derivatives was found for LAP and L929s cells, excluding a blockade of the Na+/H+ antiporter as the cause of the protection against TNF by these agents.
‘Quanta’ Ca2+ release and the control of Ca2+ entry by inositol phosphates ‐ a possible mechanism
Authors: Irvine R.F.
Published: July 2002

The release of Ca2+ from intracellular stores by sub‐optimal doses of inositol trisphosphate has been shown to be dose‐related (‘quantal’), and a simple model is proposed here to account for this phenomenon. It is suggested that there is a regulatory Ca2+‐binding site on, or associated with, the luminal domain of the InsP3 receptor, which allosterically controls Ca2+ efflux, and the affinity for Ca2+ of that site is modulated by InsP3 binding to the cytoplasmic domain of the receptor; a similar mechanism applied to the ryanodine receptor might also explain some aspects of Ca2+ ‐induced Ca2+ release. The stimulated entry of Ca2+ into a cell which occurs upon activation of inositide‐linked receptors has been variously and confusingly proposed to be regulated by InsP3, InsP4, and/or a ‘capacitative’ Ca2+ pool; the mechanism of InsP, receptor action suggested here is shown to lead to a potential reconciliation of all these conflicting proposals.
Antibodies to the β‐amyloid peptide cross‐react with conformational epitopes in human fibrinogen subunits from peripheral blood
Authors: Stem Robert A. , Trojanowski John Q. , Lee Virginia M.-Y.
Published: July 2002

Antibodies to the Alzheimer disease (AD) β‐amyloid peptide (βAP) were used to identify βAP precursor fragments in blood. The antibodies detected 3 major polypeptides with apparent molecular weights (MW) of 47‐64000 in Western blots of plasma derived clot proteins, but these proteins corresponded to human A‐α, B‐β and γ‐fibrinogen since they reacted with 2 different anti‐fibrinogen antisera, and the anti‐βAP and anti‐fibrinogen antibodies recognized purified fibrinogen and fibrin. These data are significant for efforts to develop immunochemical assays to diagnose and monitor the progression of AD.
cAMP directly facilitates Ca‐induced exocytosis in bovine lactotrophs
Authors: Sikdar Sujit K. , Zorec Robert , Mason William T.
Published: July 2002

We have used the whole cell patch clamp technique on single prolactin‐secreting bovine lactotrophs to measure plasma membrane capacitance (C
m), an index of membrane surface area, under voltage‐clamp during cytosol dialysis with Ca and cAMP. cAMP increased the magnitude and rate of Ca‐induced exocytosis (C
m increase) without affecting membrane conductance; however, cAMP had no detectable effect on C
m when intracellular Ca was low. We thus report new evidence that cAMP can facilitate Ca‐induced secretion in a synergistic fashion, by acting directly on the secretory apparatus, independently of membrane conductance activation.
Cloning of cDNA and genomic DNA for human cytochrome P‐45011β
Authors: Kawamoto Takeshi , Mitsuuchi Yasuhiro , Toda Katsumi , Miyahara Kaoru , Yokoyama Yuichi , Nakao Kazuwa , Hosoda Kiminori , Yamamoto Yasutake , Imura Hiroo , Shizuta Yutaka
Published: July 2002

A full‐length cDNA clone encoding steroid 11β‐hydroxylase (P‐45011β) has been isolated from a cDNA library derived from human adrenal tumor. The insert of the clone contains an open reading frame encoding a protein of 503 amino acid residues together with a 4 bp 5'‐untranslated region and a 576 bp 3'‐untranslated region to which a poly(A) tract is attached. The promoter region of the P‐45011β gene has also been isolated from a genomic library derived from human pre‐B cells. It contains a TATA box, a putative cAMP‐responsive element, several repeated sequences and two sequence elements similar to the consensus sequence for binding of AP‐1. A transient expression assay in Y‐1 adrenal tumor cells demonstrates that the promoter activity is remarkably enhanced by treatment of the cells with cAMP. In addition, analysis using deletion mutants containing various lengths of the 5'‐flanking region of the gene suggests that several cis‐acting elements participate in transcriptional regulation of human P‐45011β gene.
Daily amylin replacement reverses hepatic glycogen depletion in insulin‐treated streptozotocin diabetic rats
Authors: Young Andrew A. , Crocker Leslie B. , Wolfe-Lopez Deborah , Cooper Garth J.S.
Published: July 2002

In streptozotocin‐diabetic rats treated with insulin replacement, liver glycogen is some 35% depleted. Five consecutive daily subcutaneous injections with amylin dose‐dependently restored liver glycogen to normal levels. Significant increases over insulin‐only therapy occurred with amylin doses of 10, 30 and 100 , representing amylin: insulin ratios of 0.22. 0.75 and 2.79.
The assignment of the 655 nm spectral band of cytochrome oxidase
Authors: Mitchell Roy , Mitchell Peter , Rich Peter R.
Published: July 2002

The spectral characteristics of the ‘655 nm’ band of cytochrome oxidase were found to be affected by ligands of the binuclear centre, including formate and chloride, and by the resting/pulsed transition. The band titrated with near n=1 characteristics at a midpoint of about 400 mV, in contrast to haem a
3, which exhibits strong redox interaction and a titration range at significantly lower potential. Thus, although the total reduced‐oxidised difference spectrum of haem a
3, shows a trough at about 655 nm, this characteristic is absent in the low potential region. The 655 nm feature may arise from a charge transfer band of ferric high‐spin haem a
3, which is modulated by the redox state of CuB, as suggested by Beinert et al. [(1976) Biochim. Biophys. Acta 423, 339–355].
An NAD‐specific glutamate dehydrogenase from cyanobacteria Identification and properties
Authors: Chávez S. , Candau P.
Published: July 2002

