Appl Environ Microbiol, 1999 Dec, 65(12), 5600 - 3 Escherichia coli resistance to chlorine and glutathione synthesis in response to oxygenation and starvation; Saby S et al.; Reduced glutathione (GSH) levels and resistance to chlorine were measured for two isogenic Escherichia coli strains stressed by oxygenation and/or starvation . The E . coli mutant deficient in GSH was not more sensitive to the oxidant than its parent strain when the bacteria were cultured with a low oxygenation rate . Starvation or oxygenation increased the resistance of the parent strain to chlorine, while the resistance of the deficient strain remained unchanged. Appl Environ Microbiol, 1999 Dec, 65(12), 5459 - 63 Isolation and characterization of the epoxide hydrolase-encoding gene from Xanthophyllomyces dendrorhous; Visser H et al.; The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated . The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa . The amino acid sequence is similar to that of microsomal EH and belongs to the alpha/beta hydrolase fold family . The EPH1 gene was not essential for growth of X . dendrorhous in rich medium under laboratory conditions . The Eph1-encoding cDNA was functionally expressed in Escherichia coli . A sixfold increase in specific activity was observed when we used resting cells rather than X . dendrorhous . The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1 . Isolation and characterization of the X . dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system. Appl Environ Microbiol, 1999 Dec, 65(12), 5386 - 93 Reverse transcription-PCR differential display analysis of Escherichia coli global gene regulation in response to heat shock; Gill RT et al.; A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in Escherichia coli . A novel combination of primers designed specifically for the start and stop regions of E . coli genes (based on the findings of Fislage et al . {R . Fislage, M . Berceanu, Y . Humboldt, M . Wendt, and H . Oberender, Nucleic Acids Res . 25:1830-1835, 1997}) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR . The validity of the technique was demonstrated by applying it to heat shock analysis . Specifically, RT-PCR-amplified total RNA from heat-shocked and non-heat-shocked cells were hybridized with slot blots of the Kohara set (U . Kohara, K . Akiyama, and K . Isono, Cell 50:495-508, 1987; S . Chuang, D . Daniels, and F . Blattner, J . Bacteriol . 175:2026-2036, 1993) . The signals obtained for heat-shocked and control cultures of each clone were compared, and differences in intensity were evaluated by calculating induction ratios . Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation . Also, for several genes, Northern blotting and total RNA dot blotting were performed to confirm that the transcript levels in the original RNA samples were different . This technique extended previously described methods for studying global gene regulation in E . coli by incorporating a PCR amplification step in which global, mRNA-specific primers were used . In addition, the method employed here can be easily extended to study E . coli global gene regulation in response to additional environmental stimuli. Parasite Immunol, 1999 Nov, 21(11), 573 - 81 Immunogenicity of malaria transmission-blocking vaccine candidate, y230.CA14 following crosslinking in the presence of tetanus toxoid; Vincent AA et al.; Proteolytically processed 310 kDa form of Plasmodium falciparum gamete surface antigen, Pfs230, is the target of malaria transmission-blocking monoclonal antibodies . To design a recombinant malaria transmission-blocking subunit vaccine, the amino terminus of the 310 kDa surface-exposed form of Pfs230 was mapped to amino acids (aa) 522 and 584 using a series of peptides and recombinant proteins encoding distinct regions of Pfs230 . Antiserum generated against an Escherichia coli-produced recombinant protein, spanning the Pfs230 processing site and extending into the cysteine domains, r230/MBP.C (aa 443-1132), reduced parasite infectivity by 71.2-89.8% . To determine if the region spanning the cleavage site blocked malaria transmission when produced as a secreted protein by Saccharomyces cerevisiae, y230.CA14 (aa 467-584) was generated, purified, emulsified in adjuvant and used to vaccinate mice . In contrast to E . coli-produced r230/MBP.C, the immune response generated against y230 . CA14 was very weak . To enhance the response, y230.CA14 was mixed with tetanus toxoid, chemically crosslinked, repurifed, and its immunogenicty compared with unconjugated y230.CA14 . Conjugated-y230 . CA14/TT required fewer booster injections to induce an immune response against Pfs230 and the antibodies generated reacted with the surface of intact gametes and immunoprecipitated radiolabelled Pfs230 extracted from 125I surface-labelled gametes to a greater extent . After seven injections, all y230.CA14 vaccinated mice developed anti-Pfs230 antibodies and the isotype profile was the same . In addition to enhancing the initial immune response generated against y230.CA14, conjugation focuses the immune response toward epitopes within the region of Pfs230 present on the surface of the gamete. Lett Appl Microbiol, 1999 Oct, 29(4), 224 - 7 Morphological changes (including filamentation) in Escherichia coli grown under starvation conditions on silicon wafers and other surfaces; Wainwright M et al.; Using a scanning electron microscope, pleomorphism (notably filamentation) was seen when Escherichia coli was grown under starvation conditions for 14 d on microporous silicon wafers, titanium, glass and plastic discs . Under these conditions, the 'standard', rod shaped cell (1-3 microns) failed to separate after division and filaments developed, some as long as 50 microns, with many showing bulbous tips . Filamentation began to occur 5 d after the imposition of starvation conditions . Dumbbell shaped cells were also observed, although apparent 'Y' and 'V'-shaped cells proved to be artefacts, caused by overlapping rods . The implications of the appearance of pleomorphism in E . coli, when grown under starvation conditions, is discussed in relation to its pathogenicity and growth in the environment. J Appl Microbiol, 1999 Oct, 87(4), 540 - 5 The effect of alpha-lactalbumin and beta-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103; Pihlanto-Leppala A et al.; Bovine milk proteins alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were hydrolysed with seven different proteolytic enzymes, and the effect of various hydrolysates on a genetically modified luminous Escherichia coli JM103 was tested in vitro with a bioluminescence assay for bacterial growth and metabolism . Undigested proteins did not inhibit the activity of tested E . coli JM103 at a concentration as high as 0.1 g ml-1 . At the same concentrations, alpha-la hydrolysed with pepsin or trypsin and beta-lg hydrolysed with alcalase, pepsin or trypsin, showed a lower metabolic activity during the first 8 h of growth . The activity of E . coli JM103 in the presence of 25 mg ml-1 alpha-la or beta-lg hydrolysed with pepsin and trypsin was only 21% of the control after incubation for 6 h . The preliminary results indicated that ultrafiltration through 10 kDa and 1 kDa molecular mass cut-off membranes may be used to enrich bacteriostatic properties. Genes Cells, 1999 Oct, 4(10), 551 - 61 Transposition of IS1 circles; Shiga Y et al.; BACKGROUND: IS1, the smallest active transposable element in bacteria, encodes transposase . IS1 transposase promotes transposition as well as production of miniplasmids from a plasmid carrying IS1 by deletion of the region adjacent to IS1 . The IS1 transposase also promotes production of IS1 circles consisting of the entire IS1 sequence and a sequence, 6-9 bp in length, as a spacer between terminal inverted repeats of IS1 . The biological significance of the generation of IS1 circles is not known . RESULTS: Plasmids carrying an IS1 circle with a spacer sequence 6-9 bp long transposed to target plasmids at a very high frequency when transposase was produced from a co-resident plasmid . The products were target plasmids with the donor plasmid inserted at the ends of IS1 in the IS1 circle . This insertion accompanied the removal of the spacer sequence and duplication of the sequence at the target site . IS1 circles with a much longer spacer sequence transposed less frequently . The SOS response was induced in cells harbouring a plasmid with an IS1 circle owing to transposase . IS1 circles could transpose in the strain deficient in H-NS, a nucleoid-associated DNA-binding protein known to be required for the transposition of IS1 . CONCLUSIONS: IS1 circles appear to act as intermediates for simple insertion into the target DNA via cleavage of the circles which induces the SOS response . H-NS may function in promoting the assembly of an active IS1 DNA-transposase complex at the terminal inverted repeats. Eur J Biochem, 1999 Dec, 266(3), 1202 - 9 Mutational analysis of Asp51 of Anabaena azollae glutamine synthetase . D51E mutation confers resistance to the active site inhibitors L-methionine-DL-sulfoximine and phosphinothricin; Crespo JL et al.; The role of Asp51 in the catalytic activity of glutamine synthetase from the cyanobacterium Anabaena azollae has been analyzed . Five mutant enzymes, D51S, D51A, D51E, D51N and D51R, were constructed by site-directed mutagenesis and characterized . Asp51 appears to participate in the binding of ammonium ion, as affinity for this substrate was affected in all cases, although it varied according to the charge and/or size of the amino-acid residue, decreasing in the order Glu > Asn > Ser > Ala . The replacement of Asp51 by Glu (D51E) conferred besides a high resistance to the herbicides L-methionine-DL-sulfoximine and phosphinothricin, as a result of a decreased phosphorylation ability. Eur J Biochem, 1999 Dec, 266(3), 1128 - 35 Effect of modified nucleotides on Escherichia coli tRNAGlu structure and on its aminoacylation by glutamyl-tRNA synthetase . Predominant and distinct roles of the mnm5 and s2 modifications of U34; Madore E et al.; Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants . The predominant tRNAGlu modivariant in wild-type E . coli contains five modified nucleosides: Psi13, mnm5s2U34, m2A37, T54 and Psi55 . Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34 . Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others . Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D- and T-arms and in the variable region . The lack of a 2-thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520-fold the specificity of E . coli glutamyl-tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E . coli tRNAGlu . The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates . These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl-tRNA synthetase for the recognition of glutamate and ATP. Eur J Biochem, 1999 Dec, 266(3), 1081 - 9 Circular dichroism and fluorescence spectroscopic properties of the major core protein of feline immunodeficiency virus and its tryptophan mutants . Assignment of the individual contribution of the aromatic sidechains; Yelamos B et al.; The gene coding for the major capsid protein of feline immunodeficiency virus (FIV) has been cloned into the expression vector pQE60, which allows protein purification by affinity chromatography on a nitrilotriacetic acid/Ni/agarose column . The gene was expressed in Escherichia coli and the resultant soluble protein (FIV-rp24) purified to electrophoretic homogeneity . The amino-acid composition of the recombinant protein is almost identical to that predicted from the DNA sequence . This protein has two tryptophan residues at positions 40 and 126 that have been replaced by phenylalanine by site-directed mutagenesis to obtain two single mutants and a double mutant . Circular dichroism and fluorescence spectroscopy were employed to study the structural features of FIV-rp24 protein and its tryptophan mutants . The analysis of the CD spectra indicated that alpha-helix is the major secondary structural element (48-52%) and that the overall three-dimensional structure is not modified by the mutations . The fluorescence emission spectra showed that both tryptophan residues occupy a highly hydrophobic environment . Moreover, the different tyrosine fluorescence intensities of wild-type and mutant proteins are indicative of the existence of resonance energy transfer processes to nearby tryptophan . The individual contributions of each tryptophan residue to the spectroscopic properties of the wild-type protein were obtained from the spectra of all these proteins . Thermal denaturation studies indicate that the two tryptophan residues do not contribute equally to the stabilization of the three-dimensional structure. Eur J Biochem, 1999 Dec, 266(3), 1056 - 65 The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase; Rotrekl V et al.; The maize Zm-p60.1 gene encodes a beta-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides . Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit . Their role was studied by mutating them to alanine (A), serine (S), arginine (R) or aspartic acid (D) using site-directed mutagenesis, and subsequent heterologous expression in Escherichia coli . All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system . Examination of urea- and glutathione-induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology-based three-dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme . Mutating C52 to R produced a monomeric enzyme protein, too . No detectable effects on homodimer formation were apparent in C170 and C479 mutants . Given the Km values for C170A/S mutants were equal to that for the wild-type enzyme, C170 cannot participate in enzyme-substrate interactions . Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k'cat . C170 is located on a hydrophobic side of an alpha-helix packed against hydrophobic amino-acid residues of beta-strand 4, indicating participation of C170 in stabilization of a (beta/alpha)8 barrel structure in the enzyme . In C479A/D/R/S mutants, Km and k'cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action. Eur J Biochem, 1999 Dec, 266(3), 903 - 10 The NAD-linked aromatic alpha-hydroxy acid dehydrogenase from Trypanosoma cruzi . A new member of the cytosolic malate dehydrogenases group without malate dehydrogenase activity; Cazzulo Franke MC et al.; Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase . This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium . The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification . Its sequence shows high similarity to the cytosolic malate dehydrogenases . However, the enzyme has no malate dehydrogenase activity . The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T . cruzi . The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase . Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme. Eur J Biochem, 1999 Dec, 266(3), 892 - 902 Enovin, a member of the glial cell-line-derived neurotrophic factor (GDNF) family with growth promoting activity on neuronal cells . Existence and tissue-specific expression of different splice variants; Masure S et al.; Glial cell-line-derived neurotrophic factor (GDNF), neurturin and persephin are neurotrophic factors involved in neuroneal differentiation, development and maintenance . They act on different types of neuroneal cells and signal through a receptor complex composed of a specific ligand-binding subunit of the GDNF family receptor alpha (GFRalpha) family together with a common signaling partner, the cRET protein tyrosine kinase . We describe the molecular cloning, expression, chromosomal localization and functional characterization of enovin, a fourth GDNF family member almost identical to the recently described artemin . We show the occurence in most tissues of several differently spliced mRNA variants for enovin, of which only two are able to translate into functional enovin protein . Some tissues seem to express only nonfunctional transcripts . These observations may underlie a complex transcriptional regulation pattern . Enovin mRNA expression is detectable in all adult and fetal human tissues examined, but expression levels are highest in peripheral tissues including prostate, placenta, pancreas, heart and kidney . This tissue distribution pattern is in accordance with that of GFRalpha-3, which here is shown to be the preferred ligand-binding receptor for enovin (Kd = 3.1 nM) . The human enovin gene is localized on chromosome 1, region p31.3-p32 . In vitro, enovin stimulates neurite outgrowth and counteracts taxol-induced neurotoxicity in staurosporine-differentiated SH-SY5Y human neuroblastoma cells . The peripheral expression pattern of enovin and its receptor together with its effects on neuroneal cells suggest that enovin might be useful for the treatment of neurodegenerative diseases in general and peripheral neuropathies in particular. Eur J Biochem, 1999 Dec, 266(3), 865 - 77 Extensive regulation compromises the extent to which DNA gyrase controls DNA supercoiling and growth rate of Escherichia coli; Jensen PR et al.; As DNA gyrase is the only enzyme to supercoil DNA actively, we address here the question of whether it does play the expected dominant role in controlling the level of DNA supercoiling and growth rate in Escherichia coli . We modulated the expression of DNA gyrase around its wild-type level, and measured the effect on plasmid supercoiling and growth rate . As both the activity and the transcription rate of DNA gyrase are sensitive to DNA supercoiling we distinguish two types of control (with control defined as the percentage change observed on a 1% modulation of a parameter) . The first type of control, here named inherent control, quantifies the effect of a sustained modulation of the transcription rate of gyrase . At its wild-type expression level this inherent control exerted by DNA gyrase on growth rate was very low, i.e . c mu/gyrase = 0.05 - 0.00, as was the inherent control on DNA supercoiling, c aLK/gyrase = 0.2 . The second type of control, here named global control, quantifies the effect of a change in gyrase activity whilst allowing the cell to respond by readjusting gyrase transcription . Both types of control are linked via the sensitivity of gyrase transcription to DNA supercoiling, as determined from the inherent control by gyrase of the gyrase promoter activity using a chromosomal gyrB:lacZ fusion . As expected, the latter control was negative, but small, i.e . c gyr promoter/gyrase=-0.3 . The global control by gyrase of active linking number was 0.1 . These results show that although gyrase is an essential enzyme it does not have a high control, on either growth rate or DNA supercoiling . Homeostatic regulation of physiological DNA structure appears to dominate . At low degrees of DNA supercoiling, the control by DNA gyrase and by the other topoisomerases is much stronger. Eur J Biochem, 1999 Dec, 266(3), 724 - 36 Specificity of starch synthase isoforms from potato; Edwards A et al.; In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch . Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains . The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli . Significant differences in their activities are apparent which may be important in determining their specificities in vivo . These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension . To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E . coli . These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains . This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases . The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo. Antimicrob Agents Chemother, 1999 Dec, 43(12), 2960 - 3 Automated thermal cycling is superior to traditional methods for nucleotide sequencing of bla(SHV) genes; Bradford PA; Genes encoding SHV-1 and SHV-2 were sequenced by different methods . Nucleotide sequencing of the coding strand by standard dideoxy-chain termination methods resulted in errors in the interpretation of the nucleotide sequence and the derived amino acid sequence in two main regions which corresponded to nucleotide and amino acid changes that had been reported previously . The automated thermal cycling method was clearly superior and consistently resulted in the correct sequences for these genes. J Neurochem, 1999 Dec, 73(6), 2510 - 6 Characterization of wild-type and mutants of recombinant human GTP cyclohydrolase I: relationship to etiology of dopa-responsive dystonia; Suzuki T et al.; To explore the molecular etiology of two disorders caused by a defect in GTP cyclohydrolase I--hereditary progressive dystonia with marked diurnal fluctuation (HPD), also known as dopa-responsive dystonia (DRD), and autosomal recessive GTP cyclohydrolase I deficiency--we purified and analyzed recombinant human wild-type and mutant GTP cyclohydrolase I proteins expressed in Escherichia coli . Mutant proteins showed very low enzyme activities, and some mutants were eluted at a delayed volume on gel filtration compared with the recombinant wild-type . Next, we examined the GTP cyclohydrolase I protein amount by western blot analysis in phytohemagglutinin-stimulated mononuclear blood cells from HPD/DRD patients . We found a great reduction in the amount of the enzyme protein not only in one patient who had a frameshift mutation, but also in an HPD/DRD patient who had a missense mutation . These results suggest that a dominant-negative effect of chimeric protein composed of wild-type and mutant subunits is unlikely as a cause of the reduced enzyme activity in HPD/DRD patients . We suggest that reduction of the amount of the enzyme protein, which is independent of the mutation type, could be a reason for the dominant inheritance in HPD/DRD. Acta Microbiol Pol, 1999, 48(2), 197 - 201 Effect of DnaK and DnaJ proteins deprivation on Escherichia coli response to starvation; Jurkiewicz D et al.; E . coli defects in response to nutritional starvation caused by DnaK and DnaJ proteins deprivation are examined . The ability of delta dnaKdnaJ mutant to survive carbon, nitrogen and phosphorus starvation is highly impaired while delta dnaJ mutant is characterized by the diminished survival of phosphorus starvation only . delta dnaKdnaJ mutant grows slowly utilizing maltose and glycerol and delta dnaJ mutant utilizes glycerol inefficiently . The growth on alternate nitrogen sources is comparable to wild-type strain. Virus Res, 1999 Dec 15, 65(2), 155 - 60 Echovirus 9 strain barty non-structural protein 2C has NTPase activity; Klein M et al.; Non-structural protein 2C is known to play a fundamental role in the replication of picornaviruses . Sequence analyses revealed that 2C belongs to a rapidly expanding group of proteins containing a consensus sequence for nucleotide binding (NTB) . We report that echovirus 9 polypeptide 2C displays NTPase activity in vitro . In our experiments, several P2 genes were expressed in Escherichia coli as fusion proteins linked to glutathione S-transferase (GST) prior to purification close to homogeneity . In contrast to GST-2B, both GST-2C and GST-2BC showed ATPase as well as GTPase activity indicating that the site for NTB binding and splitting is located in 2C. Biochim Biophys Acta, 1999 Dec 6, 1461(2), 207 - 15 Mechanism of the ArsA ATPase; Rosen BP et al.; The ArsAB ATPase confers metalloid resistance in Escherichia coli by pumping toxic anions out of the cells . This transport ATPase shares structural and perhaps mechanism features with ABC transporters . The ArsAB pump is composed of a membrane subunit that has two groups of six transmembrane segments, and the catalytic subunit, the ArsA ATPase . As is the case with many ABC transporters, ArsA has an internal repeat, each with an ATP binding domain, and is allosterically activated by substrates of the pump . The mechanism of allosteric activation of the ArsA ATPase has been elucidated at the molecular level . Binding of the activator produces a conformational change that forms a tight interface of the nucleotide binding domains . In the rate-limiting step in the overall reaction, the enzyme undergoes a slow conformational change . The allosteric activator accelerates catalysis by increasing the velocity of this rate-limiting step . We postulate that similar conformational changes may be rate-limiting in the mechanism of ABC transporters. EMBO J, 1999 Dec 1, 18(23), 6762 - 70 Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain; Kammerer RA et al.; Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization . Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1 . The interaction with laminin mediates the integration of agrin into basement membranes . Here we characterize the binding site within the laminin-1 coiled coil in detail . Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected . By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues . Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin . The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding . Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific. EMBO J, 1999 Dec 1, 18(23), 6718 - 29 Calreticulin functions in vitro as a molecular chaperone for both glycosylated and non-glycosylated proteins; Saito Y et al.; Calreticulin (CRT) is thought to be a molecular chaperone that interacts with glycoproteins exclusively through a lectin site specific for monoglucosylated oligosaccharides . However, this chaperone function has never been directly demonstrated nor is it clear how lectin-oligosaccharide interactions facilitate glycoprotein folding . Using purified components, we show that CRT suppresses the aggregation not only of a glycoprotein bearing monoglucosylated oligosaccharides but also that of non-glycosylated proteins . Furthermore, CRT forms stable complexes with unfolded, non-glycosylated substrates but does not associate with native proteins . ATP and Zn(2+) enhance CRT's ability to suppress aggregation of non- glycoproteins, whereas engagement of its lectin site with purified oligosaccharide attenuates this function . CRT also confers protection against thermal inactivation and maintains substrates in a folding-competent state . We conclude that in addition to being a lectin CRT possesses a polypeptide binding capacity capable of discriminating between protein conformational states and that it functions in vitro as a classical molecular chaperone. EMBO J, 1999 Dec 1, 18(23), 6705 - 17 Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro; Lorsch JR et al.; Approaches have been developed for the kinetic dissection of eukaryotic translation initiation in vitro using rabbit reticulocyte ribosomes and a crude preparation of initiation factors . These new approaches have allowed the kinetics of formation of the 43S and 80S ribosomal complexes to be followed and have substantially improved the ability to follow formation of the first peptide bond . The results suggest the existence of a new step on the initiation pathway that appears to require at least one additional factor and the hydrolysis of GTP and may prepare the 80S complex for the formation of the first peptide bond . The initial kinetic framework and methods developed herein will allow the properties of individual species along the initiation pathway to be probed further and will facilitate dissection of the mechanistic roles of individual translation factors and their interplay with RNA structural elements. EMBO J, 1999 Dec 1, 18(23), 6653 - 61 Solution structure of the methyl-CpG-binding domain of the methylation-dependent transcriptional repressor MBD1; Ohki I et al.; CpG methylation in vertebrates is important for gene silencing, alterations in chromatin structure and genomic stability, and differences in the DNA-methylation status are correlated with imprinting phenomena, carcinogenesis and embryonic development . Methylation signals are interpreted by protein factors that contain shared methyl-CpG-binding domains (MBDs) . We have determined the solution structure of the MBD of the human methylation-dependent transcriptional repressor MBD1 by multi-dimensional heteronuclear NMR spectroscopy . It folds into an alpha/beta-sandwich structure with characteristic loops . Basic residues conserved in the MBD family are largely confined to one face of this fold and a flexible loop, which together form a large positively charged surface . Site-directed mutagenesis and chemical shift changes upon complexing with a methylated DNA facilitated identification of this surface as the DNA interaction site . In addition to three basic residues, conserved Tyr34 and Asp32 were shown to be important for the DNA binding. EMBO J, 1999 Dec 1, 18(23), 6642 - 52 Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli; Kurokawa K et al.; The ATP-bound but not the ADP-bound form of DnaA protein is active for replication initiation at the Escherichia coli chromosomal origin . The hydrolysis of ATP bound to DnaA is accelerated by the sliding clamp of DNA polymerase III loaded on DNA . Using a culture of randomly dividing cells, we now have evidence that the cellular level of ATP-DnaA is repressed to only approximately 20% of the total DnaA molecules, in a manner depending on DNA replication . In a synchronized culture, the ATP-DnaA level showed oscillation that has a temporal increase around the time of initiation, and decreases rapidly after initiation . Production of ATP-DnaA depended on concomitant protein synthesis, but not on SOS response, Dam or SeqA . Regeneration of ATP-DnaA from ADP-DnaA was also observed . These results indicate that the nucleotide form shifts of DnaA are tightly linked with an epistatic cell cycle event and with the chromosomal replication system. EMBO J, 1999 Dec 1, 18(23), 6599 - 609 Crystal structure of a thwarted mismatch glycosylase DNA repair complex; Barrett TE et al.; The bacterial mismatch-specific uracil-DNA glycosylase (MUG) and eukaryotic thymine-DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base-excision repair of G:U/T mismatches . Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil-DNA glycosylase enzymes, but have a very different mechanism for substrate recognition . We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate-nucleotide productively bound to a hydrolytic DNA glycosylase . The structure of this complex explains the preference for G:U over G:T mispairs, and reveals an essentially non-specific pyrimidine-binding pocket that allows MUG/TDG enzymes to excise the alkylated base, 3, N(4)-ethenocytosine . Together with structures for the free enzyme and for an abasic-DNA product complex, the MUG-substrate analogue complex reveals the conformational changes accompanying the catalytic cycle of substrate binding, base excision and product release. Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 274 - 8 Structural and functional analysis of the N-terminal extracellular region of beta-dystroglycan; Di Stasio E et al.; A protein fragment corresponding to the mouse beta-dystroglycan N-terminal extracellular region from position 654 to 750, beta-DG(654-750) was recombinantly expressed in BL21(DE3) Escherichia coli cells . Secondary structure prediction of the protein fragment reveals about 70% of random coil, as confirmed by circular dichroism analysis . Moreover, fluorescence analysis shows that the tryptophan residue in position 659 lays in a solvent-exposed fashion . These data suggest that the beta-DG(654-750) is likely to have a quite flexible structure and to be only partially folded . Interestingly, the protein still retains its biological function since using solid-phase assays we have detected binding of biotinylated beta-DG(654-750) both to native alpha-dystroglycan and to a recombinant fragment which spans the C-terminal region of alpha-dystroglycan . Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 230 - 6 A mutant streptokinase lacking the C-terminal 42 amino acids is less reactive with preexisting antibodies in patient sera; Torrens I et al.; Streptokinase (SK) is an efficacious thrombolytic drug for the treatment of myocardial infarction . Because of its immunogenicity, patients receiving SK therapy develop high anti-SK antibody (Ab) titers, which might provoke severe allergic reactions and neutralize SK activity . In this report we studied the reactivity of a synthetic 42-residue peptide resembling SKC-2 C-terminus with patient sera . SKC-2(373-414) peptide was recognized by 39 and 64% of patients, before and after SKC-2 therapy, respectively . An SKC-2 deletion mutant (mut-C42), lacking the same 42 C-terminal residues, was constructed and expressed in Escherichia coli . Recognition of mut-C42 by preexisting Abs from patient sera was 51 and 68% of reactivity to SKC-2, as assessed by direct binding and competition assays, respectively . For most of the patients, mut-C42-neutralizing activity titer (NAT) significantly decreased with respect to SKC-2-NAT . This study opens the possibility of producing a less immunogenic variant of SK, which could constitute a preferred alternative for thrombolytic therapy . Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 162 - 6 Mutations in two distinct regions of acetolactate synthase regulatory subunit from Streptomyces cinnamonensis result in the lack of sensitivity to end-product inhibition; Kopecky J et al.; Acetolactate synthase small subunit encoding ilvN genes from the parental Streptomyces cinnamonensis strain and mutants resistant either to valine analogues or to 2-ketobutyrate were cloned and sequenced . The wild-type IlvN from S . cinnamonensis is composed of 175 amino acid residues and shows a high degree of similarity with the small subunits of other valine-sensitive bacterial acetolactate synthases . Changes in the sequence of ilvN conferring the insensitivity to valine in mutant strains were found in two distinct regions . Certain point mutations were located in the conserved domain near the N terminus, while others resulting in the same phenotype shortened the protein at V(104) or V(107) . To confirm whether the described mutations were responsible for the changed biochemical properties of the native enzyme, the wild-type large subunit and the wild-type and mutant forms of the small one were expressed separately in E . coli and combined in vitro to reconstitute the active enzyme . Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 58 - 61 Nucleotide binding drives conformational changes in the isolated alpha and beta subunits of the F(1)-ATPase from Escherichia coli; Pena HN et al.; The modeling of the rotatory mechanism performed by the F(1)-ATPase complex during ATP synthesis shows that the beta, but not the alpha subunit, undergoes large conformational changes that depend on the occupancy of the catalytic site . Here we determined by fluorescence spectroscopy the changes in tertiary structure and hydrophobic exposed area of the isolated alpha and beta subunits of the F(1)-ATPase complex from Escherichia coli upon adenine nucleotide binding . The results show that in the absence of intersubunit contacts, the two subunits exhibit markedly similar conformational movements . Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 19 - 23 Recombinant soluble human CD69 dimer produced in Escherichia coli: reevaluation of saccharide binding; Childs RA et al.; We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein . We have expressed in Escherichia coli a new construct of the extracellular part (Q(65)-K(199)) of human CD69 . We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine, linked to bovine serum albumin . Monosaccharide-binding signals are not detectable . Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan . Endocr J, 1999 Aug, 46(4), 487 - 96 Temporal profiles of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in the plasma and hypothalamic paraventricular nucleus after intravenous or intraperitoneal administration of lipopolysaccharide in the rat: estimation by push-pull perfusion; Kakizaki Y et al.; Lipopolysaccharide (LPS) is known to stimulate the synthesis and secretion of various proinflammatory cytokines in both the peripheral immune cells and the brain . Yet, the relative contribution of peripheral and central cytokines to the LPS-induced activation of the hypothalamo-pituitary-adrenal axis is still poorly understood . In this study, utilizing the push-pull perfusion technique of the rat brain, we attempted to characterize in detail the temporal profiles of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha after intravenous (i.v.) or intraperitoneal (i.p.) administration of LPS in both the general circulation and the hypothalamic paraventricular nucleus (PVN), which is the primary source of corticotropin releasing hormone (CRH) . Temporal changes in plasma adrenocorticotropic hormone (ACTH) and CRH levels in the PVN were also monitored . We collected blood and perfusates every 30 min from 11:00 to 17:00 h . At 12:00 h, 1.0 or 2.5 mg/kg body weight of LPS was given via an i.v . or i.p . route, respectively . Peak ACTH response occurred 30 min after i.v . LPS and 1.5 h after ip LPS . Of the three cytokines measured in the plasma, TNF-alpha showed the fastest rise in synchrony with peak ACTH secretion after both i.v . and i.p . LPS . Although plasma IL-6 also showed a robust rise, its peak level occurred later than the ACTH peak . Elevation of plasma IL-1beta was the smallest among the three cytokines . CRH levels in the PVN reached their peaks 1 and 2.5 h after the ACTH peak following i.p . and i.v . LPS, respectively . Irrespective of the route of LPS administration, IL-6 and TNF-alpha levels in