Biochemistry, 2001 May 29, 40(21), 6326 - 34 Crystal structures of KDOP synthase in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor; Asojo O et al.; The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K(d) = 0.4 microM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 A resolution, respectively . Both the KDOPS.PEP and KDOPS.inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 A, respectively, and one subunit per asymmetric unit . The two structures are nearly identical, and superposition of their Calpha atoms indicates an rms difference of 0.41 A . The PEP in the KDOPS.PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts . This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face . Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme . The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP . The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P. Biochemistry, 2001 May 29, 40(21), 6319 - 25 Role of charged residues at the OmpF porin channel constriction probed by mutagenesis and simulation; Phale PS et al.; The channel constriction of OmpF porin, a pore protein in the bacterial outer membrane, is highly charged due to the presence of three arginines (R42, R82, and R132) and two acidic residues (D113 and E117) . The influence of these charges on ion conductance, ion selectivity, and voltage gating has been studied with mutants D113N/E117Q, R42A/R82A/R132A/D113N/E117Q, and V18K/G131K, which were designed to remove or add protein charge at the channel constriction . The crystal structures revealed no or only local changes compared to wild-type OmpF, thus allowing a comparative study . The single-channel conductance of the isosteric D113N/E117Q variant was found to be 2-fold reduced, and that of the pentuple mutant was 70% of the wild-type value, despite a considerably larger pore cross section . Ion selectivity was drastically altered by the mutations with cation/anion permeability ratios ranging from 1 to 12 . Ion flow through these and eight other mutants, which have been characterized previously, was simulated by Brownian dynamics based on the detailed crystal structures . The calculated ion selectivity and relative channel conductance values agree well with the experimental data . This demonstrates that ion translocation through porin is mainly governed by pore geometry and charge, the two factors that are properly represented in the simulations. Biochemistry, 2001 May 29, 40(21), 6216 - 26 Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: roles for aspartate 120 and glutamate 28; Trimmer EE et al.; The flavoprotein Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) . The X-ray crystal structure of the enzyme has revealed the amino acids at the flavin active site that are likely to be relevant to catalysis . Here, we have focused on two conserved residues, Asp 120 and Glu 28 . The presence of an acidic residue (Asp 120) near the N1-C2=O position of the flavin distinguishes MTHFR from all other known flavin oxidoreductases and suggests an important function for this residue in modulating the flavin reactivity . Modeling of the CH(3)-H(4)folate product into the enzyme active site also suggests roles for Asp 120 in binding of folate and in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis . In the NADH-menadione oxidoreductase assay and in the isolated reductive half-reaction, the Asp120Asn mutant enzyme is reduced by NADH 30% more rapidly than the wild-type enzyme, which is consistent with a measured increase in the flavin midpoint potential . Compared to the wild-type enzyme, the mutant showed 150-fold decreased activity in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction and in the oxidative half-reaction involving CH(2)-H(4)folate, but the apparent K(d) for CH(2)-H(4)folate was relatively unchanged . Our results support a role for Asp 120 in catalysis of folate reduction and perhaps in stabilization of the 5-iminium cation . By analogy to thymidylate synthase, which also uses CH(2)-H(4)folate as a substrate, Glu 28 may serve directly or via water as a general acid catalyst to aid in 5-iminium cation formation . Consistent with this role, the Glu28Gln mutant was unable to catalyze the reduction of CH(2)-H(4)folate and was inactive in the physiological oxidoreductase reaction . The mutant enzyme was able to bind CH(3)-H(4)folate, but reduction of the FAD cofactor was not observed . In the NADH-menadione oxidoreductase assay, the mutant demonstrated a 240-fold decrease in activity. Biochemistry, 2001 May 29, 40(21), 6205 - 15 Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies; Trimmer EE et al.; The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents . The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor . Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV . We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities . In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions . We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism . Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction . In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction . We conclude that studies of individual half-reactions catalyzed by E . coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions. J Theor Biol, 2001 May 21, 210(2), 237 - 48 DNA repair mechanism by photolyase: electron transfer path from the photolyase catalytic cofactor FADH(-) to DNA thymine dimer; Medvedev D et al.; Photolyase is an enzyme that catalyses photorepair of thymine dimers in UV damaged DNA by electron transfer reaction . The structure of the photolyase/DNA complex is unknown at present . Using crystal structure coordinates of the substrate-free enzyme from E . coli, we have recently built a computer molecular model of a thymine dimer docked to photolyase catalytic site and studied molecular dynamics of the system . In this paper, we present analysis of the electronic coupling and electron transfer pathway between the catalytic cofactor FADH(-) and the pyrimidine dimer by the method of interatomic tunneling currents . Electronic structure is treated in the extended Huckel approximation . The root mean square transfer matrix element is about 6 cm(-1), which is consistent with the experimentally determined rate of transfer . We find that electron transfer mechanism responsible for the repair utilizes an unusual folded conformation of FADH(-) in photolyases, in which the isoalloxazine ring of the flavin and the adenine are in close proximity, and the peculiar features of the docked orientation of the dimer . The tunneling currents show explicitly that despite of the close proximity between the donor and acceptor complexes, the electron transfer mechanism between the flavin and the thymine bases is not direct, but indirect, with the adenine acting as an intermediate . These calculations confirm the previously made conclusion based on an indirect evidence for such mechanism . J Mol Biol, 2001 Jun 1, 309(2), 477 - 89 Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli; Snijder HJ et al.; Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of phospholipids . Enzymatic activity is regulated by reversible dimerisation and calcium-binding . We have investigated the role of calcium by X-ray crystallography . In monomeric OMPLA, one calcium ion binds between two external loops (L3L4 site) at 10 A from the active site . After dimerisation, a new calcium-binding site (catalytic site) is formed at the dimer interface in the active site of each molecule at 6 A from the L3L4 calcium site . The close spacing and the difference in calcium affinity of both sites suggests that the L3L4 site may function as a storage site for a calcium ion, which relocates to the catalytic site upon dimerisation . A sequence alignment demonstrates conservation of the catalytic calcium site but evolutionary variation of the L3L4 site . The residues in the dimer interface are conserved as well, suggesting that all outer membrane phospholipases require dimerisation and calcium in the catalytic site for activity . For this family of phospholipases, we have characterised a consensus sequence motif (YTQ-X(n)-G-X(2)-H-X-SNG) that contains conserved residues involved in dimerisation and catalysis . Fresenius J Anal Chem, 2001 Apr, 369(7-8), 601 - 8 Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors; Watterson JH et al.; The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies . Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA . The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA . A stop-flow system of 137 microL volume permitted rapid introduction and mixing of each sample . Response times measured were on the order of seconds to minutes . Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases . The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution . The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides. Mol Genet Genomics, 2001 Mar, 265(1), 58 - 65 The ITR binding domain of the Mariner Mos-1 transposase; Auge-Gouillou C et al.; Mariner-like elements are widespread eukaryotic transposons, but Mos-1 is the only natural element that is known to be active . Little is known about the biochemistry of mariner transposition . The first step in the process is the binding of the transposase to the 5' and 3' inverted terminal repeats (ITRs) of the element . Using the 3' ITR of the element, we have determined the binding properties of a recombinant Mos-1 transposase produced in bacteria, and we have used deletion derivatives to localize the minimal ITR binding domain between amino acids 1 and 141 . Its features and structure indicate that it differs from the ITR binding domain of the transposase encoded by Tc1-related elements. Arch Biochem Biophys, 2001 Mar 15, 387(2), 180 - 7 A functional His-tagged c subunit of the Escherichia coli F-type ATPase/Synthase; Tomashek JJ et al.; The c subunit of the Escherichia coli F0 has been tagged with a hexahistidine motif at its C-terminus . The tagged subunit is capable of forming functional F0 complexes that translocate protons in the absence of the F1 complex . In the presence of F1, the two sectors associate and display all biochemical activities of the wildtype enzyme: DCCD-inhibitable ATPase activity, ATP synthase activity, and ATP-dependent proton pumping . The enzyme can be solubilized and purified as an intact complex under native conditions on immobilized-metal affinity chromatography (IMAC) resin . The purified complex can be reincorporated into liposomes and demonstrates ATP-dependent proton pumping activity . Hexahistine tags placed at the N-terminus, in contrast, were all inactive . These experiments demonstrate the feasibility of tagging the c subunit for further studies of the F0 and suggest an important role for the N-terminus of the c subunit in either assembly or function of the protein. J Exp Clin Cancer Res, 2001 Mar, 20(1), 111 - 5 Reporter gene transgenic mice as a tool for analyzing the molecular mechanisms underlying experimental carcinogenesis; Nishikawa A et al.; Carcinogenic compounds are classified into 2 categories, genotoxic and non-genotoxic, which are basically judged from in vitro genotoxicity data . However, it is well documented that genotoxicants do not necessarily exert in vivo carcinogenicity in rodents, partly because of a discrepancy between in vitro and in vivo mutagenicities . Recently, transgenic animal models with reporter genes such as lacI, lacZ and gpt have been developed as a tool for assessing in vivo mutagenicity as well as carcinogenicity . In this article, data using lacI transgenic mice and gpt delta mice are presented and their application is discussed . In lacI transgenic mice, dimethylnitrosamine (DMN) treatment significantly increased lacI mutant frequency (MF) in the liver, kidenys and lungs, but not in other non-target organs . Repeated dose ip administration of DMN was more effective than single dose treatment in the induction of lacI MF . The spectrum of mutant plaques induced by DMN was characterized by deletions as well as GC to AT base transitions . The remaining mice receiving DMN proved to have liver adenomas at a high frequency after 78 weeks . Meanwhile, dietary 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MelQx) significantly increased lacI and gpt MFs in the liver and colon . The characteristic spectrum of mutant plaques induced by MeIQx was a GC to TA base transversion in both the lacI and gpt mutations . Our results thus strongly suggest that these reporter gene transgenic animal models could offer a useful tool for analyzing molecular mechanisms underlying experimental carcinogenesis and for assessing the carcinogenic risk of environmental