Biotechnol Bioeng, 2003 Nov 20, 84(4), 467 - 73 Baculoviral polyhedrin as a novel fusion partner for formation of inclusion body in Escherichia coli; Seo JH et al.; Baculoviral polyhedrin, which originated from Autographa californica nuclear polyhedrosis virus (AcNPV), was employed for the first time as a novel fusion partner for expression of foreign proteins in an Escherichia coli system . We characterized the expression of recombinant polyhedrin protein fused to green fluorescent protein (GFP) . The polyhedrin fusion protein ( approximately 58 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 30% of the total cellular protein . The E . coli expressing polyhedrin-GFP fusion protein showed higher cell growth ( approximately 1.8-fold) and higher GFP yield ( approximately 3.5-fold) than the strain expressing soluble single GFP . Interestingly, the polyhedrin fusion portion showed almost the same characteristics as the native baculoviral polyhedrin; it was rapidly solubilized under alkaline conditions, similar to the conditions found in the insect midgut . In addition, the polyhedrin fusion portion was rapidly digested by alkaline proteases in insect Plutella xylostella midgut as well as by alpha-chymotrypsin, a protease that has similar properties to insect midgut polyhedra-associated alkaline proteases . These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for production of foreign proteins, especially harmful proteins, in E . coli expression systems . Biotechnol Bioeng, 2003 Nov 20, 84(4), 452 - 8 One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate; Oh J et al.; An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate . Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E . coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system . When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized . About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2) . The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction . Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration . Protein Sci, 2003 Nov, 12(11), 2642 - 6 Recombinant extracellular domain of the three major subunits of GABAA receptor show comparable secondary structure and benzodiazepine binding properties; Shi H et al.; The three most widely expressed subunits of the GABAA receptor are alpha(1), beta(2), and gamma(2) subunits, and the major isoform in the human brain is a pentameric receptor composed of 2alpha(1)2beta(2)1gamma(2) . Previously, we overexpressed the extracellular domain Q28-R248 of GABAA receptor alpha(1) subunit . In the present study, the homologous extracellular domains Q25-G243 of GABAA receptor beta(2) subunit and Q40-G273 of gamma(2) subunit were also obtained through overexpression in Escherichia coli . Successful production of recombinant beta(2) and gamma(2) subunit receptor protein domains facilitates the comparison of structural and functional properties of the three subunits . To this end, the secondary structures of the three fragments were measured using CD spectroscopy and the beta-strand contents calculated to be >30%, indicating a beta-rich structure for all three fragments . In addition, the benzodiazepine (BZ)-binding affinity of the recombinant fragments were measured using fluorescence polarization to be 2.16 microM, 3.63 microM, and 1.34 microM for the alpha(1), beta(2), and gamma(2) subunit fragments, respectively, indicating that all three homomeric assemblies, including that of the beta(2) subunit, generally not associated with BZ binding, can bind BZ in the micromolar range . The finding that the BZ binding affinity of these recombinant domains was highest for the gamma(2) subunit and lowest for the beta(2) subunit is consistent with results from previous binding studies using hetero-oligomeric receptors . The present results exemplify the effective approach to characterize and compare the three major subunits of the GABAA receptor, for two of which the overexpression in E . coli is reported for the first time. Protein Sci, 2003 Nov, 12(11), 2549 - 58 Modulating the binding properties of an anti-17beta-estradiol antibody by systematic mutation combinations; Lamminmaki U et al.; The anti-17beta-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties . Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants . These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in complementarity determining region CDR-H2 . The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross-reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time-resolved fluorescence based immunoassays . The double-mutant cycle method was used to address the cooperativity effects between the mutations . The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57-2 crystal structures . These analyses provided information about the steroid-binding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions . Furthermore, several combinatorial mutants with improved affinity and specificity were obtained . The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time-resolved fluorescence based immunoassay. Proc Natl Acad Sci U S A, 2003 Nov 11, 100(23), 13202 - 6 Epub 2003 Oct 22. Identification of functional similarities between proteins using directed evolution; Christ D et al.; Protein sequences are often highly redundant and evolution can change them beyond recognition . It can therefore be difficult to identify proteins with functional or structural similarities by inspection of their sequences . Here we have used an experimental evolutionary approach to detect hidden similarities between the antisense RNA-binding protein Rop and other proteins . We created an envelope of functional Rop mutants by combinatorial mutagenesis, used the compilation of mutant sequences to search a database of protein structures, and thereby identified a segment of the enzyme valyl-tRNA-synthetase (ValRS) . Further inspection revealed that the structures of the RNA-binding sites of both proteins are highly related, as indeed are the RNA ligands . From the known 3D structure of the ValRS in complex with tRNA, we were able to build a model of an RNA hairpin pair in complex with Rop that has proved to be consistent with the biochemical and NMR data for the interaction between Rop and RNA hairpins . We suggest that this approach (mutational envelope scanning), by generating sequence information de novo, can help uncover hidden similarities between proteins. Infect Immun, 2003 Nov, 71(11), 6338 - 43 PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response; Choudhary RK et al.; The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important . Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes . Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M . tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level . Recombinant protein expressed in Escherichia coli was used to screen the sera of M . tuberculosis-infected patients, as well as those of clinically healthy controls (n = 10), by enzyme-linked immunosorbent assay . The panel of patient sera comprised sera from fresh infection cases (category 1; n = 32), patients with relapsed tuberculosis (category 2; n = 30), and extrapulmonary cases (category 3; n = 30) . Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD) . However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%) . Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M . tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein. J Biol Chem, 2004 Jan 9, 279(2), 928 - 36 Epub 2003 Oct 22. Plant MAPK phosphatase interacts with calmodulins; Yamakawa H et al.; A mitogen-activated protein kinase (MAPK) phosphatase gene, designated NtMKP1, was isolated as a candidate gene for a calmodulin (CaM)-binding protein from tobacco . NtMKP1 protein has four characteristic domains conserved among plant MAPK phosphatases reported so far, namely a dual specificity protein phosphatase catalytic domain, gelsolin-like domain, putative CaM-binding domain (CaMBD), and serine-rich region, indicating that NtMKP1 is the ortholog of Arabidopsis MKP1 . The bacterially expressed NtMKP1 protein physically interacted with three plant-specific types of CaM in an overlay assay with labeled CaMs, showing high affinity to NtCaM1 and NtCaM3 but lower affinity to NtCaM13 . The peptide for the putative CaMBD bound both NtCaM1 and NtCaM3 significantly but bound NtCaM13 only slightly . Moreover, CaM overlay assays with mutated CaMBDs revealed that Trp440 and Leu443 in the middle of the basic amphiphilic alpha-helix motif (amino acids 436-453) are critical for binding CaM . In comparison with the transient accumulation of a wound-induced MAPK, WIPK transcript, a prolonged activation of NtMKP1 expression was found in response to wounding and tobacco mosaic virus-induced hypersensitive reaction . In transgenic tobacco plants overexpressing NtMKP1, wound-induced activation of SIPK, salicylic acid-induced MAPK, and WIPK was inhibited . These results suggest that plant CaMs are involved in these