The unicellular cyanobacterium Synechocystis sp. PCC 6803 presents a hexameric NAD‐specific glutamate dehydrogenase with a molecular mass of 295 kDa. The enzyme differs from the NADP‐glutamate dehydrogenase found in the same strain and is coded by a different gene. NAD‐glutamate dehydrogenase shows a high coenzyme specificity, catalyzes preferentially glutamate formation and presents Km
values for ammonium, NADH and 2‐oxoglutarate of 4.5 mM, 50 μM and 1.8 mM respectively. An animating role for the enzyme is discussed.
Autogenous translation regulation by Escherichia coli ATPase SecA may be mediated by an intrinsic RNA helicase activity of this protein
Authors: Koonin Eugene V. , Gorbalenya Alexander E.
Published: July 2002

The seven conserved motifs typical of the helicase superfamily II have been identified in the sequences of Escherichia coli protein SecA, an ATPase mediating protein translocation across the inner membrane of the bacterium, and its Bacillus subtilis homolog Div. It is hypothesized that SecA and Div possess an RNA helicase activity and may couple ATP hydrolysis both to membrane translocation of proteins, and to hairpin unwinding in their own mRNAs, leading to the known autogenous regulation of translation.
Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase)
Authors: Triebel Susanne , Bläser Jörg , Reinke Heinz , Knäuper Vera , Tschesche Harald
Published: July 2002

Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl2 and was investigated by kinetic analysis and N‐terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp15‐Leu16, Glu40‐Met41, Leu52‐Leu53 and Ala74‐Met75 peptide bonds. The PRCGVPD sequence motif (residues Pro78‐Asp84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C‐terminal cleavages. The Glu666‐Leu667, Ala506‐Glu507 and Ala398‐Leu399 bonds were hydrolysed sucessively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys497, Cys653, Cys683. Cleavage of the first C‐terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin‐like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.
Immunological identification of the α subunit of G13, a novel guanine nucleotide binding protein
Authors: Milligan Graeme , Mullaney Ian , Mitchell Fiona M.
Published: July 2002

An antiserum (13CB) was generated against a synthetic peptide, HDNLKQLMLQ, which is predicted to represent the C‐terminal decapeptide of the α subunit of the novel G‐protein, G13. Competitive ELISA indicated that the antiserum reacted with this peptide but that it showed minimal ability to recognize peptides which represent the equivalent regions of the pertussis toxin‐insensitive G‐proteins, Gq + G11, G12, G15 + G16, GL1 (also called G14) as Gz, and well as other G‐proteins. Immunoblots of human platelet membranes with antiserum 13CB identified a single 43‐kDa polypeptide, and while this immunoreactivity was abolished by the presence of the cognate peptide it was not modified by the presence of peptides corresponding to the equivalent region of other G‐proteins. Immunoreactivity corresponding to G13α was detected in a range of cell types with human platelets having the highest levels of this polypeptide.
Conservation analysis and structure prediction of the SH2 family of phosphotyrosine binding domains
Authors: Russell Robert B. , Breed Jason , Barton Geoffrey J.
Published: July 2002

Src homology 2 (SH2) regions are short (approximately 100 amino acids), non‐catalytic domains conserved among a wide variety of proteins involved in cytoplasmic signaling induced by growth factors. It is thought that SH2 domains play an important role in the intracellular response to growth factor stimulation by binding to phosphotyrosine containing proteins. In this paper we apply the techniques of multiple sequence alignment, secondary structure prediction and conservation analysis to 67 SH2 domain amino acid sciences. This combined approach predicts seven core secondary structure regions with the pattern β‐α‐β‐β‐β‐β‐α, identifies those residues most likely to be buried in the hydrophobic core of the native SH2 domain, and highlights patterns of conservation indicative or secondary structural elements. Residues likely to be involved in phosphotyrosine binding are shown and orientations of the predicted secondary structures suggested which could enable such residues to cooperate in phosphate binding. We propose a consensus pattern that encapsulates the principal conserved features or the SH2 domains. Comparison of the proposed SH2 domain or ak1 to this pattern shows only matches, suggesting that this domain may not exhibit SH2‐like properties.
Exon—intron organization of the Arabidopsis thaliana protein kinase genes CDC2a and CDC2b
Authors: Imajuku Yoshiro , Hirayama Takashi , Endoh Hideki , Oka Atsuhiro
Published: July 2002

We have previously shown by cDNA cloning that a higher plant, Arabidopsis thaliana, possesses at least two CDC2 genes (CDC2a and CDC2b) similar to the cell‐cycle‐controlling cdc2 gene of Schizosaccharomyces pombe. To understand the exon—intron organization of these genes, genomic clones were isolated and their nucleotide sequences determined. The coding and 5′‐untranslated regions of CDC2a were interrupted by seven and one introns, respectively, whilst CDC2b contained three introns within the coding portion. These intron positions partly overlapped with each other and with those of the yeast cdc2 gene, nevertheless the lengths and sequences of the corresponding introns were diverse.
p21ras contributes to HIV‐1 activation in T‐cells
Authors: Baldari Cosima T. , Macchia Giovanni , Massone Annalisa , Telford John L.
Published: July 2002