chemicals. Anim Biotechnol, 2001 May, 12(1), 1 - 19 Cloning of the full length pig PIT1 (POU1F1) CDNA and a novel alternative PIT1 transcript, and functional studies of their encoded proteins; Yu TP et al.; PIT1 is an essential regulatory gene of growth hormone (GH), prolactin (PRL) and thyrotropin beta subunit (TSHbeta) . Previously, a partial pig PIT1 cDNA and a genomic clone of the entire 3' end of the PIT1 gene was isolated, and polymorphisms at PIT1 were associated with several performance traits in the pig . In order to understand the biological function of the pig PIT1 gene and its possible application in swine genetics, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to complete the cloning of the full length cDNA for pig PIT1 . The pig PIT1 cDNA and its deduced protein sequence have approximately 90% and 95% identity, respectively, with the PIT1 cDNA and protein of other mammals (human, bovine, sheep and rodents) . Surprisingly, sequence comparison to other pig PIT1 sequences indicated only approximately 93% identity . Additional sequencing confirmed our sequence, and identified a new polymorphism in exon 4 . Phylogenetic analysis of several mammalian PIT1 sequences indicates sequencing errors may account for the discrepancies observed in the other pig sequences reported . Several PIT1 alternative spliced forms were also identified by RT-PCR . They were the delta3PIT1 (missing entire exon 3), delta4PIT1 (missing entire exon 4) and PIT1beta (additional 26 amino acids inserted in front of exon 2) transcripts . The delta4PIT1 and PIT1beta transcripts have been found to encode functionally different proteins in rodents . The delta3PIT1 transcript is a novel isoform of PIT1 . Potentially different functions between pig delta3PIT1 and PIT1 were analyzed by expressing these proteins in bacteria . The E . coli-expressed PIT1 and delta3PIT1 proteins were used with rat growth hormone (rGH) and rat prolactin (rPRL) promoter DNA in DNA mobility shift assays . The results showed that pig PIT1 can specifically bind rGH and rPRL promoter regions, but that the pig delta3PIT1 cannot, even at very high protein concentrations . Possible protein-protein interactions between delta3PIT1 and PIT1 were tested by mixing protein extracts before the gel shift assay, and the results showed that delta3PIT1 protein did not affect PIT1 binding to its target DNA . These data demonstrate the functionality of the PIT1 cDNA cloned in this study, and identify a novel delta3PIT1 transcript which encodes a protein that cannot bind rGH/rPRL target sequences. Arch Biochem Biophys, 2001 May 1, 389(1), 41 - 8 Modulation of the expression of peroxisome proliferator-activated receptor-dependent genes through disproportional expression of two subtypes in the small intestine; Mochizuki K et al.; We have reported that dietary long-chain triacylglycerols (LCT) enhance the transcription of cellular retinol-binding protein, the type II (CRBPII) gene, and the liver-type fatty acid-binding protein (L-FABP) gene in the small intestine . Because the cis elements on the CRBPII gene consisting of two AGGTCA motifs separated by a single nucleotide are known to bind not only the 9-cis-retinoic acid receptor (RXR) homodimer, but also the peroxisome proliferator-activated receptor (PPAR)-RXR heterodimer, it has been implicated that the unsaturated long-chain fatty acids, as the ligands of the PPAR, might activate the transcription of the CRBPII gene, thereby making use of the RXR-response elements (RXRE and RE3) as the PPAR-response element (PPRE) . In this study, we found that the PPARalpha mRNA level in the rat jejunum was elevated by dietary fat, whereas the PPARdelta mRNA level was reduced under this condition . Electrophoretic mobility-shift assay revealed that both PPARalpha-RXRalpha and PPARdelta-RXRalpha heterodimers, specifically and in a dose-dependent manner, bound to the two PPRE-like elements of the rat CRBPII gene as well as the known PPREs in the L-FABP and acyl-CoA oxidase genes . The binding of the PPARalpha-RXRalpha heterodimer to the CRBPII-RXRE, the CRBPII-RE3, and the PPREs of L-FABP, HMG-CoA synthase, and acyl-CoA oxidase was gradually diminished by the addition of increasing amounts of PPARdelta . The binding of the PPARdelta-RXRalpha heterodimer to CRBPII-RXRE, CRBPII-RE3, and other PPREs was also gradually reduced by the addition of increasing amounts of PPARalpha . Using Escherichia coli-expressed RXRalpha, we showed that the mutual competition for RXRalpha with PPARalpha and PPARdelta occurred at the protein level . These results suggest that the transcriptions of CRBPII, L-FABP, and the other PPAR-dependent genes in the small intestine may be coordinately regulated by the disproportional expression of PPARalpha and PPARdelta. Arch Biochem Biophys, 2001 May 1, 389(1), 31 - 40 Role of THR501 residue in substrate binding and catalytic activity of cytochrome P4501A1; Cvrk T et al.; A putative binding region for cumene hydroperoxide in the active site of cytochrome P4501A1 was identified using photoaffinity labeling . Thr501 was determined as the most likely site of modification by azidocumene used as the photoaffinity label (T . Cvrk and H . W . Strobel, (1998) Arch . Biochem . Biophys . 349, 95-104) . To evaluate further the role of this amino acid residue a site-directed mutagenesis approach was employed . P4501A1 wild type and two mutants, P4501A1Glu501 and P4501A1Phe501, were expressed in and purified from Escherichia coli and used for kinetic analysis to confirm the role of Thr501 residue in cumene hydroperoxide binding . The mutation resulted in a two- to fourfold decrease in the rate of heme degradation in the presence of 0.5 mM cumene hydroperoxide . The mutations do not prevent or significantly alter binding of the tested substrates; however, binding of 2-phenyl-2-propanol (product generated from cumene hydroperoxide) to P4501A1Glu501 and P4501A1Phe501 exhibited four- and eightfold decreases, respectively, suggesting that the mutations strongly affected the affinity of cumene hydroperoxide for the P4501A1 active site . The kinetic analysis of cumene hydroperoxide-supported reactions showed that both mutants exhibit increased Km and decreased VMax values for all tested substrates . Furthermore, the mutations affected product distribution in testosterone hydroxylation . On the basis of P4501A1Glu501 and P4501A1Phe501 characterization, it can be concluded that Thr501 plays an important role in cumene hydroperoxide/P4501A1 interaction. Arch Biochem Biophys, 2001 May 1, 389(1), 135 - 43 Expression, purification, and characterization of human type II arginase; Colleluori DM et al.; Human type II arginase, which is extrahepatic and mitochondrial in location, catalyzes the hydrolysis of arginine to form ornithine and urea . While type I arginases function in the net production of urea for excretion of excess nitrogen, type II arginases are believed to function primarily in the net production of ornithine, a precursor of polyamines, glutamate, and proline . Type II arginases may also regulate nitric oxide biosynthesis by modulating arginine availability for nitric oxide synthase . Recombinant human type II arginase was expressed in Escherichia coli and purified to apparent homogeneity . The Km of arginine for type II arginase is approximately 4.8 mM at physiological pH . Type II arginase exists primarily as a trimer, although higher order oligomers were observed . Borate is a noncompetitive inhibitor of the enzyme, with a Kis of 0.32 mM and a Kii of 0.3 mM . Ornithine, a product of the reaction catalyzed by arginase and a potent inhibitor of type I arginase, is a poor inhibitor of the type II isozyme . The findings presented here indicate that isozyme-selectivity exists between type I and type II arginases for binding of substrate and products, as well as inhibitors . Therefore, inhibitors with greater isozyme-selectivity for type II arginase may be identified and utilized for the therapeutic treatment of smooth muscle disorders, such as erectile dysfunction. Biotechnol Bioeng, 2001 Jul 20, 74(2), 89 - 95 Metabolic flux analysis for succinic acid production by recombinant Escherichia coli with amplified malic enzyme activity; Hong SH et al.; A pfl ldhA double mutant Escherichia coli strain NZN111 was used to produce succinic acid by overexpressing the E . coli malic enzyme . Escherichia coli strain NZN111 harboring pTrcML produced 6 and 8 g/L of succinic acid from 20 g/L of glucose in flask culture at 37 degrees C and 30 degrees C, respectively . When NZN111(pTrcML) was cultured at 30 degrees C with intermittent glucose feeding the final succinic acid concentration obtained was 9.5 g/L and the ratio of succinic acid to acetic acid was 13:1 . This system could not be analyzed by conventional metabolic flux analysis techniques, since some pyruvate and succinic acid were accumulated intracellularly . Therefore, a new flux analysis method was proposed by introducing intracellular pyruvate and succinic acid pools . By this new method the concentrations of intracellular metabolites were successfully predicted and the differences between the measured and calculated reaction rates could be considerably reduced . Protein Sci, 2001 Jun, 10(6), 1206 - 15 The effect of net charge on the solubility, activity, and stability of ribonuclease Sa; Shaw KL et al.; The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values . Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues . By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2 . We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved . For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH . We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not. Protein Sci, 2001 Jun, 10(6), 1137 - 49 Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine; Krell T et al.; Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase . In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins . In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair . The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive . However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme . The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed . This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role . The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability . From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms. Protein Sci, 2001 Jun, 10(6), 1124 - 9 High-resolution crystal structure of apolipoprotein(a) kringle IV type 7: insights into ligand binding; Ye Q et al.; Apolipoprotein(a) {apo(a)} consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively . Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV(1) to KIV(10)) followed by sequences similar to the kringle V and protease domains of plasminogen . The KIV domains of apo(a) differ with respect to their ability to bind lysine or lysine analogs . KIV(10) represents the high-affinity lysine-binding site (LBS) of apo(a); a weak LBS is predicted in each of KIV(5)-KIV(8) and has been directly demonstrated in KIV(7) . The present study describes the first crystal structure of apo(a) KIV(7), refined to a resolution of 1.45 A, representing the highest resolution for a kringle structure determined to date . A critical substitution of Tyr-62 in KIV(7) for the corresponding Phe-62 residue in KIV(10), in conjunction with the presence of Arg-35 in KIV(7), results in the formation of a unique network of hydrogen bonds and electrostatic interactions between key LBS residues (Arg-35, Tyr-62, Asp-54) and a peripheral tyrosine residue (Tyr-40) . These interactions restrain the flexibility of key LBS residues (Arg-35, Asp-54) and, in turn, reduce their adaptability in accommodating lysine and its analogs . Steric hindrance involving Tyr-62, as well as the elimination of critical ligand-stabilizing interactions within the LBS are also consequences of this interaction network . Thus, these subtle yet critical structural features are responsible for the weak lysine-binding affinity exhibited by KIV(7) relative to that of KIV(10). Protein Sci, 2001 Jun, 10(6), 1100 - 12 NMR and SAXS characterization of the denatured state of the chemotactic protein CheY: implications for protein folding initiation; Garcia P et al.; The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance . SAXS studies show that the denatured protein follows a wormlike chain model . Its backbone can be described as a chain composed of rigid elements connected by flexible links . A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that approximately 25% of the residues are involved in residual structures . Conformational shifts of the alpha-protons, heteronuclear (15)N-{(1)H} NOEs and (15)N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior . According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein . The first of these subdomains, spanning residues 1-70, is shown here to exhibit a restricted mobility as compared to the rest of the protein . Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts . Peptides corresponding to these sequences were characterized by NMR and their backbone (1)H chemical shifts were compared to those in the intact protein under identical denaturing conditions . For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule . The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state. J Biol Chem, 2001 Jul 20, 276(29), 27371 - 5 Epub 2001 May 21. Molecular cloning of the CA125 ovarian cancer antigen: identification as a new mucin, MUC16; Yin BW et al.; CA125 is an ovarian cancer antigen that is the basis for a widely used serum assay for the monitoring of patients with ovarian cancer; however, detailed information on its biochemical and molecular nature is lacking . We now report the isolation of a long, but partial, cDNA that corresponds to the CA125 antigen . A rabbit polyclonal antibody produced to purified CA125 antigen was used to screen a lambdaZAP cDNA library from OVCAR-3 cells in Escherichia coli . The longest insert from the 54 positive isolated clones had a 5797-base pair sequence containing a stop codon and a poly(A) sequence but no clear 5' initiation sequence . The deduced amino acid sequence has many of the attributes of a mucin molecule and was designated CA125/MUC16 (gene MUC16) . These features include a high serine, threonine, and proline content in an N-terminal region of nine partially conserved tandem repeats (156 amino acids each) and a C-terminal region non-tandem repeat sequence containing a possible transmembrane region and a potential tyrosine phosphorylation site . Northern blotting showed that the level of MUC16 mRNA correlated with the expression of CA125 in a panel of cell lines . The molecular cloning of the CA125 antigen will lead to a better understanding of its role in ovarian cancer. J Biol Chem, 2001 Jul 27, 276(30), 28380 - 7 Epub 2001 May 21. Identification of an RNA-binding Site in the ATP binding domain of Escherichia coli Rho by H2O2/Fe-EDTA cleavage protection studies; Wei RR et al.; Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis . The Rho polypeptide has a distinct RNA binding domain of known structure as well as an ATP binding domain for which a structure has been proposed based on homology modeling . Treatment of Rho with H2O2 in the presence of Fe-EDTA caused single-cut cleavage at a number of points that coincide with solvent-exposed loops in both the known and predicted structures, thereby providing support for the validity of the tertiary and quaternary structural models of Rho . The binding of ATP caused one distinct change in the cleavage pattern, a strong protection at a cleavage point in the P-loop of the ATP binding domain . Binding of RNA and single-stranded DNA (poly(dC)) caused strong protection at several accessible parts of the oligosaccharide/oligonucleotide binding (OB) fold in the RNA binding domain . RNA molecules but not DNA molecules also caused a strong, ATP-dependent protection at a cleavage site in the predicted Q-loop of the ATP binding domain . These results suggest that Rho has two distinct binding sites for RNA . Besides the site composed of multiples of the RNA binding domain, to which single-stranded DNA as well as RNA can bind, it has a separate, RNA-specific site on the Q-loop in the ATP binding domain . In the proposed quaternary structure of Rho, the Q-loops from the six subunits form the upper entrance to the hole in the ring-shaped hexamer through which the nascent transcript is translocated by actions coupled to ATP hydrolyses. J Biol Chem, 2001 Jul 27, 276(30), 27787 - 92 Epub 2001 May 21. Identification of the amino acid residue involved in rabbit hemorrhagic disease virus VPg uridylylation; Machin A et al.; The virus genome-linked protein (VPg) coding region from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was expressed in Escherichia coli by using a glutathione S-transferase-based vector . The recombinant polypeptide could be purified in good yields and was uridylylated in vitro from {alpha-32P}UTP in a reaction catalyzed by the recombinant RNA-dependent RNA polymerase from RHDV in the absence of added template RNA . The use of deletion and point mutants allowed the identification of Tyr-21 as the residue involved in uridylylation and consequently in the linkage between VPg and the viral genome . These data constitute the first report on the identity of the amino acid residue involved in VPg uridylylation in a member of the Caliciviridae family. Jpn J Ophthalmol, 2001 May-Jun, 45(3), 221 - 6 Effects of alpha(2)-adrenergic agonists on lipopolysaccharide-induced aqueous flare elevation in pigmented rabbits; Watanabe K et al.; PURPOSE: To evaluate the effects of the alpha(2)-adrenergic agonists (clonidine, apraclonidine, and guanfacine) on lipopolysaccharide (LPS)-induced aqueous flare elevation in pigmented rabbits . METHODS: Anterior uveitis was induced with an intravenous injection of LPS (0.5 microg/kg) in an ear vein . The reproducibility of experimental uveitis induced by LPS (0.5 microg/kg) was also determined . Clonidine (0.01, 0.05, 0.25, or 1%), apraclonidine (1%), or guanfacine (1%) was topically instilled in the right eye 30 and 5 minutes before and 30 minutes after LPS application (N = 6 animals, respectively) . Clonidine (0.25%) was topically administered three times at 30-minute intervals from 240 or 120 minutes before, or 120 or 240 minutes after LPS application (N = 6 animals, respectively) . Then 1 mg/kg of yohimbine was injected into an ear vein 30 minutes before each topical three-time instillation of clonidine 1%, apraclonidine 1% or guanfacine 1% (N = 6 animals, respectively) . Aqueous flare was measured with a laser flare-cell meter . Aqueous flare elevation was expressed as the area under the curve (AUC) in arbitrary units . Rabbits received the first LPS intravenous injection, and the control values of the AUC were obtained . Three months later, the alpha(2)-agonist and the second LPS administration were given to the same animals . RESULTS: The AUCs (5,184 +/- 1,255 units) after the first application of LPS were similar to those (5,033 +/- 1,290) after the second application 3 months after the first administration . Topical instillation of clonidine inhibited LPS-induced aqueous flare elevation in a dose-dependent manner (0.01-0.25%) . Topical instillation of clonidine 1%, apraclonidine 1% or guanfacine 1% inhibited LPS-induced aqueous flare elevation by 98 +/- 2.0% (mean +/- SD), 86 +/- 14% and 94 +/- 5.7%, respectively . Pretreatment with intravenous yohimbine prevented the inhibitory effect on flare elevation induced by each agent . CONCLUSION: The present findings suggested that topical instillation of some alpha(2)-agonists may have an inhibitory effect on ocular inflammation, which is mediated in part by alpha(2)-receptors. Jpn J Ophthalmol, 2001 May-Jun, 45(3), 216 - 20 Effects of scutellariae radix extract and its components (baicalein, baicalin, and wogonin) on the experimental elevation of aqueous flare in pigmented rabbits; Nagaki Y et al.; PURPOSE: To evaluate the possible inhibitory effects of hot water extract of Scutellariae radix and its major components (baicalein, baicalin, and wogonin) on experimental elevation of aqueous flare in pigmented rabbits . METHODS: To produce aqueous flare elevation in rabbits, prostaglandin E(2) (PGE(2)), 25 microg/mL, was applied to the cornea with the use of a glass cylinder, or lipopolysaccharides (LPS), 0.5 microg/kg, were injected into an ear vein . Animals were pretreated by the oral administration of 150 g/day of food containing 0.02%, 0.07%, or 0.2% (w/w) extract of Scutellariae radix for 5 days, or by intravenous injection of baicalein, baicalin, or wogonin, 60 microg/kg or 600 microg/kg, 30 minutes before experimental uveitis was induced . Aqueous flare was measured with a laser flare-cell meter . Aqueous flare intensity was expressed as the area under the curve (AUC) in arbitrary units . RESULTS: The AUC of PGE(2)- and LPS-induced aqueous flare elevation was 1,343 and 5,066 arbitrary units, respectively . Pretreatment by oral administration of 0.07% or 0.2% extract of Scutellariae radix did not inhibit PGE(2)-induced aqueous flare elevation (AUC: 1,252 and 1,210, respectively), but it did inhibit LPS-induced aqueous flare elevation (AUC: 2,248 and 1,973, respectively) . Pretreatment by intravenous injection of 600 microg/kg of baicalein, baicalin, or wogonin inhibited LPS-induced aqueous flare elevation (AUC: 2,289, 2,163, and 1,509, respectively) . Pretreatment with 60 microg/kg of wogonin also inhibited LPS-induced aqueous flare elevation (AUC: 1,980) . CONCLUSION: Hot water extract of Scutellariae radix may have an inhibitory effect on experimental anterior uveitis induced by LPS in pigmented rabbits. Curr Biol, 2001 May 1, 11(9), R364 - 6 Channel gating: twist to open; Biggin PC et al.; Recent studies of the bacterial mechanosensitive channel MscL have combined a number of different approaches to come up with a model for the channel gating mechanism. Biochem J, 2001 Jun 1, 356(Pt 2), 605 - 12 Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential; Pflanz S et al.; Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines . Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood . On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6 . Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains . The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy . For the first time the prediction of three individual membrane-proximal protein domains for gp130 has been verified experimentally . The three domains do not show intermediate-affinity or high-affinity interactions between each other . Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4. Biochem J, 2001 Jun 1, 356(Pt 2), 433 - 44 Control of the threonine-synthesis pathway in Escherichia coli: a theoretical and experimental approach; Chassagnole C et al.; A computer simulation of the threonine-synthesis pathway in Escherichia coli Tir-8 has been developed based on our previous measurements of the kinetics of the pathway enzymes under near-physiological conditions . The model successfully simulates the main features of the time courses of threonine synthesis previously observed in a cell-free extract without alteration of the experimentally determined parameters, although improved quantitative fits can be obtained with small parameter adjustments . At the concentrations of enzymes, precursors and products present in cells, the model predicts a threonine-synthesis flux close to that required to support cell growth . Furthermore, the first two enzymes operate close to equilibrium, providing an example of a near-equilibrium feedback-inhibited enzyme . The predicted flux control coefficients of the pathway enzymes under physiological conditions show that the control of flux is shared between the first three enzymes: aspartate kinase, aspartate semialdehyde dehydrogenase and homoserine dehydrogenase, with no single activity dominating the control . The response of the model to the external metabolites shows that the sharing of control between the three enzymes holds across a wide range of conditions, but that the pathway flux is sensitive to the aspartate concentration . When the model was embedded in a larger model to simulate the variable demands for threonine at different growth rates, it showed the accumulation of free threonine that is typical of the Tir-8 strain at low growth rates . At low growth rates, the control of threonine flux remains largely with the pathway enzymes . As an example of the predictive power of the model, we studied the consequences of over-expressing different enzymes in the pathway. Biochem J, 2001 Jun 1, 356(Pt 2), 425 - 32 Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics; Rais B et al.; We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions . The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH . In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH . A stoichiometric conversion of precursors into threonine was observed . We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell . The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions . A response to threonine is only observed in a narrow concentration range from 0.23 to 2 mM. Biochem J, 2001 Jun 1, 356(Pt 2), 415 - 23 An integrated study of threonine-pathway enzyme kinetics in Escherichia coli; Chassagnole C et al.; We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant . Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g . near-equilibrium reactions), as substrates of the reverse reactions . Co-operative feedback inhibition by threonine and lysine was also included as necessary . We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions. Biochem J, 2001 Jun 1, 356(Pt 2), 403 - 14 Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione; Cheng H et al.; Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue . A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene . The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1 . Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference . A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs . The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active . A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4 . Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices . Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver . This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress. Chem Res Toxicol, 2001 May, 14(5), 483 - 91 Influence of P450 3A4 SRS-2 residues on cooperativity and/or regioselectivity of aflatoxin B(1) oxidation; Xue L et al.; The major human liver drug-metabolizing cytochrome P450 enzymes P450 3A4 and P450 3A5 share >85% amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B(1) (AFB(1)) biotransformation {Gillam et al . (1995) Arch . Biochem . Biophys . 317, 74-384} . P450 3A4 prefers AFB1 3alpha-hydroxylation, which detoxifies and subsequently eliminates the hepatotoxin, over AFB1 exo-8,9-oxidation . P450 3A5, on the other hand, is a relatively sluggish 3alpha-hydroxylase and converts AFB(1) predominantly to the genotoxic exo-8,9-epoxide . Using a combination of approaches (sequence alignment, homology modeling and site-directed mutagenesis), we have previously identified several divergent residues in four of the six putative substrate recognition sites (SRSs) of P450 3A4, which when replaced individually with the corresponding amino acid of P450 3A5, resulted in a significant switch of the characteristic P450 3A4 AFB(1) regioselectivity toward that of P450 3A5 {Wang et al . (1998) Biochemistry 37, 12536-12545} . In particular, residues N206 and L210 in SRS-2 were found to be critical for AFB(1) detoxification via 3alpha-hydroxylation, and the corresponding mutants N206S and L210F most closely mimicked P450 3A5, not only in its regioselectivity of AFB(1) metabolism but also in its overall functional capacity . We have now further explored the plausible reasons for such relative inactivity of the SRS-2 mutants by examining N206S and additional mutants (L210A, L211F, L211A, and N206E) and found that the dramatically lowered activities of the N206S mutant are accompanied by a loss of cooperativity of AFB(1) oxidation . Molecular dynamics analyses with an existing P450 3A4 homology model {Szklarz and Halpert (1997) J . Comput . Aided Mol . Des . 11, 265} suggested that N206 (helix F) interacts with E244 (helix G), creating a salt bridge that stabilizes the protein structure and/or defines the active site cavity . To examine this possibility, several E244 mutants (E244A, V, N, S) were tested, of which E244S was the most notable for its relatively greater impairment of P450 3A4-dependent AFB(1) 3alpha-hydroxylation . However, the results with these E244 mutants failed to validate the N206-E244 interaction predicted from these molecular dynamics analyses . Collectively, our findings to date have led us to reconsider our original interpretations and to reexamine them in the light of AFB(1) molecular modeling analyses with a newly refined P450 3A4 homology model . These analyses predicted that F304 in SRS-4 (I-helix) plays a pivotal role in AFB(1) binding at the active site in either orientation leading to 3alpha- or exo-8,9-oxidation . Consistent with this prediction, conversion of F304 to Ala abolished P450 3A4-dependent AFB(1) 3alpha-hydroxylation and exo-8,9-oxidation. Arch Biochem Biophys, 2001 Jun 1, 390(1), 109 - 18 Characterization of a galactose specific adhesin of enteroaggregative Escherichia coli; Grover V et al.; A fimbrial adhesin was identified from an enteroaggregative Escherichia coli strain . The adhesin was purified to 740-fold by sequential chromatography on an affinity matrix and gel filtration column in the FPLC system . The homogeneity of the purified protein was established by analytical isoelectrofocussing (pI 7.25) . The native adhesin appeared as a high-molecular-weight aggregative protein as revealed by gel filtration chromatography on Superose 12HR10/30 column . However, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the adhesin was found to be 18 kDa and this was further confirmed by gel filtration chromatography on Superose 6HR 10/30 column presence of 6 M guanidine hydrochloride . The N-terminal 15-amino-acid sequence of the adhesin did not show homology with any of the previously reported fimbrial adhesins . The purified adhesin showed adhesion to human erythrocytes in the presence of Ca(2+) (5 mM) . The optimum temperature and pH for the hemadhesion activity was found to be 25 degrees C and 6.5, respectively . The inhibition study clearly suggested that the binding site of the adhesin could recognize galactose as the specific sugar . The fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside was quenched on binding to the adhesin and maximum reversal of fluorescence quenching was observed by competitive substitution titration with raffinose . The adhesin was found to contain one binding site per monomer for its specific sugar residue . The association constant and the free energy of binding were obtained as 3.98 x 10(5) M(-1) and -31.97 kJ/mol, respectively . The adherence of the bacteria to HEp-2 monolayer was inhibited in presence of galactose and this was further supported by a significant reduction in the bacterial adherence to the HEp-2 cells, pretreated with beta-D-galactosidase . Arch Biochem Biophys, 2001 Jun 1, 390(1), 42 - 50 Overexpression, biophysical characterization, and crystallization of ribonuclease I from Escherichia coli, a broad-specificity enzyme in the RNase T2 family; Padmanabhan S et al.; We have constructed a strain that overproduces ribonuclease I of Escherichia coli and we have purified large quantities of the enzyme . Data from fluorescence, CD, and DSC measurements showed that it was a very stable protein . The conformation energy determined from urea and guanidine hydrochloride denaturation experiments was 11.5 kcal mol(-1) at pH 7.5 . Thermal denaturation studies indicated that it had a T(m) of 64 degrees C at pH 4.0 . RNase I belongs to the RNase T2/S-RNase group of endoribonucleases, but near the amino terminus it has an unusually long hydrophilic segment . Part of this was removed in the deletion construct, RNase I Delta(26-38) . We have obtained crystals of both RNase I and of an enzyme-G2'p5'G complex in the P2(1) space group and oligonucleotide complexes with both wild type and mutant enzymes . The current study lays the groundwork for extensive investigation into the structure, function, and physical properties of this widely distributed group of ribonucleases . Nutr Rev, 2001 Apr, 59(4), 116 - 8 The enzymatic cleavage of beta-carotene: end of a controversy; Wolf G; The central cleavage of dietary beta-carotene to retinal was found to be the predominant mechanism whereby retinoids were formed in vivo in rats; apo-carotenals, indicative of eccentric cleavage of beta-carotene, were only a minor component (<5% of retinoids) . A gene from maize that codes for a plant carotenoid cleavage enzyme was used to isolate a homologous gene from Drosophila . This gene, when transfected into an E Coli strain capable of synthesizing and accumulating beta-carotene, caused the central cleavage of beta-carotene, forming exclusively retinoids . The enzyme that the gene codes for, beta-carotene-15,15'-dioxygenase, was purified and characterized. Arch Biochem Biophys, 2001 Feb 15, 386(2), 281 - 9 Biogenesis of volatile aldehydes from fatty acid hydroperoxides: molecular cloning of a hydroperoxide lyase (CYP74C) with specificity for both the 9- and 13-hydroperoxides of linoleic and linolenic acids; Tijet N et al.; A novel member of the plant cytochrome P450 CYP74 family of fatty acid hydroperoxide metabolizing enzymes has been cloned from melon fruit (Cucumis melo) . The cDNA is comprised of 1,446 nucleotides encoding a protein of 481 amino acids . The homology at the amino acid level to other members of the CYP74 family is 35-50%, the closest relatives being allene oxide synthases . The cDNA was expressed in Escherichia coli, and the corresponding protein was purified by affinity column chromatography . The native enzyme showed a main Soret band at 418 nm, indicative of a low spin ferric cytochrome P450, and a 447-nm peak appeared in the CO-difference spectrum . Using {U-14C}radiolabeled substrate, HPLC, UV, and GC-MS, the products of conversion of 9S-hydroperoxy-linoleic acid were identified as 9-oxo-nonanic acid and 3Z-nonenal . Kinetic analysis of this hydroperoxide lyase showed the highest rate of reaction with 9-hydroperoxy-linolenic acid followed by 9-hydroperoxy-linoleic acid and then the corresponding 13-hydroperoxides . Overall, the newly characterized cytochrome P450 enzyme is a fatty acid hydroperoxide lyase with a preference, but not absolute specificity for the 9-positional hydroperoxides of linoleic and linolenic acids. Arch Biochem Biophys, 2000 Dec 15, 384(2), 407 - 12 Characterisation of a zeta class glutathione transferase from Arabidopsis thaliana with a putative role in tyrosine catabolism; Dixon DP et al.; A glutathione transferase (GST) similar to zeta GSTs in animals and fungi has been cloned from Arabidopsis thaliana using RT-PCR . The Arabidopsis zeta GST (AtGSTZ1) was expressed in Escherichia coli as his-tagged polypeptides, which associated together to form the 50-kDa AtGSTZ1-1 homodimer . Following purification, AtGSTZ1-1 was assayed for a range of activities and compared with other purified recombinant plant GSTs from the phi, tau, and theta classes . AtGSTZ1-1 differed from the other GSTs in showing no glutathione conjugating activity toward xenobiotics and no glutathione peroxidase activity toward organic hydroperoxides . Uniquely among the plant GSTs, AtGSTZ1-1 showed activity as a maleylacetone isomerase (MAI) . This glutathione-dependent reaction is analogous to the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate, which occurs in the course of tyrosine catabolism to acetoacetate and fumarate . Thus, rather than functioning as a conventional GST, AtGSTZ1-1 appears to be involved in tyrosine degradation . In addition to the MAI activity, the AtGSTZ1-1 also catalyzed the glutathione-dependent dehalogenation of dichloroacetic acid to glyoxylic acid . This latter activity was used to demonstrate the presence of functional AtGSTZ1-1 inplanta. Arch Biochem Biophys, 2001 Apr 15, 388(2), 299 - 307 Characterization of mutants of beta histidine91, beta aspartate213, and beta asparagine222, possible components of the energy transduction pathway of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli; Bragg PD et al.; The roles of three residues (betaHis91, betaAsp213, and betaAsn222) implicated in energy transduction in the membrane-spanning domain II of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli have been examined using site-directed mutagenesis . All mutations affected transhydrogenation and proton pumping activities, although to various extents . Replacing betaHis91 or betaAsn222 of domain II by the basic residues lysine or arginine resulted in occlusion of NADP(H) at the NADP(H)-binding site of domain III . This was not seen with betaD213K or betaD213R mutants . It is suggested that betaHis91 and betaAsn222 interact with betaAsp392, a residue probably involved in initiating conformational changes at the NADP(H)-binding site in the normal catalytic cycle of the enzyme (M . Jeeves et al . (2000) Biochim . Biophys . Acta 1459, 248-257) . The introduced positive charges in the betaHis91 and betaAsn222 mutants might stabilize the carboxyl group of betaAsp392 in its anionic form, thus locking the NADP(H)-binding site in the occluded conformation . In comparison with the nonmutant enzyme, and those of mutants of betaAsp213, most mutant enzymes at betaHis91 and betaAsn222 bound NADP(H) more slowly at the NADP(H)-binding site . This is consistent with the effect of these two residues on the binding site . We could not demonstrate by mutation or crosslinking or through the formation of eximers with pyrene maleimide that betaHis91 and betaAsn222 were in proximity in domain II. Arch Biochem Biophys, 2001 Jan 15, 385(2), 392 - 6 Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase; Gakh O et al.; Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit . Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment . Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix . We conclude this as a consequence of a possible dimeric (oligomeric) structure. Arch Biochem Biophys, 2001 Jan 15, 385(2), 372 - 7 Tyrosine residue 300 is important for activity and stability of branching enzyme from Escherichia coli; Mikkelsen R et al.; Branching enzyme belongs to the alpha-amylase family, which includes enzymes that catalyze hydrolysis or transglycosylation at alpha-(1,4)- or alpha-(1,6)-glucosidic linkages . In the alpha-amylase family, four highly conserved regions are proposed to make up the active site . From amino acid sequence analysis a tyrosine residue is completely conserved in the alpha-amylase family . In Escherichia coli branching enzyme, this residue (Y300) is located prior to the conserved region 1 . Site-directed mutagenesis of the Y300 residue in E . coli branching enzyme was used in order to study its possible function in branching enzymes . Replacement of Y300 with Ala, Asp, Leu, Ser, and Trp resulted in mutant enzymes with less than 1% of wild-type activity . A Y300F substitution retained 25% of wild-type activity . Kinetic analysis of Y300F showed no effect on the Km value . The heat stability of Y300F was analyzed, and this was lowered significantly compared to that of the wild-type enzyme . Y300F also showed lower relative activity at elevated temperatures compared to wild-type . Thus, these results show that Tyr residue 300 in E . coli branching enzyme is important for activity and thermostability of the enzyme. Arch Biochem Biophys, 2001 Jan 15, 385(2), 332 - 7 An additional serine residue at the C terminus of rhodanese destabilizes the enzyme; Kramer G et al.; The rhodanese coding sequence was extended at its 3' end by three base pairs to generate mutants coding for a serine or arginine residue at the carboxyl terminus of the protein . Wild-type and mutant coding sequences were expressed in a cell-free Escherichia coli system by coupled transcription/translation . Predominantly full-length protein was formed in all cases . The amount of protein synthesized was quantified by incorporation of radioactive leucine into polypeptides . Enzymatic activity of in vitro synthesized rhodanese was determined at different temperatures . Specific enzymatic activity was calculated and is assumed to reflect the portion of the protein that is in its native three-dimensional conformation . It was observed that rhodanese extended by one serine at the C terminus lost enzymatic activity when incubated above 30 degrees C, in contrast to wild-type protein or variant rhodanese extended by an arginine residue . Similarly, variant rhodanese with an additional serine residue was more susceptible to urea denaturation than the other two rhodanese species . These results are surprising in light of the crystal structure of the protein. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1998 Jun, 20(3), 185 - 90 {Interleukin-6 gene cloning, expression and purification}; Ma X et al.; OBJECTIVE: The main purpose of this paper is to study the batch production of recombinant human interleukin-6(rhIL-6) . METHODS: The cloned rhIL-6 gene is under the control of T7 promoter of pET30a vector and expressed in E . coli . RESULTS: The ratio of expressed recombinant protein to total cell protein is more than 50% . The rhIL-6 molecular weight is 21,000, isoelectric point is 6.7 . The purity of the rhIL-6 is more than 95%, and the activity of rhIL-6, determined by IL-6 dependent mice hybridoma cell line 7TD1 and MTT assay, is 0.35 ng/ml . CONCLUSIONS: All results mentioned show that rhIL-6 meets the request of the middle scale production. Nucleic Acids Res, 2001 May 15, 29(10), E49 - 9 Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry; Patteson KG et al.; A new method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the direct analysis of the mass-silent post-transcriptionally modified nucleoside pseudouridine in nucleic acids has been developed . This method utilizes 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide to derivatize pseudouridine residues . After chemical derivatization all pseudouridine residues will contain a 252 Da 'mass tag' that allows the presence of pseudouridine to be identified using mass spectrometry . Pseudouridine residues can be identified in intact nucleic acids by obtaining a mass spectrum of the nucleic acid before and after derivatization . The mass difference (in units of 252 Da) will denote the number of pseudouridine residues present . To determine the sequence location of pseudouridine, a combination of enzymatic hydrolysis and mass spectrometric steps are used . Here, MALDI analysis of RNase T1 digestion products before and after modification are used to narrow the sequence location of pseudouridine to specific T1 fragments in the gene sequence . Further mass spectrometric monitoring of exonuclease digestion products from isolated T1 fragments is then used for exact sequence placement . This approach to pseudouridine identification is demonstrated using Escherichia coli tRNAS: This new method allows for the direct determination of pseudouridine in nucleic acids, can be used to identify modified pseudouridine residues and can be used with general modification mapping approaches to completely characterize the post-transcriptional modifications present in RNAs. BETA, 1999 Apr, 12(2), 18 - 9 Intravaginal microbicidal gel to be tested; Hanna L; AIDS: Late-stage laboratory testing will begin for Geda, an experimental microbicide, which is inserted by plunger into the vagina up to four hours before having sex . It contains the active ingredients octoxynol-9 and benzalkonium chloride . Preliminary tests show Geda kills HIV, E . coli, and hepatitis B virus . Another product with the same active ingredients, PrevenTx, is already marketed as a hand wash . Manufacturer Empyrean Bioscience is developing disinfectant and mouthwash versions of PrevenTx . Mol Genet Genomics, 2001 Apr, 265(2), 242 - 8 Suppression of gamma ray-induced illegitimate recombination in Escherichia coli by the DNA-binding protein H-NS; Shanado Y et al.; To study the mechanism of gamma-ray-induced illegitimate recombination, we examined the formation of lambdabio transducing phage in Escherichia coli after gamma-ray irradiation . We show that gamma-ray irradiation enhances the formation of lambdabio transducing phage during prophage induction . Moreover, an hns mutation synergistically enhanced the incidence of lambda-ray-induced illegitimate recombination . Next we determined the sequences at the recombination junctions of the lambdabio transducing phages induced by gamma-ray irradiation . Most of the recombination sites coincided with known hotspots . Among them, hotspot I accounted for 67% and 77% of gamma-ray-induced lambdabio transducing phages in the wild type and the hns mutant, respectively . Therefore, the recombination sites appear to occur mostly at hotspot I or at other hotspots, but rarely at non-hotspot sites . These results suggest that types of DNA damage other than the double-strand breaks induced at random sites are mainly responsible for the introduction of the site-specific or region-specific DNA double strand breaks that lead to recombination at the hotspots . The results also showed that the recombination events took place between DNA sequences possessing short stretches of homology . H-NS protein, which binds to curved DNA, suppresses illegitimate recombination in the presence and absence of gamma-ray irradiation . Models for gamma-ray-induced illegitimate recombination are discussed. Vet Res, 2001 Mar-Apr, 32(2), 131 - 44 Local and systemic effects of endotoxin mastitis on the chemiluminescence of milk and blood neutrophils in dairy cows; Mehrzad J et al.; The local and systemic effects of intramammary lipopolysaccharide (LPS) injection on the chemiluminescence (CL) of milk and blood polymorphonuclear leukocytes (PMN) were investigated in six healthy early lactation cows . Clinical signs of acute mastitis such as fever, increased heart rate and a decreased milk production were observed in all cows . Before LPS challenge, the CL activity of milk PMN was significantly lower than that of blood PMN (P < 0.01) . A significant negative correlation was found between pre-challenge milk and blood PMN CL and, the decreased milk production in unchallenged quarters . The CL activity of milk PMN from LPS-injected quarters increased following LPS challenge, whereas it remained unchanged in control quarters . The CL activity of blood PMN showed a biphasic increase, with two peaks and a valley below pre-challenge CL activity (P < 0.01) . At post-challenge hours (PCH) 6 and 12, the CL activity of milk PMN from LPS-injected quarters exceeded that of blood PMN (P < 0.05 and P < 0.001, respectively) . The decreased CL activity of blood PMN and the enhanced CL activity of milk PMN during endotoxin-induced mastitis was reflected by changes in the shape of the CL curve . In blood PMN, a decrease of the second peak of the CL curve suggests that the myeloperoxidase (MPO)-H2O2 system is impaired during endotoxin-induced mastitis . In contrast, the MPO-H2O2 system was enhanced in milk PMN from challenged quarters . The highest duration and intensity of reactive oxygen intermediate (ROI) production was observed in milk PMN from LPS-injected quarters at PCH 12 . The increased viability of PMN in LPS-injected quarters and to a lesser extent in control quarters suggests possible effects of both facilitated diapedesis and inflammatory mediators on milk PMN survival . In conclusion, our results suggest that a combination of local and systemic action of E . coli endotoxin is involved in the priming of milk PMN during mastitis. Arch Biochem Biophys, 2001 Apr 1, 388(1), 7 - 12 Effect of Li+ upon the Mg2+-dependent activation of recombinant Gialpha1; Minadeo N et al.; Although lithium salts have been used in the treatment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmacologic action of Li+ is unknown . Based on activity studies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li+ inhibition of G-proteins may account for its pharmacologic action . We used the purified alpha subunit of the recombinant inhibitory G-protein, rGialpha1, and found that Li+ at therapeutic levels significantly inhibited the formation of the GDP.AlF4-.rGialpha1 complex . Because our studies were conducted with a purified, metal-reconstituted G-protein rather than with cell membrane suspensions, our Li+ inhibition results lend additional support to the G-protein hypothesis for Li+ action. Arch Biochem Biophys, 2001 Apr 1, 388(1), 39 - 44 Role of interaction between two silkworm RecA homologs in homologous DNA pairing; Kusakabe T et al.; Recombinant BmRad51 and BmDmc1, silkworm homologs of the Escherichia coli RecA proteins catalyzing the homologous DNA pairing, were purified from E . coli cells carrying expression vectors . These possessed different enzymatic properties in the joint molecule formation between single-stranded circular DNA and homologous linear double-stranded DNA . The requirement of single-stranded circular DNA for the efficient reaction was twofold higher in BmRad51 than in BmDmc1 . Although able to mediate the joint molecule formation independently, a complex of the two enzymes formed prior to single-stranded DNA binding was found to have augmented efficiency of the pairing reaction. Arch Biochem Biophys, 2001 Apr 1, 388(1), 146 - 54 Activity of soybean lipoxygenase isoforms against esterified fatty acids indicates functional specificity; Fuller MA et al.; In soybean (Glycine max L.) vegetative tissue at least five lipoxygenase isozymes are present . Four of these proteins have been localized to the paraveinal mesophyll, a layer of cells that is thought to function in assimilate partitioning . In order to determine the role of the lipoxygenase isozymes within the soybean plant, the leaf lipoxygenases were cloned into bacterial expression vectors and expressed in Escherichia coil . The recombinant lipoxygenases were then characterized as to substrate preference, pH profiles for the most common plant lipoxygenase substrates, linoleic acid, and alpha-linolenic acid, and the reaction products with the substrates linoleic acid, alpha-linolenic acid, arachidonic acid, gamma-linolenic