the PVN showed significant rises 1-2 h after the ACTH peak, but IL-1beta in the PVN did not significantly change during the entire period of observation . The results of the present study suggest that circulating TNF-alpha may play the most important role in triggering the early, peak phase of ACTH secretion after both i.v . and i.p . LPS . Although it is possible that brain TNF-alpha, IL-6, and circulating IL-6, may be involved in the later, protracted phase of ACTH secretion induced by LPS, IL-1beta in both the brain and peripheral circulation seems to play the smallest role in ACTH secretion . This is the first study to characterize the LPS-induced temporal changes in IL-1beta, IL-6, and TNF-alpha in both plasma and PVN simultaneously in conscious, freely moving rats. RNA, 1999 Nov, 5(11), 1490 - 4 The relationship of thermodynamic stability at a G x U recognition site to tRNA aminoacylation specificity; Strazewski P et al.; The G x U pair at the third position in the acceptor helix of Escherichia coli tRNA(Ala) is critical for aminoacylation . The features that allow G x U recognition are likely to include direct interaction of alanyl-tRNA synthetase with distinctive atomic groups and indirect recognition of the structural and stability information encoded in the sequence of G x U and its immediate context . The present work investigates the thermodynamic stability and acceptor activity for a comprehensive set of variant RNAs with substitutions of the G x U pair of E . coli tRNA(Ala) . The four RNAs with Watson-Crick substitutions had a lower acceptor activity and a higher stability relative to the G x U RNA . On the other hand, the RNAs with mispair substitutions had a lower stability, but either a higher or a lower acceptor activity . Thus, the entire set of variant RNAs does not exhibit a correlation between thermodynamic stability of the free, unbound tRNA and its acceptor activity . The substantial acceptor activity of tRNAs with particular mispair substitutions may be explained by their ability to assume the conformational preferences of alanyl-tRNA synthetase . Moreover, the G x U pair may provide a point of deformability for the substrate tRNA to adapt to the enzyme's active site. RNA, 1999 Nov, 5(11), 1470 - 81 Characterization of U6 snRNA-protein interactions; Vidal VP et al.; Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs . Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs . In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome . Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA . The uridine-rich extreme 3' end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins . Interestingly, the Lsm proteins associate efficiently with the 3' half of U6, which contains the 3' stem-loop and uridine-rich 3' end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs. RNA, 1999 Nov, 5(11), 1451 - 7 Translational suppressors and antisuppressors alter the efficiency of the Ty1 programmed translational frameshift; Burck CL et al.; Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors . Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal . Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons . These mutations have previously been shown to affect the general accuracy of translational termination . We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Tyl frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect . Three antisuppressor rRNA mutations all reduced Tyl frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect . Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Tyl frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect . A mutant form of the other release factor, eRF1, also had no effect on frameshifting . Our data suggest that Ty frameshifting is under the control of the cellular translational machinery . Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease. RNA, 1999 Nov, 5(11), 1430 - 9 Identity and geometry of a base triple in 16S rRNA determined by comparative sequence analysis and molecular modeling; Babin P et al.; Comparative sequence analysis complements experimental methods for the determination of RNA three-dimensional structure . This approach is based on the concept that different sequences within the same gene family form similar higher-order structures . The large number of rRNA sequences with sufficient variation, along with improved covariation algorithms, are providing us with the opportunity to identify new base triples in 16S rRNA . The three-dimensional conformations for one of our strongest candidates involving U121 (C124:G237) and/or U121 (U125:A236) (Escherichia coli sequence and numbering) are analyzed here with different molecular modeling tools . Molecular modeling shows that U121 interacts with C124 in the U121 (C124:G237) base triple . This arrangement maintains isomorphic structures for the three most frequent sequence motifs (approximately 93% of known bacterial and archaeal sequences), is consistent with chemical reactivity of U121 in E . coli ribosomes, and is geometrically favorable . Further, the restricted set of observed canonical (GU, AU, GC) base-pair types at positions 124:237 and 125:236 is consistent with the fact that the canonical base-pair sets (for both base pairs) that are not observed in nature prevent the formation of the 121 (124:237) base triple . The analysis described here serves as a general scheme for the prediction of specific secondary and tertiary structure base pairing where there is a network of correlated base changes. RNA, 1999 Nov, 5(11), 1419 - 29 Structure of the phylogenetically most conserved domain of SRP RNA; Schmitz U et al.; The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane . Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule . All known essential interactions of SRP occur in that moiety containing domain IV . The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement . Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634-638), where all conserved nucleotides are involved in nucleotide-specific structural interactions . Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured . The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides . The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site. RNA, 1999 Nov, 5(11), 1408 - 18 Metastable structures and refolding kinetics in hok mRNA of plasmid R1; Nagel JH et al.; Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells . It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5' end . Here, experimental evidence is presented for the existence of metastable structures in the 5' leader of the hok and pnd mRNAs in vitro . The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5' ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation . The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures . Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription. Vaccine, 1999 Dec 10, 18(9-10), 850 - 9 The protective capacities of histone H1 against experimental murine cutaneous leishmaniasis; Solioz N et al.; In a murine model of experimental cutaneous leishmaniasis, we investigated the protection elicited by injection of histone H1 isolated from parasites by perchloric extraction, of a H1 recombinant protein produced in E . coli, and of H1 long and short synthetic peptides, against infection by L . major . Partial protection was achieved in most of the animals as shown by reduction in lesion size, upon immunization with histone H1 or its peptides, provided that the region 1-60 was present in the molecule . These observations argue in favor of a thorough examination of the possibility of including histone H1 described here in a cocktail vaccine against human leishmaniasis. Gene, 1999 Nov 29, 240(2), 361 - 70 The Mycobacterium tuberculosis mysB gene product is a functional equivalent of the Escherichia coli sigma factor, KatF; Mulder NJ et al.; Mycobacterium tuberculosis, the causative agent of tuberculosis, may remain dormant within its host for many years . The nature of this dormant or latent state is not known, but it may be a specialized form of the stationary growth phase . In Escherichia coli, KatF (or RpoS) is the major stationary phase sigma factor regulating an array of genes expressed in this phase of growth . A potential M . tuberculosis katF homologue was cloned using a fragment of the E . coli katF gene as a probe . DNA sequence analysis of a resultant clone showed 100% identity to a fragment of DNA encoding the M . tuberculosis mysA and mysB genes . Overexpression of mysB in M . bovis BCG resulted in an increase in katG mRNA and catalase and peroxidase activity, and an increase in sensitivity of the cells to isoniazid . An increase in katG promoter activity from a reporter vector was demonstrated when mysB was overexpressed from the same plasmid, indicating a direct relationship between MysB and katG expression. FEBS Lett, 1999 Nov 26, 462(1-2), 7 - 11 The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis; Holtz KM et al.; The proposed double in-line displacement mechanism of Escherichia coli alkaline phosphatase (AP) involving two-metal ion catalysis is based on NMR spectroscopic and X-ray crystallographic studies . This mechanism is further supported by the X-ray crystal structures of the covalent phospho-enzyme intermediate of the H331Q mutant AP and of the transition state complex between the wild-type enzyme and vanadate, a transition state analog . Kinetic and structural studies on several genetically engineered versions of AP illustrate the overall importance of the active site's metal geometry, hydrogen bonding network and electrostatic potential in the catalytic mechanism. J Natl Cancer Inst, 1999 Dec 1, 91(23), 2014 - 9 Cytosine deaminase/5-fluorocytosine-based vaccination against liver tumors: evidence of distant bystander effect; Pierrefite-Carle V et al.; BACKGROUND: The cytosine deaminase gene of Escherichia coli converts the nontoxic compound 5-fluorocytosine into 5-fluorouracil (5-FU), thereby acting as a suicide gene when introduced into cancer cells, killing the cells when they are exposed to 5-fluorocytosine . We analyzed the efficacy of using cytosine deaminase-bearing cancer cells as an autologous tumor vaccine in a rat model that mimics liver metastasis from colon carcinoma . METHODS: We introduced a plasmid vector containing the E . coli cytosine deaminase gene into a BDIX rat colon carcinoma cell line . Intrahepatic injection of the modified cells in syngeneic animals generates a single experimental liver "suicide tumor." We then analyzed the effect of 5-fluorocytosine treatment in terms of regression of cytosine deaminase-expressing cells in vivo as well as protection against wild-type cancer cells . RESULTS: Treatment with 5-fluorocytosine induced regression of cytosine deaminase-expressing (CD+) tumors, with seven of 11 treated animals being tumor free at the end of 30 days and a statistically significant difference in tumor volumes between treated and control animals (two-sided P<.0001) . Intrahepatic injection of CD+ cells followed by 5-fluorocytosine treatment rendered the treated animals resistant to challenge with wild-type tumor cells, with no (zero of seven) treated animals developing wild-type tumors in contrast to all (four of four) control animals . Moreover, in animals with established wild-type liver tumors, injection of CD+ tumor cells followed by 5-fluorocytosine treatment produced a statistically significant increase in survival time (two-sided P<.0001) . In vivo immunodepletion and immunohistologic analysis of experimental tumors indicate that natural killer cells are the major immune component involved in this antitumor effect . CONCLUSIONS and IMPLICATIONS: Taken together, these results suggest the potential use of suicide gene-modified tumor cells as therapeutic vaccines against liver metastasis from colon carcinoma. Dev Comp Immunol, 1999 Oct-Dec, 23(7-8), 553 - 62 Induction of mosquito hemolymph proteins in response to immune challenge and wounding; Han YS et al.; The rapid induction of proteins in the hemolymph of the mosquito, Anopheles gambiae, was examined after wounding or injection of immune elicitors (Escherichia coli, lipopolysaccharide, laminarin, zymosan) . One-dimensional gel electrophoresis revealed at least six hemolymph polypeptides >25 kDa that consistently appeared after any breech of the cuticle . All of these polypeptides appeared in the hemolymph within 30 min and reached a maximum concentration after approx . 