Activation of T‐cells infected by HIV‐1 results in activation of long terminal repeal (LTR)‐dependent viral transcription and ultimately the production of infectious virus. Although fulI T‐cell activation requires a complex series of intracellular signals, including protein kinase C activation, calcium mobilisation, and less‐well defined lymphokine‐induced signals, the HIV‐1 LTR can be activated by subsets of these signals. We have studied the interaction of these signals in the human lymphoma line, Jurkat, in activation of the HIV‐1 LTR. The HIV promoter was induced by IL‐1 and phorbol ester activation of PKC but not by a calcium ionophore. The constitutively active form of Ha‐ras could replace phorbol ester stimulation of the HIV promoter and of a synthetic promoter containing NFκB binding sites.
Observation of the FeIV=O stretching Raman band for a thiolate‐ligated heme protein Compound I of chloroperoxidase
Authors: Egawa Tsuyoshi , Miki Hideho , Ogura Takashi , Makino Ryu , Ishimura Yuzuru , Kitagawa Teizo
Published: July 2002

The FeIV=O stretching vibration has never been identified for a cysteine‐coordinated heme enzyme. In this study, resonance Raman and visible absorption spectra were observed simultaneously for transient species in the catalytic reaction of chloroperoxidase with hydrogen peroxide by using our original apparatus for mixed‐flow and Raman/absorption simultaneous measurements. For the first intermediate, the FeIV=O stretching Raman band was observed at 790 cm−1, which shifted to 756 cm−1 with the 18O derivative, but the ν4 band was too weak to be identified. This suggested the formation of an oxoferryl porphyrin π cation radical. The second intermediate gave an intense ν4 band at 1,372 cm−1 but no oxygen isotope‐sensitive Raman band, suggesting oxygen exchange with bulk water.
Calcium administration stimulates the expression of calcium‐binding protein regucalcin mRNA in rat liver
Authors: Shimokawa Noriaki , Yamaguchi Masayoshi
Published: July 2002

The distribution and expression of mRNA encoding the Ca2+‐binding protein regucalcin in rats were investigated by Northern blot analyses. Liver regucalcin cDNA (0.6 kb) was used as a probe. The analyses of total RNAs extracted from various tissues of rat indicated that regucalcin mRNA was mainly present in liver but only slightly in kidney with a size of 1.8 kb. The expression level decreased with increasing age (3, 10 and 25 weeks). A single intraperitoneal administration of calcium chloride (15 mg Ca/100 g body weight) induced a remarkable increase in regucalcin mRNA in liver; the level was about 200% of control at 30 min after the administration. Subsequently, the expression level began to decrease with time and was about 40% of control level at 120 min after the administration. The increase in regucalcin mRNA levels at 30 min after calcium administration was dose‐dependent. These observations show that the expression of regucalcin mRNA is specific in liver of various tissues, and that it is regulated by Ca2+ administration. Regucalcin may have a role as regulatory protein for calcium homeostasis in liver cells.
Preparation of a stable subunit of Japanese elderberry (Sambucus sieboldiana) bark lectin and its application for the study of cell surface carbohydrates by flow cytometry
Authors: Kaku Hanae , Shibuya Naoto
Published: July 2002

A stable subunit of Sambucus sieboldiana bark lectin (MSSA) was prepared by selective reduction of disulfide bridges between the subunits and alkylation with 4‐vinylpyridine. Amino acid analysis of MSSA revealed that 1.4 cysteine residues per subunit were selectively modified, MSSA failed to agglutinate rabbit erythrocytes and precipitate fetuin. However, MSSA retained the ability to bind to fetuin, as detected by ELISA. Neu5Acα2‐6lactose inhibited the binding to fetuin of both SSA and MSSA. Flow cytometric analysis showed that human histocytic lymphoma U937 cells were clearly stained with FITC‐labeled MSSA (FITC‐MSSA) without any detectable agglutination and that this staining was almost completely inhibited by the addition of Neu5Acα2‐6lactose (2 mM). Treatment of U937 cells with native FITC‐SSA at the sub‐agalutinating concentration (0.3,μg/ml) showed much poorer fluorescence intensity than that of MSSA, suggesting that MSSA is an invaluable tool for the detection of cell surface glycoconjugates containing NeuAcα2‐6Gal/GalNAc sequences by flow cytometry.
DNA unwinding activity of replication protein A
Authors: Georgaki Anthi , Strack Bettina , Podust Vladimir , Hübscher Ulrich
Published: July 2002

Replication protein A (RP‐A) is a heterotrimeric complex conserved in cukaryotic cells. It binds to single‐stranded DNA and is essential for initiation and elongation of DNA replication. In this communication we give evidence that this protein can unwind DNA independent of magnesium and ATP, two essential cofactors for bona fide DNA helicase activity. RP‐A can unwind up to at least 350 basepairs and appears to be required in stoichiometric amounts. The reaction is extremely sensitive to NaCl and MgCl2. This activity of RF‐A is suggestive for a possible unwinding function in initiation of DNA replication in eukaryotes.
Angiotensin II induces formation of the early growth response gene‐1 protein in rat vascular smooth muscle cells
Authors: Sachinidis Agapios , Weisser Peter , Ko Yon , Schulte Kerstin , zu Brickwedde Katharina Meyer , Neyses Ludwig , Vetter Hans
Published: July 2002