acid, and the triacylglycerol trilinolein . All five enzymes were shown to be (13S)-lipoxygenases against linoleic acid . The results of these assays also indicate that two of these isozymes are highly active against esterified fatty acid groups, such as those found in triacylglycerols . Lipid analysis of leaves from plants subjected to sink limitation conditions indicates that the soybean leaf lipoxygenases are active in vivo against both free fatty acids and esterified lipids, and that the quantities of lipoxygenase products found in leaf tissue show a positive correlation with the level of lipoxygenase in the leaf . Implications for the putative role of these enzymes in the paraveinal mesophyll are discussed. Vet Q, 2001 Apr, 23(2), 62 - 6 The effect of apramycin on colonization of pathogenic Escherichia coli in the intestinal tract of chicks; Leitner G et al.; The purpose of the present study was to examine the effect of apramycin sulphate on the colonization of pathogenic E . coli in the intestines of chicks . Apramycin treatment (0.5g/l in the drinking water) of 3-to 5-week-old Leghorn chicks for 24 or 48 hours resulted in a reduction, to an undetectable level, in the number of coliforms in the digestive tract for at least the first 24 h . Per os inoculation of E . coli (O2:K1) after 24 to 48 h of treatment resulted in a significant decrease in colony forming units (cfu) in the digestive tract of the treated chicks . Food deprivation from the time of inoculation did not significantly change the results . However, food and water deprivation caused bacteraemia in a number of the control chicks but not in the treated chicks . Comparison of the level of protection between Leghorn and broiler (Anak strain) chicks revealed that there was a significantly higher (P<0.05) level of bacteraemia in the broiler than in the Leghorn chicks . Chicks treated with 0.25 g/l or 0.125 g/l apramycin for 24 or 48 h before E . coli inoculation showed significantly lower cfu in the colon and caecum than untreated control chicks, but significantly higher cfu were found in the colon than in chicks treated with 0.5 g/l apramycin . Although in vitro preincubation of apramycin with ileum cells did not decrease the percentage of cells to which the bacteria adhered, the number of bacteria adhered per cell decreased significantly . Taken together, our in vitro and in vivo results show that apramycin is effective against E . coli by preventing colonization of the gut by the bacteria, which could lead to a reduction of colibacillosis in poultry. J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 371 - 80 Regulation of PTS gene expression by the homologous transcriptional regulators, Mlc and NagC, in Escherichia coli (or how two similar repressors can behave differently); Plumbridge J; NagC and Mlc are paralogous transcriptional repressors in E.coli . Unexpectedly they possess almost identical amino acid sequences in their helix-turn-helix (H-T-H), DNA binding motif and they bind to very similar consensus operator targets . Binding to each others sites can be demonstrated in vitro but no cross regulation can be detected in vivo with physiological amounts of the two proteins . Although both proteins are involved in regulating the expression of PTS genes, the characteristics of their repression and induction are very different . NagC is a dual-function, activator-repressor which co-ordinates the metabolism of the amino sugars, N-acetylglucosamine (GlcNAc) and glucosamine, by repressing the divergent nagE-BA operons and by activating the glmUS operon . Repression (and activation) by NagC requires that NagC binds simultaneously to two operators, thus forming a DNA loop . This chelation effect allows use of lower affinity sites which would not individually bind the repressor . The specific inducer for NagC is GlcNAc-6-P, the product of GlcNAc transport by the PTS and a key compound in amino sugar metabolism . Mlc represses several genes implicated in the uptake of glucose; ptsG, ptsHI and manXYZ, and malT, the activator of the mal regulon . Glucose behaves like the inducer but growth on glucose only produces an overall increase in expression for ptsG and ptsHI . All Mlc repressed genes are also controlled by cAMP/CAP, so that glucose affects their transcription in two opposing ways: increasing expression by acting as the inducer for Mlc but decreasing expression by lowering the cAMP/CAP concentration . The Mlc protein is not directly responsive to glucose per se but to the activity status of the PTS . Displacement of Mlc from its binding sites occurs during growth on glucose and other PTS sugars and involves sequestration of the repressor to membranes by binding to dephosphorylated PtsG. J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 355 - 9 Routes for fructose utilization by Escherichia coli; Kornberg HL; There are three main routes for the utilization of fructose by Escherichia coli . One (Route A) predominates in the growth of wild-type strains . It involves the functioning of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and a fructose operon, mapping at min . 48.7, containing genes for a membrane-spanning protein (fruA), a 1-phosphofructose kinase (fruK) and a diphosphoryl transfer protein (fruB), under negative regulation by a fruR gene mapping at min . 1.9 . A second route (Route B) also involves the PTS and membrane-spanning proteins that recognize a variety of sugars possessing the 3,4,5-D-arabino-hexoseconfiguration but with primary specificity for mannose(manXYZ), mannitol (mtlA) and glucitol (gutA) and which, if over-produced, can transport also fructose . A third route (Route C), functioning in mutants devoid of Routes A and B, does not involve the PTS: fructose diffuses into the cell via an isoform (PtsG-F) of the major glucose permease of the PTS and is then phosphorylated by ATP and a manno(fructo)kinase (Mak+) specified by a normally cryptic 1032 bp ORF (yajF) of hitherto unknown function (Mak-o), mapping at min . 8.8 and corresponding to a peptide of 344 amino acids . Conversion of the Mak-o to the Mak+ phenotypeinvolves an A24D mutation in a putative regulatory region. J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 329 - 46 The complete phosphotranferase system in Escherichia coli; Tchieu JH et al.; We here tabulate and describe all currently recognized proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and their homologues encoded within the genomes of sequenced E . coli strains . There are five recognized Enzyme I homologues and six recognized HPr homologues . A nitrogen-metabolic PTS phosphoryl transfer chain encoded within the rpoN and ptsP operons and a tri-domain regulatory PTS protein encoded within the dha (dihydroxyacetone catabolic) operon, probably serve regulatory roles exclusively . In addition to several additional putative regulatory proteins, there are 21 (and possibly 22) recognized Enzyme II complexes . Of the 21 Enzyme II complexes, 7 belong to the fructose (Fru) family, 7 belong to the glucose (Glc) family, and 7 belong to the other PTS permease families . All of these proteins are briefly described, and phylogenetic data for the major families are presented. Arch Biochem Biophys, 2001 Jan 1, 385(1), 179 - 85 The catalytic mechanism of 1-aminocyclopropane-1-carboxylic acid oxidase; Charng YY et al.; It has been proposed that 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase catalyzes the oxidation of ACC to ethylene via N-hydroxyl-ACC as an intermediate . However, due to its chemical instability the putative intermediate has never been isolated . Here, we have shown that a purified recombinant ACC oxidase can utilize alpha-aminoisobutyric acid (AIB), an analog of ACC, as an alternative substrate, converting AIB into CO2, acetone, and ammonia . We chemically synthesized the putative intermediate compound, N-hydroxyl-AIB (HAIB), and tested whether it serves as an intermediate in the oxidation of AIB . When {1-(14)C}AIB was incubated with ACC oxidase in the presence of excess unlabeled HAIB as a trap, no labeled HAIB was detected . By comparing the acetone production rates employing HAIB and AIB as substrates, the conversion of HAIB to acetone was found to be much slower than that of using AIB as substrate . Based on these observations, we conclude that ACC oxidase does not catalyze via the N-hydroxylation of its amino acid substrate . ACC oxidase also catalyzes the oxidation of other amino acids, with preference for the D-enantiomers, indicating a stereoselectivity of the enzyme. Arch Biochem Biophys, 2001 Feb 1, 386(1), 62 - 8 Analysis of the amino terminus of maize branching enzyme II by polymerase chain reaction random mutagenesis; Hong S et al.; Maize endosperm branching enzyme II (mBEII) plays a pivotal role in determining the quality of starch by catalyzing the synthesis of the alpha-1,6-branch points . While the central (alpha/beta)8-barrel and the C-terminal domains of mBEII have been analyzed previously, the possible role of its amino terminus in catalysis is still poorly understood . Because the amino terminus of mBEII shares very little sequence homology with other amylolytic enzymes, the Met1-Gly276 region of mBEII was randomly mutagenized under error-prone PCR conditions . Subsequent screening by a heterologous complementation system, utilizing an Escherichia coli strain devoid of the endogenous glycogen branching enzyme (glgB-), led to the recovery of mBEII mutants with altered iodine-staining patterns and reduced branching enzyme activities . The NR-625 mutant enzyme, which lacks the N-terminal 39 residues of mBEII due to a frameshift mutation introduced during the random mutagenesis, retained more than 70% of the wild-type activity . The chain transfer pattern and substrate preference of the truncated enzyme were almost identical to those of the wild-type mBEII . It appears that the N-terminal 39 residues of mBEII are neither required for catalysis nor involved in chain transfer . On the other hand, the Gln-to-Arg substitution at position 270 of mBEII resulted in the loss of more than 90% of branching activity . The Gln270 of mBEII, located at the beginning of the (alpha/beta)8-barrel domain, may be required for maximum enzyme activity. Arch Biochem Biophys, 2001 Feb 1, 386(1), 106 - 16 A drug-unresponsive and protease-resistant CNOX protein from human sera; Sedlak D et al.; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ammonium sulfate fractionation were employed in series to purify and concentrate a 12.5-kDa protein fragment with a periodic (24-min period) proteinase K-resistant and drug-unresponsive NADH oxidase (CNOX) activity from pooled sera from healthy volunteers . The activity was unresponsive to capsaicin to distinguish it from the previously isolated cancer-associated NOX form (tNOX) . Polyclonal antisera generated to the CNOX fragment cross-reacted with 20.5- to 24-kDa proteins of human sera, human lymphocytes, and plasma membranes from Escherichia coli with the molecular weight depending on source and conditions of treatment with proteinase K. Dis Markers, 2000, 16(1-2), 3 - 13 Creating hybrid genes by homologous recombination; Wang PL; Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution . In vitro recombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties . In vivo recombination in both E . coli and yeast is greatly enhanced by double-strand breaks; for E . coli, mutant strains are often necessary to obtain high efficiency . Intra- and inter-molecular recombination in vivo have distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins . Recently in vivo recombination has been used to generate diversity for directed evolution, creating a large phage display antibody library . Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins. Zhonghua Fu Chan Ke Za Zhi, 1999 Jul, 34(7), 417 - 9 {The construction and expression of 6B11scFv anti-idiotypic antibody and human granulocyte-macrophage-colony stimulating factor in Escherichia coli}; Yang F et al.; OBJECTIVE: To enhance the antigenicity of an anti-idiotypic single chain antibody 6B11 mimicking ovarian cancer antigen . METHODS: Using DNA recombinant technique, a recombinant fusion protein expression vector was constructed . This vector linked 6B11scFv cDNA and human GM-CSF with a linker . Protein productions were induced with the temperature change in E . Coli and analyzed with western blot . RESULTS: The expressed fusion protein can be reacted with both anti-human antibody and COC166-9(AB1 of 6B11) . CONCLUSION: The recombinant fusion protein keeps the activities of both components and may be used as tumor vaccine for ovarian cancer. Chin Med Sci J, 1997 Sep, 12(3), 143 - 7 The application of a novel thrombin sensitive site in genetic engineering; Hong M et al.; To obtain high efficiency of cleavage of thrombin in fusion protein containing alpha-ANP fused to Ref peptides, the linker sequence designed as VIAGR which was different from GVRGPR formerly used was studied . Plasmid pHL carrying the fusion gene Ref-NT-ANP downstream from PL promoter was derived from expression vector pLY1 by inserting the fragment of NT-ANP into it . The expression of fusion gene was induced at 42 degrees C, and the interested protein Ref-NT-ANP accumulated as inclusion bodies was isolated by gradient centrifuge and then dissolved in 7 mol/L guanidinehydrochloride (Gdn-HCl) . After dilution, renaturation and dialyzation, the cleavage of thrombin was examined using samples with 1.1 mol/L Gdn-HC1 and samples free of Gdn-HCl respectively . Digestion result showed that the novel-adopted cleavage sequence was highly sensitive to thrombin when the substrate dissolved in 1.1 mol/L Gdn-HCl . The time needed for 87% cleavage (the ratio of substrate to thrombin was 50 micrograms/u) was less than 24 hours . This sequence described here which was specifically recognized by thrombin might be broadly applied in other fusion systems. Chin Med Sci J, 1997 Dec, 12(4), 212 - 5 Evidence of increased endogenous carbon monoxide production in newborn rat endotoxicosis; Shi Y et al.; Carbon monoxide is thought to serve as a new endogenous mediator in the pathogenesis of sepsis and septic shock . In newborn rat endotoxicosis, carbon monoxide levels in the circulation as well as liver, kidney and lung were found to be significantly increased (P < 0.05) . Moreover, the elevations of carbon monoxide correlated with enhanced nitric oxide production as indicated by nitrite/nitrate levels (P < 0.05) . Our present data showed for the first time that endogenously produced carbon monoxide was increased during the course of shock-like states, which suggested that the role of carbon monoxide in sepsis and septic shock might worth further study. Glia, 2001 Jun, 34(4), 272 - 82 Inducible ablation of astrocytes shows that these cells are required for neuronal survival in the adult brain; Cui W et al.; To study the function of astrocytes in the adult brain, we have targeted the expression of E . coli nitroreductase (NTR) to the astrocytes of transgenic mice under the control of the GFAP promoter . The astrocytes expressing NTR were selectively ablated after administration of the prodrug CB1954, resulting in motor discoordination . Histological examination showed that the region most affected in the brain was the cerebellum, in which the Bergmann glia were eliminated and the granular neurons had degenerated . Specific effects were also noted on the dendrites of the Purkinje cells, and the junction between these neurons and granular layer was disrupted . Astrocyte ablation was associated with a dramatic decrease in the expression of glutamate transporters, which may account for the degeneration of granular neurons since the excitotoxic effects of glutamate result in a similar phenotype . These results provide the first evidence that astrocytes are important for the survival of neurons in the adult brain in vivo . J Med Virol, 2001 Jun, 64(2), 125 - 32 Conformational antigenic determinants generated by interactions between a bacterially expressed recombinant peptide of the hepatitis E virus structural protein; Zhang JZ et al.; A 23 kDa peptide locating to amino acid residues 394 to 604 of the major Hepatitis E Virus (HEV) structural protein was expressed in E . coli . This peptide was found to interact naturally with one another to form homodimers and it was recognized strongly and commonly in its dimeric form by HEV reactive human sera . The antigenic activity associated with the dimeric form was abrogated when the dimer was dissociated into monomer and the activity was reconstituted after the monomer was re-associated into dimer again . The dimeric form of the peptide elicited a vigorous antibody response in experimental animals and the resulting antisera were found to cross-react against HEV, effecting an efficient immune capture of the virus . These results attributed the antigenic activity associated with the dimeric form of the peptide to conformational antigenic determinants generated as a result of interaction between the peptide molecules . It is suggested that some of these antigenic determinants may be expressed by the HEV capsid and raised the possibility of this bacterially expressed peptide as an HEV vaccine candidate . Clin Infect Dis, 2001 Jun 15, 32(12), 1706 - 9 Epub 2001 May 21. Enteroaggregative Escherichia coli as a major etiologic agent in traveler's diarrhea in 3 regions of the world; Adachi JA et al.; Enteroaggregative Escherichia coli (EAEC) has been reported to cause traveler's diarrhea and persistent diarrhea in children in developing countries and in immunocompromised patients . To clarify the prevalence of EAEC in traveler's diarrhea, we studied 636 US, Canadian, or European travelers with diarrhea: 218 in Guadalajara, Mexico (June--August 1997 and 1998), 125 in Ocho Rios, Jamaica (September 1997--May 1998), and 293 in Goa, India (January 1997--April 1997 and October 1997--February 1998) . Stool samples were tested for conventional enteropathogens . EAEC strains were identified by use of the HEp-2 assay . EAEC was isolated in 26% of cases of traveler's diarrhea (ranging from 19% in Goa to 33% in Guadalajara) and was second only to enterotoxigenic E . coli as the most common enteropathogen in all areas . Identification of EAEC reduced the number of cases for which the pathogen was unknown from 327 (51%) to 237 (37%) and explained 28% of cases with unknown etiology . EAEC was a major cause of traveler's diarrhea in 3 geographically distinct study areas. J Biol Chem, 2001 Jul 20, 276(29), 26807 - 13 Epub 2001 May 18. Redox-mediated transcriptional activation in a CooA variant; Thorsteinsson MV et al.; CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO at a reduced (Fe(II)) heme moiety with resulting conformational changes that promote DNA binding . In this study, we report a variant of CooA, M124R, that is active in transcriptional activation in a redox-dependent manner . Where wild-type CooA is active only in the Fe(II) + CO form, M124R CooA is active in both Fe(II) + CO and Fe(III) forms . Analysis of the pH dependence of the activity of Fe(III) M124R CooA demonstrated that the activity was also coordination state-dependent with a five-coordinate, high-spin species identified as the active form and Cys(75) as the retained ligand . In contrast, the active Fe(II) + CO forms of both wild-type and M124R CooA are six-coordinate and low-spin with a protein ligand other than Cys(75), so that WT and Fe(III) M124R CooA are apparently achieving an active conformation despite two different heme coordination and ligation states . A hypothesis to explain these results is proposed . This study demonstrates the utility of CooA as a model system for the isolation of functionally interesting heme proteins. Mol Microbiol, 2001 May, 40(3), 684 - 90 Activation and repression of transcription initiation by a distant DNA structural transition; Sheridan SD et al.; Negative superhelical tension can drive local transitions to alternative DNA structures . Long regions of DNA may contain several sites that are susceptible to forming alternative structures . Their relative propensities to undergo transition are ordered according to the energies required for their formation . These energies have two components - the energy needed to drive the transition and the energy relieved by the partial relaxation of superhelicity that the transition provides . This coupling can cause a complex competition among the possible transitions, in which the formation of one energetically favourable alternative structure may inhibit the formation of another within the same domain . In principle, DNA structural competitions can affect the structural and energetic requirements for the initiation of transcription at distant promoter sites . We have tested this possibility by examining the effects of structural transitions on transcription initiation from promoter sites in the same superhelical domain . Specifically, we describe the effects of the presence of a Z-DNA-forming DNA sequence on the basal levels of expression of two supercoiling-sensitive promoters of Escherichia coli, ilvPG and gyrA . We demonstrate transcriptional repression of the ilvPG promoter and activation of the gyrA promoter . We present evidence that this regulation is effected by the superhelically induced B- to Z-DNA transition in a manner that is both orientation and distance independent . We discuss the mechanism of topological coupling between left-handed Z-DNA and the regulation of promoter activity . We also discuss the possibility that the coupling of DNA structural transitions and transcriptional activity might be used as a general regulatory mechanism for gene expression. Mol Microbiol, 2001 May, 40(3), 669 - 83 Diversity in the Bordetella virulence regulon: transcriptional control of a Bvg-intermediate phase gene; Deora R et al.; The BvgAS signal transduction system controls the expression of at least three distinct phenotypic phases that lie along a continuum of gene expression states . The Bvg+ phase is characterized by the expression of adhesins and toxins, whereas the Bvg- phase is characterized by motility in Bordetella bronchiseptica and the expression of vrg loci in Bordetella pertussis . The Bvg-intermediate (Bvgi) phase is characterized by the absence of Bvg-repressed phenotypes, the expression of some, but not all, Bvg-activated virulence factors and the presence of a recently discovered set of antigens and phenotypes that are unique to this phase . We report here the transcriptional regulation of bipA, the first-identified Bvgi phase gene . We have mapped the bipA promoter and identified numerous BvgA binding sites in the transcriptional control region . Based on these data, we present a model in which phase-dependent expression of bipA results from the spatial distribution and relative affinities of multiple BvgA binding sites relative to the start site of transcription. Mol Microbiol, 2001 May, 40(3), 621 - 33 Molecular analysis of the pRA2 partitioning region: ParB autoregulates parAB transcription and forms a nucleoprotein complex with the plasmid partition site, parS; Kwong SM et al.; The partitioning locus (par) of plasmid pRA2 belongs to a recently discovered subgroup of plasmid partitioning systems that are evolutionarily distinct from the P1, F and R1/NR1 prototypes . The pRA2 par region was effective in stabilizing both pRA2 and F mini-replicons . Analysis of the nucleotide sequence revealed three potential coding regions that were designated parA, parB and parC . Through mutagenesis, parA and parB were found to be essential for partitioning function, whereas parC did not appear to be required . Using transcriptional reporter systems, it was demonstrated in vivo that ParB repressed par promoter activity by 60-fold and that ParA had little effect on transcriptional activity . Primer extension analysis revealed that the par transcriptional start point was located 47 nucleotides upstream of the parA translational start codon . Based on this information, putative -10 and -35 transcriptional signals were identified, and their subsequent deletion resulted in a dramatic reduction in promoter activity . The par promoter region was also demonstrated to exert incompatibility towards a plasmid with an active pRA2 par system . Nested deletions in this region allowed the incompatibility determinant, designated parS, to be localized . Recombinant ParA and ParB proteins were overexpressed and purified by affinity chromatography . Through in vitro binding experiments, purified ParB was shown to interact specifically with the par promoter region . DNase I footprinting revealed that ParB not only binds to the conserved sequence 5'-TCA AA(T/C) (G/C)CT CAA (A/T)A, which is present in three copies in the par promoter region, but also binds to the pRA2 partitioning site, parS . It appears that ParB has a dual role in pRA2 partitioning, being responsible for both the regulation of par transcription and the formation of a partition nucleoprotein complex at parS. Mol Microbiol, 2001 May, 40(3), 596 - 609 The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues; Skouloubris S et al.; Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of short-chain amides to produce ammonia and the corresponding organic acid . Such an