stress-activated MAPK cascades via NtMKP1. J Biol Chem, 2003 Dec 26, 278(52), 52546 - 50 Epub 2003 Oct 22. Escherichia coli DNA polymerase V subunit exchange: a post-SOS mechanism to curtail error-prone DNA synthesis; Shen X et al.; DNA polymerase V consisting of a heterotrimer composed of one molecule of UmuC and two molecules of UmuD' (UmuD'2C) is responsible for SOS damage-induced mutagenesis in Escherichia coli . Here we show that although the UmuD'2C complex remains intact through multiple chromatographic steps, excess UmuD, the precursor to UmuD', displaces UmuD' from UmuD'2C by forming a UmuDD' heterodimer, while UmuC concomitantly aggregates as an insoluble precipitate . Although soluble UmuD'2C is readily detected when the two genes are co-transcribed and translated in vitro, soluble UmuD2C or UmuDD'C are not detected . The subunit exchange between UmuD'2C and UmuD offers a biological means to inactivate error-prone polymerase V following translesion synthesis, thus preventing mutations from occurring on undamaged DNA. Biochim Biophys Acta, 2003 Oct 13, 1623(2-3), 143 - 53 Characterization of the N-terminal domain of NrtC, the ATP-binding subunit of ABC-type nitrate transporter of the cyanobacterium Phormidium laminosum; Nagore D et al.; The N-terminal domain of NrtC, the ATP-binding subunit of nitrate/nitrite ABC-transporter in the cyanobacterium Phormidium laminosum, has been expressed in Escherichia coli as a histidine-tagged fusion protein (His(6)NrtC1) . Binding of ATP to the pure His(6)NrtC1 was characterized using the nucleotide analogue TNP-ATP {2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate} . Fluorescence assays showed that His(6)NrtC1 specifically binds Mg(2+) TNP-ATP with high affinity, binding being dependent on protein concentration . The presence of ATP prevents the covalent modification of His(6)NrtC1 by fluorescein 5'-isothiocyanate (FITC), suggesting that this probe reacts at the nucleotide-binding site of NrtC . The active form of the truncated NrtC is a dimer that shows high affinity for TNP-ATP (K(d)=0.76+/-0.1 microM) . Evidence for the presence of two nucleotide-binding sites per dimer protein is given . Our results indicate that nucleotide binding is strongly dependent on the dimerization of NrtC and that the N-terminal domain of the protein contains the binding site for ATP . No ATPase activity catalyzed in vitro by the truncated subunit was detected. FEBS Lett, 2003 Oct 23, 553(3), 408 - 12 The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase; Bittel C et al.; Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing . The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli . Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties . They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase. FEBS Lett, 2003 Oct 23, 553(3), 397 - 402 Growth rate regulation of lac operon expression in Escherichia coli is cyclic AMP dependent; Kuo JT et al.; In contrast to the ribosomal RNA gene expression increasing with growth rate, transcription of the lac operon is downregulated by cell growth rate . In continuous culture, growth rate regulation of lac promoter was independent of carbon substrate used and its location on the chromosome . Since the lac operon is activated by cyclic adenosine monophosphate (cAMP), which decreases with increasing cell growth rate, expression of plac-lacZ reporter fusion was analyzed in cya mutant under various growth conditions . The results demonstrated that expression of plac-lacZ in cya mutant was both lower and growth rate independent . In addition, ppGpp (guanosine tetraphosphate) was not involved in the mechanism of growth rate regulation of the lac promoter . Thus, the results of this study indicate that cAMP mediates the growth rate-dependent regulation of lac operon expression in Escherichia coli. FEBS Lett, 2003 Oct 23, 553(3), 351 - 4 Determination of the cleavage sites in SulA, a cell division inhibitor, by the ATP-dependent HslVU protease from Escherichia coli; Nishii W et al.; HslVU is an ATP-dependent protease from Escherichia coli and known to degrade SulA, a cell division inhibitor, both in vivo and in vitro, like the ATP-dependent protease Lon . In this study, the cleavage specificity of HslVU toward SulA was investigated . The enzyme was shown to produce 58 peptides with various sizes (3-31 residues), not following the 'molecular ruler' model . Cleavage occurred at 39 peptide bonds preferentially after Leu in an ATP-dependent manner and in