The effect of angiotensin II (Ang II) on the early growth response gene‐1 (Egr‐1) mRNA, on the Egr‐1 protein and on the phosphoinositide PI turnover signalling system was investigated in the presence and absence of EXP3174, a potent non‐peptide Ang II receptor antagonist. Ang II induced an accumulation of 3.4 kb Egr‐1 mRNA and the 80 kDa Egr‐1 protein, with a maximum at 30 min and 60 min, respectively. EXP3174 blocked the Ang II‐induced increase of inositolphosphates, Egr‐1 mRNA and the Egr‐1 protein, suggesting the involvement of the PI signalling system by the expression of the Egr‐1 gene.
Primary structure of a cardioactive neuropeptide from the tobacco hawkmoth, Manduca sexta
Authors: Cheung Clement C. , Loi Poh Kheng , Sylwester Andy W. , Lee Terry D. , Tublitz Nathan J.
Published: July 2002

The amino acid sequence of the first of a family of insect cardioregulatory peptides from the tobacco hawkmoth, Manduca sexta, has been determined using a combination of Edman degradation microsequencing and mass spectroscopy. This peptide contains 9 amino acid residues and an observed mass for the monoisotopic protonated molecule of 956.4 Da. There are two cysteines at positions 3 and 9 forming a disulfide bridge and the carboxyl‐terminus is amidated. The structure of this peptide, Pro‐Phe‐Cys‐Asn‐Ala‐Phe‐Thr‐Gly‐Cys‐NH2, is identical to a peptide recently isolated from crabs called crustacean cardioactive peptide (CCAP) and we propose that this peptide be named Manduca CCAP.
The effect of cyclic AMP analogues and glucagon on cholesteryl ester synthesis and hydrolysis in cultured hamster hepatocytes
Authors: Hoang Van-Quyen , Suckling Keith E. , Cho-Chui Y.S. , M. Botham Kathleen
Published: July 2002

Two cyclic AMP analogues, 8‐chloro cyclic AMP and 8‐(4 ehlorophenylthio) cyclic AMP, were found to increase the incorporation of [3H]oleate into cholereryl ester in cultured hamster hapatocytes (30–40%), while incorporation into triacylglycerol was unaffected. An increase of a similar magnitude was observed in the presence of glucagon and the phosphodiesterase inhibitor, theophylline. The cyclic AMP analogues also stimulated the activity of neutral cholesteryl ester hydrolase in the cells, and this effect was mimicked by glucagon and theophylline. These results show that cyclic AMP can affect the cholesteryl ester cycle in hamster hepatocytes, and support the idea that the enzymes involved may be co‐ordinately regulated.
Developmental extinction of liver lipoprotein lipase mRNA expression might be regulated by an NF‐1‐like site
Authors: Schoonjans Kristina , Staels Bart , Devos Piet , Szpirer Josiane , Szpirer Claude , Deeb Samir , Verhoeven Guido , Auwerx Johan
Published: July 2002

The molecular mechanism underlying the extinction of lipoprotein lipase (LPL) expression in rat liver during development was investigated. A mouse (BWTG3) and a rat (7777) hepatoma, both of which exhibit characteristics of fetal hepatocytes, were found to contain LPL mRNA, whereas the more differentiated human (Hep G2 and Hep 3B) or rat (Fa32) hepatoma cell lines did not. Somatic cell hybrids between LPL‐producing hepatoma cells and non‐LPL‐producing cells, such as adult rat hepatocytes or fibroblasts, exhibited extinction of LPL gene expression. Assay of expression of nested deletions in the 5' regulatory sequences of the LPL gene in the Hep G2 cell line and in BWTG3 cells localized sequences involved in the suppression of LPL production to a region between − 591 and − 288 relative to the transcription initiation site. A site with sequence homology to a glucocorticoid responsive element (GRE) was shown not to play an important role in the extinction process. A novel transcription factor, termed RF‐1‐LPL, was shown to bind to an NF‐1‐like site in this region. In contrast to neonatal animals, in adult animals an additional protein complex (RF‐2‐LPL), was formed on the NF‐1‐like site, suggesting that this sequence might recruit a trans‐acting factor involved in the extinction of LPL gene expression in adult rat liver.
Pectate lyase from Bacillus subtilis: molecular characterization of the gene, and properties of the cloned enzyme
Authors: Nasser W. , Awadé A.C. , Reverchon S. , Robert-Baudouy J.
Published: July 2002

Pectate lyases (PL) initiate soft‐rot diseases in plants by cleaving pectin which is the major component of the plant cell wall. The present paper reports the first cloning and characterization of a pectate lyase (pel) gene from the Bacillus genus. This gene was isolated from a Bacillus subtilis genomic library constructed in pUC18 as vector and Escherichia coli as host. By Southern hybridization this gene was shown to be present in a single copy in the B. subtilis genome. The nucleotide sequence of a 1.6 kb‐pair HindIII restriction fragment, which confers pectate lyase activity to E. coli, indicated a 1,260 bp open reading frame encoding a 420 amino acid polypeptide which includes a 21 amino acid signal sequence. The 45,605 Da deduced protein displays homologies to PLs from Erwinia chrysanthemi. The B. subtilis PL cloned in E.coli was located in the periplasm. It was purified to homogeneity in a one‐step procedure from the E. coli periplasmic fluid after overproduction using the pT7 system. Biochemical properties of the purified enzyme were similar to those found for the PL isolated from B. subtilis extracellular media.
Molecular evolution of P450 superfamily and P450‐containing monooxygenase systems
Authors: Degtyarenko Kirill N. , Archakov Alexander I.
Published: July 2002