6 h . No proteins were specifically induced by bacteria or bacterial or fungal cell wall products, however two constitutively expressed proteins were repressed by these injections . Patterns of hemolymph proteins were further analyzed by two-dimensional electrophoresis . Seven spots were enhanced or induced 2 h after injection in four replicate experiments . An additional two spots demonstrated some variability between replicates, but were generally responsive to injection . These rapidly induced polypeptides are candidates for regulating and initiating the mosquito's responses to pathogens and wounding. Bioorg Khim, 1999 Aug, 25(8), 630 - 3 {DNA methyltransferases for detection of the level of methylation of cytosine in the DNA CCWGG sequence}; Shevchuk TV et al.; An assay for the cytosine methylation level in the eukaryotic DNA CCWGG sequence is proposed . The method is based on the ability of DNA methylase BstNI to methylate DNA containing in a CCWGG site a nonmodified or 5-methylated cytosine to yield N4-methyl- or N4,5'-dimethylcytosine, respectively. Bioorg Khim, 1999 Aug, 25(8), 623 - 9 {Production of recombinant hIL-4delta2-a native isoform of human interleukin-4 in Escherichia coli cells}; Ptitsyn LR et al.; Expression plasmids containing the synthetic gene hil-4 delta 2 was constructed to produce human interleukin-4 in Escherichia coli cells . Strains TG1 (pBTIL-4 delta 2) and BL21 (DE3) (pETIL-4 delta 2) produced the recombinant protein as inclusion bodies, and its production level was up to 30% of the total cell protein . The renatured hIL-4 delta 2 inhibited IL-4-stimulated T cell proliferation, and this effect was enhanced by cyclosporin A. Cell Death Differ, 1999 Nov, 6(11), 1117 - 24 The proteolytic procaspase activation network: an in vitro analysis; Van de Craen M et al.; In general, apoptotic stimuli lead to activation of caspases . Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members . We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts . Caspase-8 processed all procaspases examined . Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6 . However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases . This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway . Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor . Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade . Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases . Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated . The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis. J Biochem (Tokyo), 1999 Dec, 126(6), 1080 - 9 Misfolded membrane-bound cytochrome P450 activates KAR2 induction through two distinct mechanisms; Zimmer T et al.; Using the mRNA differential display technique and Western blot analysis, the present study demonstrates that induction of KAR2 occurs when misfolded membrane-bound cytochrome P450, mutated in its cytosolically exposed domain, is expressed in Saccharomyces cerevisiae . Using various KAR2 promoter constructs in front of the Escherichia coli beta-galactosidase reporter gene, we found a fast and strong induction through the heat shock element (HSE), which was enhanced several fold by its adjacent GC-rich region . Additionally, a less pronounced induction was detected for the UPR element (UPRE) . As expected, this response was absent in the ire1 disruptant strain . However, the HSE-mediated induction was enhanced upon disruption of IRE1 suggesting that the HSE pathway can compensate for the lack of a functional UPR pathway . Western blotting confirmed that Kar2p levels were increased to the same extent in the ire1 disruptant and in the non-disruptant strain . Removal of the P450 membrane-spanning region also abolished the UPRE-mediated induction of KAR2 transcription, but the HSE-mediated response remained . The data show for the first time that the transcription of KAR2 is significantly induced in response to a misfolded membrane-bound endoplasmic reticulum protein, and identifies the HSE and UPRE regions as KAR2 promoter elements responding to the misfolded cytosolic P450 domain and to the membrane-integrated mutant P450, respectively. J Biochem (Tokyo), 1999 Dec, 126(6), 1013 - 9 Purification, molecular cloning, and genomic organization of human brain long-chain acyl-CoA hydrolase; Yamada J et al.; An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol . The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g . a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA . Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates . In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue . The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity . The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog . The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram . These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase. Bioorg Med Chem, 1999 Oct, 7(10), 2209 - 13 Engineering of a novel biochemical pathway for the biosynthesis of L-2-aminobutyric acid in Escherichia coli K12; Fotheringham IG et al.; L-2-Aminobutyric acid was synthesised in a transamination reaction from L-threonine and L-aspartic acid as substrates in a whole cell biotransformation using recombinant Escherichia coli K12 . The cells contained the cloned genes tyrB, ilvA and alsS which respectively encode tyrosine aminotransferase of E . coli, threonine deaminase of E . coli and alpha-acetolactate synthase of B . subtilis 168 . The 2-aminobutyric acid was produced by the action of the aminotransferase on 2-ketobutyrate and L-aspartate . The 2-ketobutyrate is generated in situ from L-threonine by the action of the deaminase, and the pyruvate by-product is eliminated by the acetolactate synthase . The concerted action of the three enzymes offers significant yield and purity advantages over the process using the transaminase alone with an eight to tenfold increase in the ratio of product to the major impurity. Bioorg Med Chem, 1999 Oct, 7(10), 2139 - 44 Incremental truncation as a strategy in the engineering of novel biocatalysts; Ostermeier M et al.; The application and success of combinatorial approaches to protein engineering problems have increased dramatically . However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity . To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries . For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA . Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins . In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology. Plant Mol Biol, 1999 Sep, 41(2), 279 - 91 Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.) Brill EM, Abrahams S, Hayes CM, Jenkins CL, Watson JM. A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase (CAD) cDNA probe from tobacco (Nicotiana tabacum cv . Samsun) . Two distinctly different cDNA clones (54% identical) were isolated and identified as putative CAD-encoding cDNAs by comparison of their nucleotide sequences with those of CAD-encoding DNA sequences from other plant species . One of the cDNAs, MsaCad2, was found to be 99.4% identical at the nucleotide level to the previously isolated lucerne cad cDNA which encodes a CAD isoform involved in lignin biosynthesis . The other cDNA, MsaCad1, has not been reported previously in lucerne, and encodes a protein related to the ELI3 class of elicitor-inducible defence-related plant proteins . The MsaCad1- and MsaCad2-encoded proteins were expressed in Escherichia coli and CAD1 was shown to be active with a range of cinnamyl, benzyl and aliphatic aldehyde substrates, while CAD2 was specific for the cinnamyl aldehydes only . Each of the respective genes is present as one or two copies . The MsaCad1 gene is expressed most actively in stem and floral tissue, whereas MsaCad2 is most actively expressed in stem, hypocotyl and root tissue . In stem tissue, expression of both genes occurs predominantly in internodes 4 and 5 (from the apex) . MsaCad2, in contrast to MsaCad1, is not significantly expressed in the top three internodes of the stem . Both MsaCad1 and MsaCad2 are wound-inducible, and the wound-responsiveness of each gene is modulated by salicylic acid. Plant Mol Biol, 1999 Sep, 41(2), 171 - 80 Age-dependent wound induction of a myrosinase-associated protein from oilseed rape (Brassica napus); Andreasson E et al.; In order to study the expression of the induced form of myrosinase-associated protein (iMyAP), a genomic clone encoding the protein was isolated from Brassica napus . The coding portion of the gene was found to consist of five exons separated by one long intron of 938 bp and three shorter introns of ca . 100 bp . A 1.9 kb promoter fragment including the 5'-untranslated region was cloned in front of the coding portion of the Escherichia coli iudA gene and transformed into Arabidopsis thaliana . Expression was observed in hypocotyls of 4-day seedlings, but in 7-day seedlings the iMyAP promoter did not direct expression . In flowering plants, only the abscission zone of the young silique displayed promoter activity . In contrast, mechanical wounding of 7-day seedlings induced a systemic expression in all cells of the cotyledons . Wounding of 14-day seedlings gave rise to systemic induced expression mainly in the vascular tissue . However, mechanical wounding and wounding by flea beetles (Phyllotreta undulata) of 4-week old plants only gave rise to a local induction of the promoter, suggesting that the systemic signal system is age-dependent . Methyl jasmonate also induced iMyAP expression . In situ and northern analysis of iMyAP transcripts in young leaves of B . napus showed that the induction was high after 1 h and absent after 24 h . Comparison of the effect of different types of wounding on the iMyAP promoter induction in transgenic Arabidopsis showed that similar degrees of local induction were achieved regardless of the degree of macerated tissue left on the plant. Int J Parasitol, 1999 Sep, 29(9), 1451 - 5 Identification of an asparagine amidohydrolase from the filarial parasite Dirofilaria immitis; Tsuji N et al.; The nematode cuticle is a complex extracellular structure which is secreted by an underlying syncytium of hypodermal cells . Recent studies have demonstrated that the cuticle of parasitic nematodes is a dynamic structure with important absorptive, secretory, and enzymatic activities . In addition, the cuticle serves as a protective barrier against the host . A 48-h third stage larval Dirofilaria immitis cDNA library was immunoscreened with sera raised against larval cuticles . One clone, L3MC4 that reacted strongly with the anti-cuticle antisera was sequenced . The composite cDNA sequence comprises 2073 bp coding for a full-length protein of 590 amino acids . GenBank analysis showed that DiAsp had significant similarity to a Caenorhabditis elegans gene-product (54% identity) and to other asparaginases at the amino acid level . Escherichia coli-expressed recombinant DiAsp (rDiAsp) catalysed the hydrolysis of asparagine to aspartate and ammonia . Antibodies raised against D . immitis larval cuticles reacted with rDiAsp in immunoblots . This is the first report of identification of a cDNA clone encoding an asparaginase enzyme from a parasitic nematode. Int J Parasitol, 1999 Sep, 29(9), 1437 - 46 Up-regulation of extracellular copper/zinc superoxide dismutase mRNA after transmission of the filarial parasite Acanthocheilonema viteae in the vertebrate host Meriones unguiculatus; Lattemann CT et al.; The gene encoding the cytoplasmic copper/zinc superoxide dismutase (AVSOD1) from the filarial parasite Acanthocheilonema viteae was isolated from a genomic DNA library using a degenerate oligonucleotide probe . Additionally, cDNAs of the AVSOD1 and the secreted extracellular SOD (AVSOD2) were both cloned by RT-PCR, and the AVSOD2 was expressed at high levels in E . coli . The amino acid sequence of the AVSOD1 is 89.5 and 87.5% identical to that of the corresponding enzymes of Brugia pahangi and Onchocerca volvulus, respectively . In contrast, the AVSOD2 shows a lower degree of identity to the other filarial SODs and is extensively glycosylated . RT-PCR studies demonstrate the expression of both SOD subtypes in all developmental stages of A . viteae and indicate up-regulation of the AVSOD2 expression after transmission from the vector to the definitive host . This suggests an enhanced requirement for SOD activity in post-infective larval stages and adults of A . viteae . ELISAs performed with purified recombinant AVSOD2 show that the AVSOD2 is not a major target for the immune system in naturally infected jirds. Br Poult Sci, 1999 Sep, 40(4), 552 - 4 Effect of dietary xylitol on growth