a processive fashion . Interestingly, the central and C-terminal regions of SulA, which are known to be important for the function of SulA, such as inhibition of cell division and molecular interaction with certain other proteins, were shown to be preferentially cleaved by HslVU, as well as by Lon, despite the fact that the peptide bond specificities of the two enzymes were distinct from each other. FEBS Lett, 2003 Oct 23, 553(3), 271 - 6 Protein refolding assisted by self-assembled nanogels as novel artificial molecular chaperone; Nomura Y et al.; Molecular chaperone-like activity for protein refolding was investigated using nanogels of self-assembly of cholesterol-bearing pullulan . Nanogels effectively prevented protein aggregation (i.e . carbonic anhydrase and citrate synthase) during protein refolding from GdmCl denaturation . Enzyme activity recovered in high yields upon dissociation of the gel structure in which the proteins were trapped, by the addition of cyclodextrins . The nanogels assisted protein refolding in a manner similar to the mechanism of molecular chaperones, namely by catching and releasing proteins . The nanogels acted as a host for the trapping of refolded intermediate proteins . Cyclodextrin is an effector molecule that controls the binding ability of these host nanogels to proteins . The present nanogel system was also effective at the renaturation of inclusion body of a recombinant protein of the serine protease family. FEBS Lett, 2003 Oct 23, 553(3), 232 - 8 Solution structure of epiregulin and the effect of its C-terminal domain for receptor binding affinity; Sato K et al.; Epiregulin (EPR), a novel member of epidermal growth factor (EGF) family, is a ligand for ErbB-1 and ErbB-4 receptors . The binding affinity of EPR for the receptors is lower than those of other EGF-family ligands . The solution structure of EPR was determined using two-dimensional nuclear magnetic resonance spectroscopy . The secondary structure in the C-terminal domain of EPR is different from other EGF-family ligands because of the lack of hydrogen bonds . The structural difference in the C-terminal domain may provide an explanation for the reduced binding affinity of EPR to the ErbB receptors. J Food Prot, 2003 Oct, 66(10), 1927 - 31 Construction and preliminary evaluation of an Aspergillus flavus reporter gene construct as a potential tool for screening aflatoxin resistance; Brown RL et al.; Effective preharvest strategies to eliminate aflatoxin accumulation in crops are not presently available . The molecular biology of aflatoxin biosynthesis has been extensively studied, and genetic and molecular tools such as reporter gene systems for the measurement of fungal growth have been developed . A reporter construct containing the Aspergillus flavus beta-tubulin gene promoter fused to Escherichia coli beta-glucuronidase (GUS) has been shown to be a reliable tool for the indirect measurement of fungal growth in maize kernels . Since cost-saving alternative methods for the direct measurement of aflatoxin levels are needed to facilitate more widespread field and laboratory screening of maize lines, a new reporter gene construct involving the promoter region of the omtA gene of the aflatoxin biosynthetic pathway was constructed and tested . Expression of GUS activity by this construct (omtA::GUS) was correlated with aflatoxin accumulation in culture . In the fungal transformant GAP26-1, which harbors this construct, aflatoxin production and GUS expression on sucrose-containing medium showed the same temporal pattern of toxin induction . Furthermore, GUS expression by GAP26-1 was shown to be associated with aflatoxin accumulation in maize kernels inoculated with this strain . Our results suggest that this and other reporter gene pathway promoter constructs may provide superior alternatives to direct aflatoxin quantification with respect to time, labor, and materials for the screening of maize lines for resistance to aflatoxin accumulation. Biotechnol Lett, 2003 Sep, 25(18), 1499 - 504 Enhanced ribosome and tRNA contents in Escherichia coli expressing a truncated Vitreoscilla hemoglobin mutant analyzed by flow field-flow fractionation; Andersson CI et al.; The ribosome and tRNA levels of Escherichia coli cells, transformed with a native or mutated Vitreoscilla hemoglobin genes (vhb), were investigated using asymmetrical flow field-flow fractionation (AFFFF) . Mutagenesis of rhb by error-prone PCR was carried out to alter the growth