amidase, AmiE, has been detected previously in Helicobacter pylori . Analysis of the complete H . pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF . The corresponding AmiF protein is 34% identical to its AmiE paralogue . Because gene duplication is widely considered to be a fundamental process in the acquisition of novel enzymatic functions, we decided to study and compare the functions of the paralogous amidases of H . pylori . AmiE and AmiF proteins were overproduced in Escherichia coli and purified by a two-step chromatographic procedure . The two H . pylori amidases could be distinguished by different biochemical characteristics such as optimum pH or temperature . AmiE hydrolysed propionamide, acetamide and acrylamide and had no activity with formamide . AmiF presented an unexpected substrate specificity: it only hydrolysed formamide . AmiF is thus the first formamidase (EC 3.5.1.49) related to aliphatic amidases to be described . Cys-165 in AmiE and Cys-166 in AmiF were identified as residues essential for catalysis of the corresponding enzymes . H . pylori strains carrying single and double mutations of amiE and amiF were constructed . The substrate specificities of these enzymes were confirmed in H . pylori . Production of AmiE and AmiF proteins is dependent on the activity of other enzymes involved in the nitrogen metabolism of H . pylori (urease and arginase respectively) . Our results strongly suggest that (i) the H . pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral gene duplication; and (ii) the production of these enzymes is regulated to maintain intracellular nitrogen balance in H . pylori. Am J Hum Genet, 2001 Jun, 68(6), 1506 - 13 Epub 2001 May 15. Impaired heme binding and aggregation of mutant cystathionine beta-synthase subunits in homocystinuria; Janosik M et al.; During the past 20 years, cystathionine beta-synthase (CBS) deficiency has been detected in the former Czechoslovakia with a calculated frequency of 1:349,000 . The clinical manifestation was typical of homocystinuria, and about half of the 21 patients were not responsive to pyridoxine . Twelve distinct mutations were detected in 30 independent homocystinuric alleles . One half of the alleles carried either the c.833 T-->C or the IVS11-2A-->C mutation; the remaining alleles contained private mutations . The abundance of five mutant mRNAs with premature stop codons was analyzed by PCR-RFLP . Two mRNAs, c.828_931ins104 (IVS7+1G-->A) and c.1226 G-->A, were severely reduced in the cytoplasm as a result of nonsense-mediated decay . In contrast, the other three mRNAs-c.19_20insC, c.28_29delG, and c.210_235del26 (IVS1-1G-->C)-were stable . Native western blot analysis of 14 mutant fibroblast lines showed a paucity of CBS antigen, which was detectable only in aggregates . Five mutations-A114V (c.341C-->T), A155T (c.463G-->A), E176K (c.526G-->A), I278T (c.833T-->C), and W409_G453del (IVS11-2A-->C)-were expressed in Escherichia coli . All five mutant proteins formed substantially more aggregates than did the wild-type CBS, and no aggregates contained heme . These data suggest that abnormal folding, impaired heme binding, and aggregation of mutant CBS polypeptides may be common pathogenic mechanisms in CBS deficiency. Cancer Res, 2001 May 15, 61(10), 3913 - 8 Molecular nature of ultraviolet B light-induced deletions in the murine epidermis; Horiguchi M et al.; Depletion of the stratospheric ozone layer leads to an increase in ambient UV loads, which are expected to raise skin cancer incidences . Tumor development in the skin could be a multistep process in which various genetic alterations, such as point mutations and deletions, occur successively . Here, we demonstrate that UVB irradiation efficiently induces deletions in the epidermis using a novel transgenic mouse, gpt delta . In this mouse model, deletions in lambda DNA integrated in the chromosome are preferentially selected as Spi(-) (sensitive to P2 interference) phages, which can then be subjected to molecular analysis . The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2) . After 4 weeks, lambda phage was rescued from the genomic DNA of the epidermis by in vitro packaging reactions . The mutant frequencies of Spi(-) with large deletions in the epidermis increased >15-fold at a UVB dose of 0.5 kJ/m(2) over the control . Molecular sizes of most of the large deletions were >1000 bp . More than one-half of the large deletions occurred between short direct-repeat sequences from 1 to 6 bp, and the remainder had flush ends . In the unirradiated mouse, almost all of the Spi(-) mutants were 1-bp frameshifts in runs of identical bases . These results suggest that UVB irradiation induces deletions in the murine epidermis, and most of the deletions are generated through end-joining of double strand breaks in DNA. J Steroid Biochem Mol Biol, 2001 Apr, 77(1), 83 - 6 High metabolization of catecholestrogens by type 1 estrogen sulfotransferase (hEST1); Faucher F et al.; Recently, two types of estrogen sulfotransferase, chronologically named types 1 and 2 estrogen sulfotransferase (hEST1 and hEST2), have been described . Since hEST2 selectively catalyzes the sulfonation of ethinyl estradiol as well as that of estrone (E1) and estradiol (E2), but poorly the sulfonation of catecholestrogens, we wanted to assess the ability of hEST1 to metabolize these compounds . We overexpressed hEST1 in Escherichia coli in fusion with GST, then purified the enzyme using a glutathione affinity column, and obtained GST-free enzyme by digestion with thrombin . Using {35S}-phosphosadenosine phosphosulfate (PAPS) as cofactor, we showed that hEST1 efficiently metabolizes the transformation of 2-OH-E2 and 2-OH-E1 . However, the transformation of 4-OH-E1 and 4-OH-E2 is much less efficient . Our results also show that hEST1 metabolizes more efficiently E2 than E1 . Since hEST1 mRNA is produced from the same gene as MPST using different alternative promoters and since it is expressed in most breast cancer cells (MCF-7, ZR-75-1, T47-D, MDA-231, and MDA-418), studies of the expression and activity of hEST1 will be most important to have a better knowledge about its involvement in the control of the genotoxicity of estrogens and catecholestrogens. Eur J Biochem, 2001 May, 268(10), 2912 - 23 Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products; Singh SP et al.; Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (earlier designated as GST-2) is related to sigma class GSTs and was previously described as an indirect flight muscle-associated protein with no known catalytic properties . We now report that DmGSTS1-1 isolated from Drosophila or expressed in Escherichia coli is essentially inactive toward the commonly used synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB), but has relatively high glutathione-conjugating activity for 4-hydroxynonenal (4-HNE), an electrophilic aldehyde derived from lipid peroxidation . 4-HNE is thought to have signaling functions and, at higher concentrations, has been shown to be cytotoxic and involved in the etiology of various degenerative diseases . Drosophila strains carrying P-element insertions in the GstS1 gene have a reduced capacity for glutathione conjugation of 4-HNE . In flies with both, one, or none of the GstS1 alleles disrupted by P-element insertion, there is a linear correlation between DmGSTS1-1 protein content and 4-HNE-conjugating activity . This correlation indicates that in adult Drosophila 70 +/- 6% of the capacity to conjugate 4-HNE is attributable to DmGSTS1-1 . The high abundance of DmGSTS1-1 (approximately 2% of the soluble protein in adult flies) and its previously reported localization in tissues that are either highly aerobic (indirect flight muscle) or especially sensitive to oxidative damage (neuronal tissue) suggest that the enzyme may have a protective role against deleterious effects of oxidative stress . Such function in insects would be analogous to that carried out in mammals by specialized alpha class glutathione S-transferases (e.g . GSTA4-4) . The independent emergence of 4-HNE-conjugating activity in more than one branch of the glutathione S-transferase superfamily suggests that 4-HNE catabolism may be essential for aerobic life. Eur J Biochem, 2001 May, 268(10), 2838 - 46 The interaction of human apolipoprotein C-I with sub-micellar phospholipid; Atcliffe BW et al.; Mature human apolipoprotein C-I (apoC-I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family . Amphipathic helical regions within apoC-I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC-I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein . To examine apoC-I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC-I with yields of approximately 4-5 mg per L of culture . The purified product has properties similar to plasma-derived apoC-I including self-association in the lipid-free state and induced alpha-helical content in the presence of egg-yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles . We chose the short-chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro-PCho), to examine the interaction of apoC-I with submicellar phospholipid . Circular dichroism spectroscopy and cross-linking experiments show that apoC-I acquires helical content and remains self-associated at submicellar concentrations of Hex2Gro-PCho (4 mM) . Sedimentation equilibrium studies of apoC-I at submicellar levels of Hex2Gro-PCho and analysis of the effects of apoC-I on the 1H NMR spectrum of Hex2Gro-PCho indicate micelle induction by apoC-I, and establish the capacity of apoC-I to assemble individual phospholipid molecules. Mol Cell Neurosci, 2001 May, 17(5), 842 - 54 Neuronal targeting of cardiotrophin-1 by coupling with tetanus toxin C fragment; Bordet T et al.; Cardiotrophin-1 (CT-1) is a potent neurotrophic factor for motoneurons but its clinical use in motor neuron diseases is precluded by side effects on the heart and liver . We explored the possibility of targeting CT-1 to neurons by coupling with the tetanus toxin fragment TTC . Genetic fusion proteins between CT-1 or GFP and TTC were produced in Escherichia coli and assayed in vitro . In contrast to uncoupled CT-1 or GFP, TTC-coupled proteins bound with high affinity to cerebral neurons and spinal cord motoneurons and were rapidly internalized . Glia, hepatocytes, or cardiomyocytes did not show detectable binding or uptake of TTC-coupled proteins . Similar to CT-1, TTC-coupled CT-1 induced IL-6 secretion by KB cells, activated Reg-2 gene expression, and promoted motoneuron survival in a dose-dependent manner . In vivo studies will test whether TTC-coupled CT-1 might be targeted to degenerating spinal cord or brain-stem motoneurons and migrate trans-synaptically to cortical motoneurons, which are also affected in amyotrophic lateral sclerosis . J Chromatogr A, 2001 Apr 27, 915(1-2), 97 - 106 Affinity chromatography of polyhistidine tagged enzymes . New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions; Mateo C et al.; New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins . The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp . strain T2) has been used to check the performance of these new chromatographic media . IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC . However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E . coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits . In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole) . The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole . In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports) . Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface . However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface. J Chromatogr A, 2001 Apr 20, 914(1-2), 257 - 64 High-efficiency capillary isoelectric focusing of protein complexes from Escherichia coli cytosolic extracts; Shen Y et al.; High-efficiency capillary isoelectric focusing (cIEF) separations of protein complexes obtained from soluble protein fractions are demonstrated . Size-exclusion chromatography was used as a first dimension separation to fractionate putative protein complexes with apparent molecular masses of up to 1,500,000 from an Escherichia coli cytosolic fraction . Non-denaturing cIEF separations using highly hydrophilic polymer-coated capillaries constituted the second dimension . The conditions developed produced reproducible and high-efficiency separations, corresponding to approximately 2 x 10(6) theoretical plates and peak capacities of approximately 10(3) for pH 3-10 cIEF separations in 65 cm long capillaries . Combination of the two non-denaturing separation dimensions permitted isolation and analysis of individ