and inflammatory responses in immune stimulated chickens; Takahashi K et al.; It has been argued that stimulation of the immune system depresses performance . Accordingly, an experiment was conducted to determine the effect of dietary xylitol (150 g/kg diet) on growth and selected inflammatory responses in male broiler chickens . During the final 6 d of the experimental periods, chicks were injected with antigens: Escherichia coli lipopolysaccharide (LPS) on days 1, 3 and 5 and with Sephadex-G50 superfine on days 2 and 4 to stimulate macrophage functions . The immune stimulation reduced body weight gain and food intake, but enhanced alpha 1 acid glycoprotein (AGP) concentration and interleukin (IL-1) like activity in plasma . Feeding the xylitol diet partially, but significantly, prevented the reductions in body weight gain and food intake, without affecting the early stage of inflammatory responses triggered by LPS and Sephadex injections. Endocrinology, 1999 Dec, 140(12), 5999 - 6002 Specific progesterone binding to a membrane protein and related nongenomic effects on Ca2+-fluxes in sperm; Falkenstein E et al.; Rapid, nongenomic effects of steroids are supposed to be transmitted by membrane receptors unrelated to the classic intracellular steroid receptors . In this context, a putative progesterone membrane binding protein (mPR) has been identified, recently . Here we show that expression of mPR-cDNA in CHO cells leads to increased microsomal progesterone binding . This result is mirrored by effects of an antibody raised against the recombinant E . coli mPR which suppressed the rapid progesterone-initiated Ca2+ increase in sperm . Our results support the assumption that mPR represents the first steroid membrane receptor or a part of it involved in rapid, nongenomic steroid signalling. Endocrinology, 1999 Dec, 140(12), 5505 - 15 Endotoxin-induced inhibition of growth hormone receptor signaling in rat liver in vivo; Mao Y et al.; The bacterial lipopolysaccharide endotoxin induces a catabolic response characterized by resistance to multiple anabolic hormones . The objective of this study was to determine the effects of endotoxin on the GH signaling pathway in rat liver in vivo . After the iv injection of Escherichia coli endotoxin (1 mg/kg), there was a progressive decrease in liver STAT5 (signal transducer and activator of transcription-5) tyrosine phosphorylation in response to GH (40% decrease 6 h after endotoxin), which occurred in the absence of a change in abundance of the STAT5 protein . Endotoxin resulted in a rapid 40-fold increase in liver Janus family kinase-2 (JAK2) messenger RNA, followed by a 2-fold increase in JAK2 protein abundance . This was associated with a 50% decrease in phosphorylated/total JAK2 after GH stimulation . GH receptor abundance was unchanged, suggesting a postreceptor site of endotoxin-induced GH resistance . Rat complementary DNAs for three members of the suppressor of cytokine signaling gene family were cloned {cytokine-inducible sequence (CIS), suppressor of cytokine signaling-2 (SOCS-2), and SOCS-3} and, using these probes, messenger RNAs for SOCS-3 and CIS were shown to be increased 10- and 4-fold above control values, respectively, 2 h after endotoxin infusion . The finding of endotoxin inhibition of in vivo STAT5 tyrosine phosphorylation in response to a supramaximal dose of GH in the absence of a change in GH receptor abundance or total GH-stimulated JAK2 tyrosine phosphorylation provides the first demonstration of acquired postreceptor GH resistance . We hypothesize that this may occur through a specificity-spillover mechanism involving the induction of SOCS genes by cytokines released in response to endotoxin and subsequent SOCS inhibition of GH signaling. Crit Care Med, 1999 Nov, 27(11), 2518 - 24 Ileal mucosal oxygen consumption is decreased in endotoxemic rats but is restored toward normal by treatment with aminoguanidine; King CJ et al.; OBJECTIVE: We sought to test the hypothesis that ileal mucosal oxygen consumption is impaired in endotoxemic rats . METHODS: Male Sprague-Dawley rats were injected intravenously with either Escherichia coli lipopolysaccharide (5 mg/kg) or a similar volume of vehicle . A segment of ileum was excised 8 hrs later, and the serosal and muscular layers of the bowel were stripped away from the mucosa . A strip of mucosa was mounted in a polarographic chamber containing air-saturated Krebs-Henseleit buffer plus 20 mM glucose, PO2 being monitored during a 10-min period . Some rats were injected intraperitoneally with the inducible nitric oxide synthase inhibitor, aminoguanidine (30 mg/kg per dose), or a similar volume of vehicle, at 1, 3 and 6 hrs after injection of lipopolysaccharide . RESULTS: In an initial experiment, the rate of oxygen consumption was significantly lower for mucosal samples from endotoxemic rats as compared with control rats (0.76+/-0.11 ng-atoms vs . 1.42+/-0.22 ng-atoms of 0/min per microg dry weight, respectively; n = 8 per group; p<.05) . The rate of mucosal oxygen consumption was higher in aminoguanidine-treated as compared with vehicle-treated endotoxemic rats (1.25+/-0.11 ng-atoms and 0.73+/-0.07 ng-atoms of 0/min per microg, respectively; n = 7 and n = 6, respectively; p<.05) . CONCLUSION: Endotoxemia is associated with diminished intestinal mucosal oxygen utilization due to an intrinsic acquired derangement in cellular respiration that is caused, at least in part, by an aminoguanidine-inhibitable mechanism. Crit Care Med, 1999 Nov, 27(11), 2474 - 9 Modulation of systemic hemodynamics by exogenous L-arginine in normal and bacteremic sheep; Lorente JA et al.; OBJECTIVE: To investigate whether exogenous L-arginine, the substrate for nitric oxide synthase, modulates systemic hemodynamics in sepsis . DESIGN: Prospective, controlled study in a sheep model of sepsis . SETTING: Animal research facility in a university hospital . SUBJECTS: Adult sheep weighing between 35 and 55 kg . INTERVENTIONS: Adult sheep sedated and mechanically ventilated, were monitored with a pulmonary arterial catheter and an ileal tonometer . Four groups of sheep were studied: nonseptic, septic, nonseptic treated with L-arginine, and septic treated with L-arginine . Sepsis was induced by the intravenous administration of Escherichia coli (1.5x10(8) colony-forming units/kg for 30 mins) . L-arginine was administered as an intravenous bolus (200 mg/kg for 10 mins) before the septic challenge followed by 200 mg/kg/hr for 300 mins . MEASUREMENTS AND MAIN RESULTS: Sepsis induced a state of acidosis, hyperlactatemia, hypoxemia, and gastric intramucosal acidosis . During the first 30 mins after the septic challenge, there was a decrease in cardiac index and blood pressure, and an increase in systemic vascular resistance . Thereafter, blood pressure returned to baseline values, and systemic vascular resistance fell . Treatment with L-arginine in nonseptic sheep did not induce any biochemical or hemodynamic effect . In septic sheep, treatment with L-arginine was associated with a greater increase in systemic vascular resistance during the first 30 mins, and a more marked decrease in blood pressure and systemic vascular resistance after 180 mins . CONCLUSIONS: Exogenous administration of L-arginine does not induce hemodynamic effects in normal animals, exacerbates the acute vasoconstriction associated with the intravenous infusion of E . coli and potentiates the sepsis-induced vasodilation . Our results suggest that a) nitric oxide production is not constitutively modulated by exogenous L-arginine, b) L-arginine probably enhances the sepsis-induced sympathetic discharge, and c) L-arginine becomes rate-limiting for the formation of nitric oxide at approximately 3 hrs after the initiation of the septic challenge. Biotechnol Bioeng, 1999, 66(4), 247 - 57 Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification; Johansson HO et al.; In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution . This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt) . The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO) . This polymer thermoseparates in water, with a cloud point at 14 degrees C . The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C . The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%) . The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning . The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range . The partitioning of hydrophobic proteins can be directed with addition of salt . Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system . Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate . The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations . This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract . Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase . By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase . This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered . Cytometry, 1999 Jun 1, 36(2), 123 - 30 Characterization of the simultaneous binding of Escherichia coli endotoxin to Kupffer and endothelial liver cells by flow cytometry; Catala M et al.; BACKGROUND: The triggering of cellular responses during endotoxic shock is initiated for the binding of endotoxin (lipopolysaccharide; LPS) to the cell surface . Kupffer and endothelial liver cells, involved in the removal of endotoxin from blood circulation, show in vitro a rapid response to LPS in the absence of serum . METHODS: A double-labeling fluorescent assay was designed to evaluate the binding properties of Escherichia coli O111:B4 LPS to individual endothelial and Kupffer cells in suspension, where both populations occurred in the same relative proportion as in liver . After immunolabeling of the Kupffer cell population with the monoclonal antibody ED1 conjugated to R . phycoerythrin, the binding characteristics of LPS labeled with fluorescein to both endothelial and Kupffer cells were simultaneously studied by flow cytometry in serum-free conditions . RESULTS: Specific and saturable binding of endotoxin was observed with both populations, showing properties of a receptor-mediated process . The Kupffer cell population showed a faster capacity and a higher affinity for LPS binding . The Hill coefficients indicated positive cooperativity in the LPS interaction with both populations . CONCLUSIONS: Specific endotoxin binding to liver sinusoidal cells occurs in a serum-independent manner, particularly at high LPS concentrations . Flow cytometry is a fast, precise, and efficient technique to evaluate the simultaneous interaction of a ligand with two different cell types. Nature, 1999 Oct 28, 401(6756), 935 - 8 A triple beta-spiral in the adenovirus fibre shaft reveals a new structural motif for a fibrous protein; van Raaij MJ et al.; Human adenoviruses are responsible for respiratory, gastroenteric and ocular infections and can serve as gene therapy vectors . They form icosahedral particles with 240 copies of the trimeric hexon protein arranged on the planes and a penton complex at each of the twelve vertices . The penton consists of a pentameric base, implicated in virus internalization, and a protruding trimeric fibre, responsible for receptor attachment . The fibres are homo-trimeric proteins containing an amino-terminal penton base attachment domain, a long, thin central shaft and a carboxy-terminal cell attachment or head domain . The shaft domain contains a repeating sequence motif with an invariant glycine or proline and a conserved pattern of hydrophobic residues . Here we describe the crystal structure at 2.4 A resolution of a recombinant protein containing the four distal repeats of the adenovirus type 2 fibre shaft plus the receptor-binding head domain . The structure reveals a novel triple beta-spiral fibrous fold for the shaft . Implications for folding of fibrous proteins (misfolding of shaft peptides leads to amyloid-like fibrils) and for the design of a new class of artificial, silk-like fibrous materials are discussed. Nature, 1999 Oct 28, 401(6756), 932 - 5 A kinetic proofreading mechanism for disentanglement of DNA by topoisomerases; Yan J et al.; Cells must remove all entanglements between their replicated chromosomal DNAs to segregate them during cell division . Entanglement removal is done by ATP-driven enzymes that pass DNA strands through one another, called type II topoisomerases . In vitro, some type II topoisomerases can reduce entanglements much more than expected, given the assumption that they pass DNA segments through one another in a random way . These type II topoisomerases (of less than 10 nm in diameter) thus use ATP hydrolysis to sense and remove entanglements spread along flexible DNA strands of up to 3,000 nm long . Here we propose a mechanism for this, based on the higher rate of collisions along entangled DNA strands, relative to collision rates on disentangled DNA strands . We show theoretically that if a type II topoisomerase requires an initial 'activating' collision before a second strand-passing collision, the probability of entanglement may be reduced to experimentally observed levels . This proposed two-collision reaction is similar to 'kinetic proofreading' models of molecular recognition. Biotechnol Bioeng, 1999, 66(3), 189 - 94 Analysis and use of endogenous nuclease activities in Escherichia coli lysates during the primary isolation of plasmids for gene therapy; Monteiro GA et al.; Two important issues in the downstream processing of plasmids for gene therapy are the stability of plasmids in the process streams, and the presence of contaminating host RNA . Results with a 4.8-kb plasmid harbored in a non-nuclease-deficient strain of Escherichia coli show that, in spite of the harsh conditions during alkaline lysis, a fraction of endogenous nucleases remains active, degrading both RNA and genomic and plasmid DNA . Although it is possible to minimize plasmid degradation by decreasing temperature and reducing processing times, the presence of endogenous nucleases can be used advantageously to purify the plasmid streams . The kinetics of nucleic acid degradation showed that, by controlling the incubation at 37 degrees C, it was possible to degrade RNA selectively, while maintaining plasmid integrity . A reduction of 40% in RNA content was obtained, corresponding to a 1.5-fold increase in plasmid purity using high-performance liquid chromatography (HPLC) . This strategy is simple and straightforward, and the increase in processing time and the associated plasmid loss (9%) are fully justified by the purity increase . Furthermore, the use of endogenous RNase activity is clearly advantageous over alternative procedures, such as the addition of external RNase, in terms of cost, validation, and compliance with guidelines from regulatory agencies . Arch Biochem Biophys, 1999 Oct 15, 370(2), 231 - 5 Substrate specificity of human glycinamide ribonucleotide transformylase; Antle VD et al.; The nucleotide substrate specificity of human glycinamide ribonucleotide transformylase, a chemotherapeutic target, has been examined . The enzyme accepts the sarcosyl analog of glycinamide ribonucleotide, carbocyclic glycinamide ribonucleotide, and two phosphonate derivatives of carbocyclic glycinamide ribonucleotide with V/K values, relative to that obtained for beta-glycinamide ribonucleotide, of 1, 27, 1.4, and 2.9%, respectively . Several other analogs of carbocyclic glycinamide ribonucleotide, namely a truncated phosphonate and 2',3'-dideoxy- and 2',3'-dideoxy-2',3'-didehydro-carbocyclic glycinamide ribonucleotide, were inhibitors of the enzyme, competitive against glycinamide ribonucleotide, with Ki values approximately 100 times higher than the Km for -glycinamide ribonucleotide . Although the results of the present study parallel those obtained previously with the avian enzyme (V . D . Antle, D . Liu, B . R . McKellar, C . A . Caperelli, M . Hua, and R . Vince (1996) J . Biol . Chem . 271, 6045-6049), quantitative differences between the two enzyme species have been uncovered. Clin Cancer Res, 1999 Oct, 5(10 Suppl), 3088s - 3094s Development of a hyperimmune anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer; Winthrop MD et al.; Radioimmunotherapy (RIT) has demonstrated potential for improving clinical cancer therapy . Optimizing the approach has proven difficult thus far . Antibody phage display libraries provide unique molecules that could improve RIT . A phage display library of single chain antibody fragments (scFv) against the MUC-1 mucin molecule, which is expressed on 90% of human breast cancers, was produced from the spleen cells of MUC-1 hyperimmunized BALB/c mice . Increased serum IgG levels, 15 times baseline, were detected following the third immunization . RNA from the spleen cells was isolated, cDNA was made, and variable heavy and variable light immunoglobulin chain gene regions were amplified using PCR technology . The variable heavy and variable light chain gene regions were combined with a flexible linker, ligated into the pCANTAB 5E phagemid vector, and electroporated into TG1 Escherichia coli cells . A library of 10(7) initial colonies was compiled . Forty-six of 288 colonies screened for reactivity demonstrated binding to MUC-1-expressing MCF-7 breast cancer cell membrane fragments . Anti-MUC-1 library diversity evaluated by BstNI digest demonstrated that 52% of the anti-MUC-1 scFv binding MCF-7 possessed individual banding patterns representative of approximately 5 x 10(5) colonies likely able to recognize distinct epitopes present on MUC-1 positive human breast cancers . In summary, the anti-MUC-1 scFv antibody phage library contains diverse scFv molecules, which should provide unique characteristics and epitope recognition . These molecules will be used in the development of pretargeting RIT strategies designed to improve the clinical outcome of patients with breast cancer. Res Microbiol, 1999 Sep, 150(7), 457 - 63 Temperature-dependent flagellar antigen phase variation in Escherichia coli; Ratiner YA; A new kind of flagellar phase (H antigen) variation which is dependent on the temperature of growth is described for Escherichia coli strains, all but one of which belong to serogroup O148, isolated in different geographical regions . At 37 degrees C the strains simultaneously displayed two different H antigen specificities, H40 and H53, while in cultures grown at 30 degrees C only a single flagellar antigen, H53, was detected . It was shown that the bacteria possess two separate flagellin genes, fliC40 and flkA53 . An element controlling the temperature-dependent expression of fliC was localized in the region of flkA53 . Relevant problems in H antigen serotyping in E . coli are discussed. Science, 1999 Nov 26, 286(5445), 1724 - 8 Visualization of dioxygen bound to copper during enzyme catalysis; Wilmot CM et al.; X-ray crystal structures of three species related to the oxidative half of the reaction of the copper-containing quinoprotein amine oxidase from Escherichia coli have been determined . Crystals were freeze-trapped either anaerobically or aerobically after exposure to substrate, and structures were determined to resolutions between 2.1 and 2.4 angstroms . The oxidation state of the quinone cofactor was investigated by single-crystal spectrophotometry . The structures reveal the site of bound dioxygen and the proton transfer pathways involved in oxygen reduction . The quinone cofactor is regenerated from the iminoquinone intermediate by hydrolysis involving Asp383, the catalytic base in the reductive half-reaction . Product aldehyde inhibits the hydrolysis, making release of product the rate-determining step of the reaction in the crystal. Science, 1999 Nov 26, 286(5445), 1722 - 4 Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation; Sambongi Y et al.; F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient . An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis . The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor . These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis . Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1. Life Sci, 1999, 65(17), 1773 - 86 Differential effect of chronic antidepressant treatments on lipopolysaccharide-induced depressive-like behavioural symptoms in the rat; Shen Y et al.; In the present study we observed that lipopolysaccharide (LPS) administration provoked a characteristic reduction in body weight gain, food consumption, saccharin (but not water) consumption and nocturnal locomotor activity . It has been previously suggested that the ability of LPS to suppress the consumption of, and preference for, a palatable solution such as saccharin without altering water consumption, may represent an anhedonic response . The results of the present study demonstrate that chronic treatment with the tricyclic antidepressant (TCA) desipramine (7.5 mg/kg; i.p.) prevented LPS-induced anorexia, loss of body weight, the antidipsogenic effect and hypoactivity . In contrast, chronic treatment with the antidepressants paroxetine (7.5 mg/kg; i.p.) and venlafaxine (10 mg/kg; i.p.) failed to alter any of the LPS-induced behavioural responses . Furthermore, chronic treatment with desipramine (and to a lesser extent paroxetine) reduced the consumption of, and preference for, saccharin suggesting that these antidepressant treatments induce an "anhedonic" response in their own right . In conclusion, chronic desipramine treatment attenuated LPS-induced depressive-like behavioural symptoms in the rat . However, chronic treatment with paroxetine and venlafaxine did not significantly alter LPS-induced behavioural responses . The results of the present study support the hypothesis that TCA's may exert part of their anti-depressive efficacy through their effects on the immune system . However, this property does not appear to be shared by newer antidepressants which possess a better side effect profile than the TCA's . The suppressive effect of TCA's on proinflammatory cytokine secretion is discussed as a mechanism by which these agents alter LPS-induced behavioural responses. Biochimie, 1999 Nov, 81(11), 1015 - 23 In vitro assembly of yeast 5S rRNA and a fusion protein containing ribosomal protein L5 and maltose binding protein; Pakhomova ON et al.; Binding of yeast ribosomal protein L5 with 5S rRNA has long been considered a promising model for studying molecular mechanisms of protein-RNA interactions . However, in vitro assembly of a ribonucleoprotein (RNP) complex from purified yeast ribosomal protein L5 (also known as L1, L1a, or YL3) and 5S rRNA proved to be difficult, thus limiting the utility of this model . In the present report, we present data on the successful in vitro assembly of a RNP complex using a fusion (MBP-L5) protein consisting of the yeast ribosomal protein L5 fused to the carboxyl terminus of the E . coli maltose-binding protein (MBP) . We demonstrated that: 1) the MBP-L5 protein binds yeast 5S rRNA but not 5.8S rRNA in vitro; 2) the MBP protein itself does not bind yeast 5S rRNA; 3) formation of the RNP complex is proportional to the concentration of MBP-L5 protein and 5S rRNA; and 4) the MBP moiety of the fusion protein in the RNP complex can be removed with factor Xa . The electrophoretic mobility of the resultant RNP complex is indistinguishable from that of L5-5S rRNA complex isolated from the ribosome . Using this new experimental approach, we further showed that the RNA binding capability of a mutant L5 protein is decreased by 60% compared to the wild-type protein . Additionally, the mutant RNP complex migrates slower than the wild-type RNP complex suggesting that the mutant RNP complex has a less compact conformation . The finding provides a probable explanation for an earlier observation that the 60S ribosomal subunit containing the mutant protein is unstable. Biochimie, 1999 Oct, 81(10), 995 - 1002 Initiation of Escherichia coli ribosomes on matrix coupled mRNAs studied by optical biosensor technique; Karlsson M et al.; The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, has been used to study the initiation of protein synthesis by E . coli ribosomes on surface coupled mRNA . mRNA was first periodate oxidized and then hydrazide coupled to the surface of a CM5 sensor chip . The formation of initiation complexes on the surface coupled mRNA was monitored in real-time with a BIACORE 2000 instrument . Mature 70S*mRNA*fMet-tRNA(Met) initiation complexes were assembled on mRNA by sequential introduction of the 30S and 50S subunits supplemented with appropriate initiation factors and fMet-tRNA(Met) . We show that the formation of 70S*mRNA complexes on the surface coupled mRNA proceeds efficiently only in the presence of tRNA . Moreover, 70S*mRNA*fMet-tRNA(Met) complexes formed with fMet-tRNA(Met) are more stable than similar complexes formed with deacylated tRNAs . The efficient formation and slow dissociation of mature 70S*mRNA*fMet-tRNA(Met) initiation complexes are most easily explained by the stabilization of the interaction of the ribosomal subunits by fMet-tRNA(Met) . This work demonstrates the feasibility