behavior of microaerobically cultivated native VHb-expressing E . coli . A VHb mutant, pVMT1, was identified, which was able to reach a remarkably high final A600 of 15, the value of which being 160% higher than that of a VHb control carrying pVHb8 (A600 5.8) . AFFFF revealed that cells expressing mutant vhbs showed up to a doubling in the number of active 70S ribosomes cell(-1), an almost 3-fold increase in the number of tRNAs cell(-1), and up to a 26% increase in the mass fraction of active 70S ribosomes. Genome Inform Ser Workshop Genome Inform, 2002, 13, 214 - 23 In silico metabolic pathway analysis and design: succinic acid production by metabolically engineered Escherichia coli as an example; Lee SY et al.; The intracellular metabolic fluxes can be calculated by metabolic flux analysis, which uses a stoichiometric model for the intracellular reactions along with mass balances around the intracellular metabolites . In this study, we have constructed in silico metabolic pathway network of Escherichia coli consisting of 301 reactions and 294 metabolites . Metabolic flux analyses were carried out to estimate flux distributions to achieve the maximum in silico yield of succinic acid in E . coli . The maximum in silico yield of succinic acid was only 83% of its theoretical yield . The lower in silico yield of succinic acid was found to be due to the insufficient reducing power, which could be increased to its theoretical yield by supplying more reducing power . Furthermore, the optimal metabolic pathways for the production of succinic acid could be proposed based on the results of metabolic flux analyses . In the case of succinic acid production, it was found that pyruvate carboxylation pathway should be used rather than phosphoenolpyruvate carboxylation pathway for its optimal production in E . coli . Then, the in silico optimal succinic acid pathway was compared with conventional succinic acid pathway through minimum set of wet experiments . The results of wet experiments indicate that the pathway predicted by in silico analysis is more efficient than conventional pathway. J Biol Chem, 2004 Jan 16, 279(3), 1950 - 5 Epub 2003 Oct 21. The donor subsite of trehalose-6-phosphate synthase: binary complexes with UDP-glucose and UDP-2-deoxy-2-fluoro-glucose at 2 A resolution; Gibson RP et al.; Trehalose is an unusual non-reducing disaccharide that plays a variety of biological roles, from food storage to cellular protection from environmental stresses such as desiccation, pressure, heat-shock, extreme cold, and oxygen radicals . It is also an integral component of the cell-wall glycolipids of mycobacteria . The primary enzymatic route to trehalose first involves the transfer of glucose from a UDP-glucose donor to glucose-6-phosphate to form alpha,alpha-1,1 trehalose-6-phosphate . This reaction, in which the configurations of two glycosidic bonds are set simultaneously, is catalyzed by the glycosyltransferase trehalose-6-phosphate synthase (OtsA), which acts with retention of the anomeric configuration of the UDP-sugar donor . The classification of activated sugar-dependent glycosyltransferases into approximately 70 distinct families based upon amino acid sequence similarities places OtsA in glycosyltransferase family 20 (see afmb.cnrs-mrs.fr/CAZY/) . The recent 2.4 A structure of Escherichia coli OtsA revealed a two-domain enzyme with catalysis occurring at the interface of the twin beta/alpha/beta domains . Here we present the 2.0 A structures of the E . coli OtsA in complex with either UDP-Glc or the non-transferable analogue UDP-2-deoxy-2-fluoroglucose . Both complexes unveil the donor subsite interactions, confirming a strong similarity to glycogen phosphorylases, and reveal substantial conformational differences to the previously reported complex with UDP and glucose 6-phosphate . Both the relative orientation of the two domains and substantial (up to 10 A) movements of an N-terminal loop (residues 9-22) characterize the more open "relaxed" conformation of the binary UDP-sugar complexes reported here. J Biol Chem, 2004 Jan 9, 279(2), 901 - 9 Epub 2003 Oct 21. Cys32 and His105 are the critical residues for the calcium-dependent cysteine proteolytic activity of CvaB, an ATP-binding cassette transporter; Wu KH et al.; CvaB, a member of the ATP-binding cassette transporter superfamily, is the central membrane transporter of the colicin V secretion system in Escherichia coli . Cys32 and His105 in the N-terminal domain of CvaB were