This paper reviews the classification of the P450 superfamily which is mainly based on sequence homology. The widely accepted classification by Nebert et al. [(1991) DNA Cell Biol. 10, 1‐14] as well as the results of a ‘two‐step’ multiple sequence alignment technique show that the molecular evolution of P450s, in contrast to that of many protein families, does not follow phylogeny. The data suggest that during the evolution of P450s, gene duplications and gene fusions, horizontal gene transfer and intron loss events have occurred. ‘Weak’ and ‘strong’ hierarchies in the clustering of P450 sequences were revealed. A novel evolutionary tree of the P450 superfamily has been constructed using a multiple alignment of consensus sequences. The simple classification of known P450‐containing monooxygenase systems into three‐, two‐ and one‐component systems is further discussed. Particularly, the multidomain enzyme, nitric oxide synthase (NOS), should be classified as an example of a eukaryotic one‐component P450 system since its N‐terminal (haem) domain exhibits similarity with microsomal P450s.
Cloning, sequence and expression of the gene encoding the malolactic enzyme from Lactococcuslactis
Authors: Ansanay Virginie , Dequin Sylvie , Blondin Bruno , Barre Pierre
Published: July 2002

Many lactic acid bacteria can carry out malolactic fermentation. This secondary fermentation is mediated by the NAD‐ and Mn2+‐dependent malolactic enzyme, which catalyses the decarboxylation of l‐malate to l‐lactate. The gene we call mleS, coding for malolactic enzyme, was isolated from Lactococcus
lactis. The mleS gene consists of one open reading frame capable of coding for a protein with a calculated molecular mass of 59 kDa. The amino acid sequence of the predicted MleS gene product is homologous to the sequences of different malic enzymes. Bacterial and yeast cells expressing the malolactic gene convert l‐malate to l‐lactate.
Tetrahydrobiopterin‐dependent nitrite oxidation to nitrate in isolated rat hepatocytes
Authors: Kim Young-Myeong , Lancaster Jack R.
Published: July 2002

We have found that isolated rat hepatocytes and cell extracts catalyze the stoichiometric conversion of nitrite (either as a product of the oxidation of endogenously synthesized nitric oxide or added as sodium nitrite) to nitrate, which in extracts requires the presence of tetrahydrobiopterin and is inhibited by cyanide but apparently not by carbon monoxide. The reaction is sensitive to heat denaturation and does not involve oxidation of NO2
− by a peroxidative or radical oxygen mechanism. These results indicate the presence of a hitherto undescribed mammalian mechanism of inorganic nitrogen oxide oxidation that may be a protective mechanism against one potentially damaging effect of endogenous 'NO production (NO2
− formation), and also that assays of NO formation based on NO2
− determination alone may be an inaccurate measurement of this activity.
Differential expression of dystrophin, utrophin and dystrophin‐associated proteins in peripheral nerve
Authors: Matsumura Kiichiro , Yamada Hiroki , Shimizu Teruo , Campbell Kevin P.
Published: July 2002

The dystrophin‐glycoprotein complex is a novel laminin receptor in skeletal muscle. Dystrophin‐associated proteins are comprised of an extracellular glycoprotein of 156 kDa (156DAG), transmembrane glycoproteins of 50 kDa (50DAG), 43 kDa (43DAG) and 35 kDa (35DAG), and a cytoskeletal protein of 59 kDa (59DAP). The laminin‐binding 156DAG and 43DAG are encoded by a single gene and are now called α‐ and β‐dystroglycan, respectively. In neuromuscular junctions, utrophin, an autosomal homologue of dystrophin, is associated with sarcolemmal proteins identical or immunologically homologous to the dystrophin‐associated proteins. Here we demonstrate the co‐localization of Dp116 (a 116 kDa protein product of the DMD gene), full‐size utrophin, α‐ and β‐dystroglycan, 59DAP and 35DAG in a thin rim surrounding the outermost layer of myelin sheath of peripheral nerve fibers. The α‐dystroglycan in peripheral nerve had molecular weight of 120 kDa instead of 156 kDa, suggesting different levels of glycosylation between skeletal muscle and peripheral nerve. In sharp contrast to skeletal muscle, however, full‐size dystrophin and 50DAG were undetectable in peripheral nerve. Our results demonstrate the varied expression of the components of the dystrophin/utrophin‐glycoprotein complex between skeletal muscle and peripheral nerve suggesting the complex may exist in varied compositions and have varied functions in these two tissues.
Differential contribution by conserved glutamate residues to an ion‐selectivity site in the L‐type Ca2+ channel pore
Authors: Mikala Gabor , Bahinski Anthony , Yatani Atsuko , Tang Shaoqing , Schwartz Arnold
Published: July 2002