of the BIACORE technique for studying the initiation of protein synthesis. Biochimie, 1999 Oct, 81(10), 973 - 80 Isoleucine 10 is essential for DNA gyrase B function in Escherichia coli; Brino L et al.; DNA gyrase is an essential enzyme that regulates the DNA topology in bacteria . It belongs to the type II DNA topoisomerase family and is responsible for the introduction of negative supercoils into DNA at the expense of hydrolysis of ATP molecules . The aim of the present work was to study the contribution of I10, one of the most important residues responsible for the stabilization of GyrB dimer and involved in the ATP-binding step, in the ATP-hydrolysis reaction and in the DNA supercoiling mechanism . We constructed MBP-tagged GyrB mutants I10G and Delta4-14 . Our results demonstrate that both mutations severely affect the DNA-dependent ATPase activity and DNA supercoiling . Mutation of Y5 residue involved in the formation of ATPase catalytic site (Y5G mutant) had only little effect on the DNA-dependent ATPase activity and DNA supercoiling . Interestingly, the DNA-relaxation activity of MBP-GyrB mutants and wild type was completely inhibited by ATP . Binding of ADPNP to MBP-tagged mutants was significantly decreased . ADPNP had no effect on DNA-relaxation activity of MBP-tagged mutants but was able to inhibit MBP-tagged wild type enzyme . Our results demonstrate that GyrB N-terminal arm, and specially I10 residue is essential for ATP binding/hydrolysis efficiency and DNA transfer through DNA gyrase. J Biol Chem, 1999 Dec 3, 274(49), 35139 - 46 Expression of alpha2,8/2,9-polysialyltransferase from Escherichia coli K92 . Characterization of the enzyme and its reaction products; Shen GJ et al.; The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages . The enzyme catalyzing this polymerizing reaction has been cloned from the genomic DNA of E . coli K92 . The 1.2-kilobase polymerase chain reaction fragment was subcloned in pRSET vector and the protein was expressed in the BL21(DE3) strain of E . coli with a hexameric histidine at its N-terminal end . The enzyme was isolated in the supernatant after lysis of the cells and fractionated by ultracentrifugation . Western blotting using anti-histidine antibody showed the presence of a band that migrated at about 47.5 kDa on both reducing and nonreducing SDS-polyacrylamide gel electrophoresis, indicating a monomeric enzyme . Among the carbohydrate acceptors tested, N-acetylneuraminic acid and the gangliosides G(D3) and G(Q1b) were preferred substrates . The cell-free enzyme reaction products obtained were characterized by NMR and mass spectrometry, which indicated the presence of both alpha2,9- and alpha2,8-linked polysialyl structure . The K92 neuS gene was used to transform the K1 strain of E . coli, the capsule of which contains only -8-NeuAcalpha2- linkages . Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages . Our results suggest that the neuS gene product of E . coli K92 catalyzes the synthesis of polysialic acid with alpha2,9- and alpha2,8-linkages in vitro and in vivo. J Biol Chem, 1999 Dec 3, 274(49), 35067 - 73 A homolog of old yellow enzyme in tomato . Spectral properties and substrate specificity of the recombinant protein; Strassner J et al.; A cDNA was isolated and characterized from a tomato shoot cDNA library, the deduced amino acid sequence of which exhibited similarity with yeast Old Yellow Enzymes (OYEs) and related enzymes of bacterial and plant origin . Sequence identity was particularly high with 12-oxophytodienoate 10,11-reductase (OPR) from Arabidopsis thaliana . The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein in Escherichia coli and was purified from bacterial extracts . The protein was found to be a flavoprotein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds, including 12-oxophytodienoic acid . Thus, the tomato enzyme was termed LeOPR . The catalytic efficiency of LeOPR was highest with N-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates . Photoreduction of the LeOPR-bound FMN resulted in the formation of a red, anionic semiquinone prior to the formation of the fully reduced flavin dihydroquinone . Spectroscopic characterization of LeOPR revealed the formation of charge transfer complexes upon titration with para-substituted phenolic compounds, a distinctive feature of the enzymes of the OYE family . The ligand binding properties were compared between LeOPR and OYE, and the findings are discussed with respect to structural differences between the active sites of OYE and LeOPR. J Biol Chem, 1999 Dec 3, 274(49), 34851 - 8 Variability among the sites by which curaremimetic toxins bind to torpedo acetylcholine receptor, as revealed by identification of the functional residues of alpha-cobratoxin; Antil S et al.; alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom, was produced recombinantly (ralpha-Cbtx) from Escherichia coli . It was indistinguishable from the snake toxin . Mutations at 8 of the 29 explored toxin positions resulted in affinity decreases for Torpedo receptor with DeltaDeltaG higher than 1.1 kcal/mol . These are R33E > K49E > D27R > K23E > F29A >/= W25A > R36A >/= F65A . These positions cover a homogeneous surface of approximately 880 A(2) and mostly belong to the second toxin loop, except Lys-49 and Phe-65 which are, respectively, on the third loop and C-terminal tail . The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites . When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in binding to muscular-type nicotinic acetylcholine receptor . These are Lys-23/Lys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and variable extent Trp-25/Trp-29 and Phe-29/Phe-32 . In addition, however, the short and long toxins display three major differences . First, Asp-38 is important in Ea in contrast to the homologous Glu-38 in alpha-Cbtx . Second, all of the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea . Third, the C-terminal tail may be specifically critical in alpha-Cbtx . Therefore, the functional sites of long and short curaremimetic toxins are not identical, but they share common features and marked differences that might reflect an evolutionary pressure associated with a great diversity of prey receptors. J Biol Chem, 1999 Dec 3, 274(49), 34832 - 7 Role of iron and superoxide for generation of hydroxyl radical, oxidative DNA lesions, and mutagenesis in Escherichia coli; Nunoshiba T et al.; We measured the generation of hydroxyl radical (OH(.)) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method . A specific signal corresponding to OH(.) generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected in the strain deficient in sodA sodB fur . We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload . The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain . The mutation spectrum in the strain was found to induce GC --> TA and AT --> CG transversions predominantly . The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport . Thus, excess iron and excess superoxide were responsible for OH(.) generation, oxidative DNA lesion formation, and hypermutability in E . coli. J Biol Chem, 1999 Dec 3, 274(49), 34745 - 50 Covalent labeling of adenylyl cyclase cytosolic domains with gamma-methylimidazole-2',5'-dideoxy-{gamma-(32)P}3'-ATP and the mechanism for P-site-mediated inhibition; Doronin S et al.; A truncated first cytosolic domain of type V adenylyl cyclase (VC(1)) and a truncated second cytosolic domain of type II adenylyl cyclase (IIC(2)) were used alone and in the readily reversible complex (VC(1).IIC(2)) to evaluate interactions with each other and with reversible and irreversible P-site ligands . Enzyme activity was used to assess formation and dissolution of VC(1).IIC(2) . The data suggest that binding of 2',5'-dideoxy-3'-ATP to VC(1) and IIC(2) prevented formation of VC(1).IIC(2) and that 2',5'-dideoxy-3'-ATP dissociation occurred slowly . To enable configuration specific cross-linking to the catalytic site, 2',5'-dideoxyadenosine 3'-{gamma-(1-methylimidazole)-triphosphate} (gamma-MetIm-2', 5'-dd-3'-ATP) and 2',5'-dd-adenosine 3'-(gamma-azidoanilido)-triphosphate (gamma-azidoanilido-2', 5'-dd-3'-ATP) were synthesized, the former also as its gamma-(32)P-labeled analog . gamma-Azidoanilido-2',5'-dd-3'-ATP exhibited an inhibitory potency comparable with that of 2', 5'-dd-3'-ATP . gamma-MetIm-2',5'-dd-{gamma-(32)P}3'-ATP labeled the individual VC(1) and IIC(2) domains comparably and covalently to approximately 20% within 1 h . Formation of VC(1).IIC(2) resulted in reduced labeling of VC(1) but enhanced labeling of IIC(2) . The data imply that formation of the catalytically active VC(1).IIC(2) complex affects the interaction of each domain with the 2', 5'-dd-3'-ATP, the binding of which also affects the interaction between the two cytosolic domains, leading to a pseudo-irreversible inhibition. J Biol Chem, 1999 Dec 3, 274(49), 34706 - 10 Matrix metalloproteinase homologues from Arabidopsis thaliana . Expression and activity; Maidment JM et al.; Five genes potentially encoding novel matrix metalloproteinases (MMPs) have been identified on the Arabidopsis thaliana data base . The predicted proteins have a similar domain structure to mammalian MMP-7, with a propeptide and catalytic domain but no C-terminal hemopexin-like domain . Four of the A . thaliana MMPs (At-MMPs) have a predicted C-terminal transmembrane domain . The At-MMPs are differentially expressed in flower, leaf, root, and stem tissues from 14-day-old plants . The cDNA for one of the At-MMPs (At1-MMP) was cloned and expressed in Escherichia coli . Following refolding and purification, the proenzyme At1-MMP was shown to undergo autolytic activation in the presence of an organomercurial with a concomitant decrease in M(r) . In contrast to this, trypsin-treatment led to the formation of an inactive product . The activated At1-MMP digested myelin basic protein, but was unable to digest gelatin or casein . Three peptide substrates for MMPs were also cleaved by At1-MMP . The enzyme activity of At1-MMP was inhibited by human tissue inhibitors of metalloproteinases 1 and 2 and the hydroxamate inhibitor BB-94. J Biol Chem, 1999 Dec 3, 274(49), 34539 - 42 Direct NMR observation of the thioredoxin-mediated reduction of the chloroplast NADP-malate dehydrogenase provides a structural basis for the relief of autoinhibition; Krimm I et al.; The chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) catalyzing the reduction of oxaloacetate into L-malate is regulated by light . Its activation results from the thioredoxin-mediated reduction of two disulfides, located, respectively, in N- and C-terminal sequence extensions typical of all NADP-dependent light-regulated forms . Site-directed mutagenesis studies and the resolution of the three-dimensional structure of the oxidized (inactive) Sorghum vulgare enzyme showed that the C-terminal Cys(365)-Cys(377) disulfide constrains the C-terminal extension to fold into the active site where it acts as an internal inhibitor . In the present study, two-dimensional proton NMR spectra of an engineered NADP-MDH rendered monomeric by a 33-amino acid deletion at the N terminus (38 kDa) revealed that a 15-amino acid-long C-terminal peptide (Ala(375) to C-terminal Val(389)) acquired an increased mobility upon reduction, allowing its direct sequence-specific NMR assignment . The location of the flexible peptide in the sequence suggests that the first part of the C-terminal peptide is still folded near the core of the enzyme, so that cysteines 365 and 377 remain in proximity to allow for an efficient reoxidation/inactivation of the enzyme. Structure Fold Des, 1999 Nov 15, 7(11), 1395 - 406 Crystal structure of Escherichia coli PurE, an unusual mutase in the purine biosynthetic pathway; Mathews II et al.; BACKGROUND: Conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins - PurK and PurE . PurE has recently been shown to be a mutase that catalyzes the unusual rearrangement of N(5)-carboxyaminoimidazole ribonucleotide (N(5)-CAIR), the PurK reaction product, to CAIR . PurEs from higher eukaryotes are homologous to E . coli PurE, but use AIR and CO(2) as substrates to produce CAIR directly . RESULTS: The 1.50 A crystal structure of PurE reveals an octameric structure with 422 symmetry . A central three-layer (alphabetaalpha) sandwich domain and a kinked C-terminal helix form the folded structure of the monomeric unit . The structure reveals a cleft at the interface of two subunits and near the C-terminal helix of a third subunit .