In voltage‐gated cation channels, it is thought that residues responsible for ion‐selectivity are located within the pore‐lining SS1‐SS2 segments. In this study, we compared the ion permeation properties of mutant calcium channels in which highly conserved glutamate residues, located at analogous positions in the SS2 regions of all four motifs, were individually replaced. All of the mutants exhibited a loss of selectivity for divalent over monovalent cations. However, the permeation properties of the individual mutants varied in a position dependent manner. The results provide strong evidence that these glutamate residues, positioned at equivalent locations in the aligned sequences, play significantly different roles in forming the selectivity barrier of the calcium channel, and are probably arranged in an asymmetrical manner inside the ion‐conducting pore.
Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum
Authors: Holloway Daniel E. , Marsh E.Neil G.
Published: July 2002

between mutE and a region of β‐methylaspartase which contains an active‐site serine residue.
Dipeptidyl peptidase IV (CD 26) and aminopeptidase N (CD 13) catalyzed hydrolysis of cytokines and peptides with N‐terminal cytokine sequences
Authors: Hoffmann T. , Faust J. , Neubert K. , Ansorge S.
Published: July 2002

A number of natural cytokines are characterized as having dipeptidyl peptidase (DP) IV susceptible N‐terminal peptide sequences. Here we demonstrate that oligopeptides with sequences analogous to the N‐terminal part of human IL‐1β, IL‐2, TNF‐β and murine IL‐6 were hydrolyzed by purified DP IV and aminopeptidase N (AP‐N). The rate of DP IV‐catalyzed hydrolysis of these peptides was negatively correlated with their chain length. In contrast to these results, no degradation was found under our conditions for the intact recombinant cytokines, IL‐1α, IL‐1β, IL‐2, G‐CSF and for natural IL‐2, independent of whether DP IV and AP‐N were used separately or in combination.
Preliminary X‐ray crystallographic studies of photosynthetic reaction center from a thermophilic sulfur bacterium, Chromatium tepidum
Authors: Katayama Naoko , Kobayashi Masayuki , Motojima Fumihiro , Inaka Koji , Nozawa Tsunenori , Miki Kunio
Published: July 2002

A membrane protein complex, photosynthetic reaction center purified from the thermophilic purple sulfur bacterium, Chromatium tepidum has been crystallized from a phosphate butter containing a detergent, n‐octyl‐β‐D‐glucopyranoside and a precipitant, polyethylene glycol 4000. The crystals diffracted X‐rays beyond 3Å resolution with synchrotron radiation and are suitable for high‐resolution X‐ray crystallographic studies. The crystals belong to the orthorhombic space group P212121 with unit‐cell dimensions of a = 136Å, b = 197Å, and c = 82Å. Assuming that they contain one reaction center complex in the asymmetric unit, V
M was calculated to be 4.3 Å3/Da, which agrees with the values obtained in the membrane protein complexes.
Ca2+ influx drives agonist‐activated [Ca2+]i oscillations in an exocrine cell
Authors: Martin Shaun C. , Shuttleworth Trevor J.
Published: July 2002

In current models describing agonist‐induced oscillations in [Ca 2+]i, Ca2+ entry is generally assumed to have a simple sustaining role, replenishing Ca2+ lost from the cell and recharging intracellular Ca2+ stores. In cells from the avian nasal gland, a model exocrine cell, we show that inhibition of Ca2+ entry by La3+, SK&F 96365, or by membrane depolarization, rapidly blocks [Ca2+]i oscillations but does so without detectible depletion of agonist‐sensitive Ca2+ stores. As the rate of Mn2+ quenching during [Ca2+]i oscillations is constant, Ca2+ entry is not directly contributing to the [Ca2+]i changes and, instead, appears to be involved in inducing the repetitive release of Ca2+ from internal stores. Together, these data contradict current models in that (i) at the low agonist concentrations where [Ca2+]i oscillations are seen, generated levels of Ins(1,4,5)P3 are themselves inadequate to result in a regenerative [Ca2+]i signal, and (ii) Ca2+ entry is necessary to actually drive the intrinsic oscillatory mechanism.
Comparison of the kinetic properties of MGDG synthase in mixed micelles and in envelope membranes from spinach chloroplast
Authors: Maréchal Eric , Block Maryse A. , Joyard Jacques , Douce Roland
Published: July 2002

We have applied the ‘membrane partition’ kinetic modelling approach proposed by Heirwegh et al. [(1988)] Biochem. J. 254, 101–108] to MGDG synthase in isolated envelope vesicles. Comparison of the kinetic parameters obtained for MGDG synthase assayed in purified envelope membranes and in mixed‐micelles demonstrates that the latter are relevant to the situation in envelope membranes and that MGDG synthase has a very high affinity for dilinoleoylglycerol. Our results provide additional evidence for the hypothesis that the high affinity of the envelope MGDG synthase for dilinoleoylglycerol could be responsible for the presence of C18 fatty acids at both the sn‐1 and sn‐2 position of the glycerol backbone in MGDG.
Assembly of Alzheimer‐like filaments from full‐length tau protein
Authors: Crowther R.A. , Olesen O.F. , Smith M.J. , Jakes R. , Goedert M.
Published: July 2002

The principal fibrous component of neurofibrillary pathology in Alzheimer's disease, the paired helical filament, is formed from hyperphosphorylated microtubule‐associated protein tau. Here we show that recombinant tau protein either in a non‐phosphorylated state or following phosphorylation with brain extract can be assembled in vitro into filaments resembling those seen in Alzheimer's disease.
YKC1 encodes the depolarization‐activated K+ channel in the plasma membrane of yeast
Authors: Zhou Xin-Liang , Vaillant Brian , Loukin Stephen H. , Kung Ching , Saimi Yoshiro
Published: July 2002

Our previous patch‐clamp studies showed that depolarization activates a K+‐specific current in the plasma membrane of the binding yeast, Saccharomyces cerevisiae [Gustin et al. (1986) Science 233, 1195–1197]. The yeast Genome Sequencing Project has now uncovered on the left arm of chromosome X an open reading frame (ORF) that predicts a 77‐kDa protein reminiscent of a shaker‐like α subunit with 6 membrane spans followed by a subunit with 2 spans. We found that deleting this ORF removes the yeast K+ current. Furnishing the ORF from plasmids restores or even greatly amplifies this current. These manipulations have no effects on the 40‐pS mechanosensitive conductance also native to this membrane. Thus, this ORF, named YKC1 here, likely encodes a structure for the K+‐specific channel of the yeast plasma membrane. This and other K+ channel subunits are compared and the possible uses of this gene in research are discussed. YKC1 has recently been shown by others to induce in frog oocytes a K+ current. Its activation is coupled to E
K+
and its outward rectification depends on external divalent cations. We found the YKC1 channel in its native membrane activates at low voltages largely independent of E
K+
and it remains so despite removal of divalents by chelation.
Dictyostelium protein kinase C‐delta‐like protein is localized in the cell nucleus
Authors: Wang Yingcai , Rubin Hila , Ravid Shoshana
Published: July 2002

Summry— The molecular mechanism whereby protein kinase C (PKC) molecules transduce signals into the cell nucleus is unknown. In this study, we provide evidence that Dictyostelium discoideum contains PKCδ‐like protein that is localized in the nucleus. The Dictyostelium PKCδ‐like protein has an apparent molecular mass of 76 kDa. This protein is already highly expressed in vegetative Dictyostelium cells. The expression level remained constant up to 12 h of development, and sharply decreased after 16 h. The PKCδ‐like protein is phosphorylated in vivo in response to cAMP and phorbol ester stimulation. Immunofluorescent studies, as well as subcellular fractionation experiments, have indicated that Dictyostelium PKCδ‐like protein is permanently located in the nucleus. Our results may indicate that PKCδ‐like protein in Dictyostelium functions as a link between cAMP and the tumor‐promoting phorbol esters, and events that take place in the nucleus.
Motor function analysis of myelin mutant mice using a rotarod
Authors: Kuhn P.L. , Petroulakis E. , Zazanis G.A. , McKinnon R.D.
Published: July 2002

We have examined motor control in normal and shiverer mutant mice using the rotarod assay, a forced motor activity which tests for balance and co‐ordination. Shiverer mice carry a deletion of the myelin basic protein (MBP) gene, resulting in CNS dysmyelination and characteristics motor dysfunction. Homozygous mutant mice had a significant increase in cumulative falls from the rotatod relative to heterozygous mice. Non‐acclimated animals of both genotypes showed progressive improvement in performance when tested on successive days. The rotarod test also discriminited shiverer mutants from animals that received gene therapy intervention. Shiverer animals carrying an MBP transgene showed gene‐dosage‐dependent improvements in motor function, and mutants which received thalamic transplants of wild type oligodendrocyte precursor cells showed improvement relative to sham operated and non‐transplanted controls. Thus the rotarod is a sensitive measure of motor function in hypomyelinated mice, and may be useful for assessing the results of experimental manipulations including transgenic gene therapy and cell transplantation.
Intolerance to neuroleptic drugs: the art of avoiding extrapyramidal syndromes
Authors: Gerlach J , Peacock L
Published: July 2002

Akathisia, dystonia, dyskinesia and parkinsonism, the four main categories of neuroleptic-induced extrapyramidal syndromes (EPS), represent major disadvantages in antipsychotic therapy. In vulnerable patients, acute EPS may progress into potentially irreversible forms such as tardive dystonia and tardive dyskinesia. In the psychiatric clinic, these EPS are often insufficiently recognised or permitted to exist without treatment. In order to ensure a better EPS diagnosis, a simple examination procedure is described. EPS rating scales may serve as an aid in this process. Guidelines are given to prevent and treat EPS. Thus, EPS are best prevented by a course of neuroleptic medication involving as little antidopaminergic D2 effect as possible, including the use of the lowest effective dose (sometimes obtained by addition of a benzodiazepine or carbamazepine) and with antipsychotic drugs which produce low D2 receptor blockade. Treating EPS also consists of using the lowest effective dose and antipsychotics with a low D2 dopamine receptor occupancy. At present, clozapine is the only drug that produces antipsychotic benefits at doses that cause far less D2 receptor antagonism in the basal ganglia of the brain than that seen with standard neuroleptics; however, newer drugs, such as olanzepine, seroquel and sertindole, are on the way.
Quality of life measurement in the medical setting
Authors: Bech P
Published: July 2002

Quality of life became a paradigm in the medical setting two decades ago. Although ‘humanistic’ psychologists still maintain that what can be specified and measured is precisely what is not quality of life, time has shown that the major components of quality of life in health and disease can be measured with an acceptable internal as well as external validity. Quality of life in randomized clinical trials is now the third dimension to be measured, the other dimensions are efficacy of the drug and adverse drug reactions. Among the major areas of external validity quality of life scales (eg the PCASEE questionnaire and the Psychological General Well-Being Schedule) have discriminating validity (the discrimination between treatments), predictive-validity (the prediction of relapse of illness), and teological validity (defining goal of treatment).
IL‐10 attenuates IFN‐α‐activated STAT1 in the liver: involvement of SOCS2 and SOCS3
Authors: Shen Xuening , Hong Feng , Nguyen Van-Anh , Gao Bin
Published: July 2002

Interleukin‐10 (IL‐10) has been used in the treatment of viral hepatitis in interferon‐α (IFN‐α) non‐responders while patients who have high levels of IL‐10 are poorly responsive to IFN‐α. The mechanism underlying such controversial functions of IL‐10 remains unknown. Here we demonstrated that injection of IL‐10 into mice attenuated IFN‐α‐induced signal transducer and activator transcription factor (STAT)1 tyrosine phosphorylation in the liver. Reverse transcriptase‐polymerase chain reaction assay demonstrated that mouse liver expressed high levels of IL‐10 receptor 2 (IL‐10R2) but low levels of IL‐10R1. Injection of IL‐10 into mice activated STAT3 but not STAT1 tyrosine phosphorylation and induced suppressor of cytokine signal 2 (SOCS2), SOCS3, and cytokine‐inducible SH2 protein (CIS) mRNA expression in the liver. Furthermore, overexpression of SOCS2 or SOCS3 inhibited IFN‐α‐induced reporter activity in hepatic cells. These findings suggest that IL‐10 inhibits IFN‐α‐activated STAT1 in the liver, at least in part, by inducing SOCS2, SOCS3, and CIS expression, which may be responsible for the resistance of IFN‐α therapy in patients who have high levels of IL‐10 and recommends that IL‐10 treatment for viral hepatitis should be cautious.
Interaction of human Ku70 with TRF2
Authors: Song Kyuyoung , Jung Donghae , Jung Yusun , Lee Seong-Gene , Lee Inchul
Published: July 2002

Ku, a heterodimer of 70‐ and 80‐kDa subunits, plays a general role in the metabolism of DNA ends in eukaryotic cells, including double‐strand DNA break repair, V(D)J recombination, and maintenance of telomeres. We have utilized the yeast two‐hybrid system to identify Ku70‐interacting proteins other than Ku80. Two reactive clones were found to encode the dimerization domain of TRF2, a mammalian telomeric protein that binds to duplex TTAGGG repeats at chromosome ends. This interaction was confirmed using bacterial fusion proteins and co‐immunoprecipitations from eukaryotic cells overexpressing TRF2. The transfected TFR2 colocalized with Ku70.
Molecular evidence of a unique lipoamide dehydrogenase in plastids: analysis of plastidic lipoamide dehydrogenase from Arabidopsis thaliana1
Authors: Lutziger Isabelle , Oliver David J.
Published: July 2002

Lipoamide dehydrogenase is a subunit of the α‐ketoacid dehydrogenases and the glycine decarboxylase complex in mitochondria, and the pyruvate dehydrogenase complex in plastids. We report here the unexpected finding of two plastidic isoforms of lipoamide dehydrogenase from Arabidopsis thaliana that are different from the mitochondrial form of the enzyme. The cDNA clones were confirmed by sequence alignment analysis and their location verified by chloroplast import assay. They are single copy genes that appear to be expressed in parallel in different tissues with highest level in developing siliques. Phylogenetic analysis gives further exemplary evidence for the plastidic evolution derived from cyanobacteria.
The open reading frame of the Na+‐dependent glutamate transporter GLAST‐1 is expressed in bone and a splice variant of this molecule is expressed in bone and brain
Authors: Huggett Jim , Vaughan-Thomas Anne , Mason Deborah
Published: July 2002

Previously we have reported expression of an mRNA with homology to the Na+‐dependent glutamate transporter, GLAST‐1, in bone. Here we demonstrate that the complete open reading frame of GLAST‐1 mRNA and corresponding 69 kDa protein are expressed in rat bone in vivo. We have also discovered a novel splice variant (GLAST‐1a), lacking exon 3, expressed in rat bone and brain. A 55 kDa protein detected by anti‐GLAST antibody in rat cerebellum corresponds to the molecular weight of unglycosylated GLAST‐1a. This has led us to propose that GLAST‐1a has an opposite orientation in the cell membrane to GLAST‐1.
SRC transcriptional activation in a subset of human colon cancer cell lines
Authors: Dehm Scott , Senger Mary-Anne , Bonham Keith
Published: July 2002

Human SRC encodes the non‐receptor tyrosine kinase pp60c‐Src, which is activated in many human colon cancer cell lines (HCCLs) and tumors. We found that both c‐Src protein and mRNA levels were elevated in a subset of HCCLs. Increased c‐Src mRNA and protein levels correlated strongly with increased c‐Src kinase activity. Nuclear run‐on analysis and c‐Src mRNA half‐life determination demonstrated increased mRNA levels were due to increased transcription of the SRC gene. We also observed decreased c‐Src mRNA stability in cell lines that displayed SRC transcriptional activation. Our findings provide the first evidence that SRC transcriptional activation is an important determinant of c‐Src expression and activity in HCCLs.