identified as critical residues for both colicin V secretion and cysteine proteolytic activity . By inhibiting degradation with N-ethylmaleimide and a mixture of protease inhibitors, a stable wild-type N-terminal domain (which showed cysteine protease activity when activated) was purified . Such protease activity was Ca2+- and concentration-dependent and could be inhibited by antipain, N-ethylmaleimide, EDTA, and EGTA . At low concentrations, the Ca2+ analogs Tb3+ and La3+ (but not Fe3+) significantly enhanced proteolytic activity, suggesting that the size of the cations is important for activity . Together with comparisons of the sequences of members of the cysteine protease family, these results indicate that Cys32 and His105 are the critical residues in the CvaB N-terminal domain for the calcium-dependent cysteine protease activity and secretion of colicin V. J Biol Chem, 2003 Dec 26, 278(52), 52551 - 8 Epub 2003 Oct 21. Caspase 3-mediated proteolysis of the N-terminal cytoplasmic domain of the human erythroid anion exchanger 1 (band 3); Mandal D et al.; The N-terminal cytoplasmic domain of the anion exchanger 1 (AE1 or band 3) of the human erythrocyte associates with peripheral membrane proteins to regulate membrane-cytoskeleton interactions, with glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase and aldolase, with the protein-tyrosine kinase p72syk, with hemoglobin and with hemichromes . We have demonstrated that the N-terminal cytoplasmic domain of band 3 (CDB3) is a substrate of the apoptosis executioner caspase 3 (1) . CDB3 has two non-conventional caspase 3 cleavage sites, TATD45 and EQGD205 (2) . In vitro treatment of recombinant CDB3 with caspase 3 generated two fragments, which could be blocked by pretreatment with the caspase 3 inhibitor Z-DEVD-fmk (3) . Recombinant CDB3 in which the caspase 3 cleavage sites Asp45 and Asp205 were mutated, was resistant to proteolysis (4) . Proteolytically derived fragments crossreactive with polyclonal anti-band 3 antibody appeared with simultaneous cleavage of poly (ADP-ribose) polymerase and procaspase 3 in staurosporine (STS)-treated HEK293 cells transiently transfected with CDB3 (5) . In vivo cleavage of CDB3 could be blocked by pretreatment of cells with Z-DEVD-fmk or in cells transfected with mutant CDB3 (D45A, D205A) (6) . Co-transfection experiments showed that STS-mediated cleavage of CDB3 diminished its interaction with the N-terminal domain of protein 4.2, confirming that such cleavage interferes with the interaction of CDB3 with cytoskeletal proteins (7) . Active caspase 3 was observed in aged red cells but not in young cells . This red cell caspase 3 could cleave band 3 present in inside-out vesicles prepared from young erythrocytes arguing in favor of a physiological role of caspase 3 in aged erythrocytes. J Biol Chem, 2004 Jan 9, 279(2), 1468 - 73 Epub 2003 Oct 21. An Arabidopsis RNA lariat debranching enzyme is essential for embryogenesis; Wang H et al.; An embryo-defective mutant of Arabidopsis thaliana was isolated that arrests development at a variety of stages, from as early as the globular stage of embryogenesis to as late as formation of an abnormal bent cotyledon stage embryo . Defects in the suspensor, a normally transient structure derived from the fertilized egg, were often associated with the arrested embryo . The lesion was within a gene encoding a protein with domains characteristic of lariat debranching enzymes, which has been named AtDBR1 (for Arabidopsis thaliana Debranching enzyme 1) . Cleavage of the 2'-5'-phosphodiester bond found in excised intron lariats ("debranching") is essential for turnover of intronic sequences as well as generation of some small nucleolar RNAs . The mutation within AtDBR1 was confirmed by complementation as being responsible for the embryo-lethal phenotype, and the activity of the encoded protein in cleavage of 2'-5'-phosphodiester bonds was verified using an in vitro debranching assay. J Biol Chem, 2004 Jan 2, 279(1), 42 - 50 Epub 2003 Oct 21. Regulatory mechanism of Dictyostelium myosin light chain kinase A; Tokumitsu H et al.; In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase . MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II . Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A . Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues . Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase . By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation . Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity . Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme. Biochem J, 2004 Jan 1, 377(Pt 1), 249 - 55 Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event; Cole A et al.; Phosphorylation of the endogenous GSK3alpha (glycogen synthase kinase-3alpha) at Tyr279 and GSK3beta at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo{3,4- d }pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein) . GSK3beta transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not . GSK3beta expressed in insect Sf 21 cells or E . coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216 in vitro after incubation with MgATP . The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone . Wild-type GSK3beta was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3beta lacking phosphate at Tyr216 . Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme. Curr Microbiol, 2003 Sep, 47(3), 180 - 5 Construction of secretory expression system suitable to express glucagon under the control of PL promoter; Wen C et al.; It was reported that PL promoter and alkaline phosphatase (phoA) signal peptide were used to construct secretory expression plasmid suitable to express glucagon and {Des-His1} glucagon in E . coli BL21 herein . Expression studies showed these two peptides could be expressed and secreted into the culture medium . The expression yield of recombinant glucagon reached 3.46 mg/L/OD600 unit of cells in shake flask . The yield of {Des-His1} glucagon was found to be higher than that of glucagon . In addition, some factors involved in secretion were studied too. Anal Chem, 2003 Jul 15, 75(14), 3451 - 9 Genetic engineering of an allosterically based glucose indicator protein for continuous glucose monitoring by fluorescence resonance energy transfer; Ye K et al.; Real-time monitoring of blood glucose could vastly reduce a number of the long-term complications associated with diabetes . In this article, we present a novel approach that relies on a glucose-binding protein engineered such that a 20% reduction in fluorescence due to the fluorescence resonance energy transfer occurs as a result of glucose binding . This change in fluorescence provides a signal for the optical detection of glucose . The novel glucose indicator protein (GIP) was created by fusing two fluorescent reporter proteins (green fluorescent proteins) to each end of an Escherichia coli glucose-binding protein in such a manner that the spatial separation between the fluorescent moieties changes when glucose binds, thus generating a distinct optical signal that can be used for glucose detection . By placing the GIP within a dialysis hollow fiber sensor, a microsensor has been developed for continuous monitoring of glucose . The sensor had a response time to sudden glucose changes within 100 s and was reversible . The sensor was shown to have an optional range on the order of 10 microM of glucose. Can J Microbiol, 2003 Jun, 49(6), 391 - 8 Magnetic bead hybridization to detect enterotoxigenic Escherichia coli strains associated with cattle in environmental water sources; Tsai YL et al.; A magnetic capture hybridization - polymerase chain reaction (MCH-PCR) method was used to increase the detection sensitivity of the enterotoxin gene LTIIa, used as a biomarker for waste in environmental samples . The samples were collected from cow lagoons of different farms and from environmental waters . Total DNA was extracted from colonies grown on mTEC medium or directly from environmental samples . The cow-specific Escherichia coli LTIIa gene was used as a DNA marker . A LTIIa-specific oligonucleotide probe was designed to capture the LTIIa marker during the MCH, followed by PCR . Varying levels of humic acid were added to the DNA extracts to evaluate the sensitivity and effectiveness of MCH-PCR . The minimal detection limit of MCH-PCR for the LTIIa gene was 2.5 ag/muL DNA . In the presence of humic acid, MCH-PCR was able to increase the detection sensitivity 10 000-fold over that of conventional PCR . The MCH-PCR could also detect one cell with the LTIIa DNA marker in a 1-L seeded environmental water sample . Results in this study indicate that MCH-PCR is more sensitive than nested PCR in testing environmental samples. Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12724 - 8 Epub 2003 Oct 20. Dynamic structures in Escherichia coli: spontaneous formation of MinE rings and MinD polar zones; Huang KC et al.; In Escherichia coli, division site selection is regulated in part by the Min-protein system . Oscillations of the Min proteins from pole to pole every approximately 40 sec have been revealed by in vivo studies of GFP fusions . The dynamic oscillatory structures produced by the Min proteins, including a ring of MinE protein, compact polar zones of MinD, and zebra-striped oscillations in filamentous cells, remain unexplained . We show that the Min oscillations, including mutant phenotypes, can be accounted for by in vitro-observed interactions involving MinD and MinE, with a crucial role played by the rate of nucleotide exchange . Recent discoveries suggest that protein oscillations may play a general role in proper chromosome and plasmid partitioning. Environ Pollut, 2004, 127(2), 303 - 11 Effects of environmental concentrations of atrazine on hemocyte density and phagocytic activity in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata); Russo J et al.; Immunotoxicological effects of environmentally relevant concentrations (10, 23, 50, 100 microg/l) of atrazine were studied in Lymnaea stagnalis . Individual hemolymph sampling was performed at 0, 24, 48, 72, 96, 168, 336, 504 and 672 h during exposure . Every atrazine concentration induced a significant increase in the mean number of circulating hemocytes, without any concentration-response relation . A peak (1.6-fold increase) of hemocyte density was observed after 96 h of exposure . After 504 h, the number of hemocytes remained higher only in the snails exposed to the two highest concentrations . Granulocytes contributed most to the increase in hemocyte density in herbicide-exposed snails . Both short- (24 and 96 h) and long-term (504 h) exposures resulted in significant inhibition of hemocyte phagocytic activity upon E . coli . Over the long-term, phagocytosis recovered for the two lowest concentrations . After 504 h of exposure, every herbicide level resulted in a significant reduction of reactive oxygen species production in E . coli-stimulated hemocytes, which was not observed for short-term exposures. Biosens Bioelectron, 2003 Nov 15, 19(2), 131 - 5 A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs; Soldatkin AP et al.; A novel urea biosensor based on immobilised recombinant urease as sensitive element and ion sensitive field effect transistor as transducer was developed . Recombinant urease from E . coli with an increased Km was photoimmobilised in PVA/SbQ (poly(vinyl alcohol) containing styrylpyridinium) membrane and has demonstrated quite good performance as biosensitive element . Enzymatic field effect transistors based on such a bioselective element were studied in model buffer solutions . This biosensor demonstrated an extended dynamic range up to 80 mM, a quite good reproducibility (standard deviation of the sensor responses was approximately 2.5%, n= 20 for urea concentration 10 mM) and a high stability . Such characteristics fit with the analytical requirements needed for urea control in plasma and liquids used during renal dialysis. Pathol Biol (Paris), 2003 Oct, 51(8-9), 512 - 5 {Revised prevalence of afa+ Escherichia coli strains in acute pyelonephritis of children}; Licznar P et al.; Two hundred E . coli strains isolated from children with pyelonephritis were investigated for the presence of six virulence factors . The used primers amplified adhesin pap and sfa, toxin haemolysin (hly) and cytotoxic necrotizing factor 1 (cnf1) and aerobactin (aer) . For afimbrial adhesin, the previously used set of primers could not allow to detect the newly reported afa operons (Le Bouguenec et al., 2001) . With a new set of primers specific for the afa operon family the prevalence of afa+ strains increased from 3.5% to 13.5% . Combinations of three or more factors in a same strain were found in 48.5% . Thirty two different urovirulent genotypes were observed; two strains contained the six studied factors. J Mol Biol, 2003 Oct 31, 333(4), 831 - 44 A novel binding protein for a member of CyP40-type Cyclophilins: N.crassa CyPBP37, a growth and thiamine regulated protein homolog to yeast Thi4p; Faou P et al.; Cyclophilins belong to the family of peptidyl-prolyl cis/trans isomerases (PPIases), which are ubiquitous and highly conserved enzymes capable of cis/trans isomerizing Xaa-Pro peptide bonds . Members of the CyP40-type cyclophilins have originally been described as components of hormone receptor complexes . Here, we describe NcCyP41, a CyP40 ortholog from Neurospora crassa, its expression in Escherichia coli and subsequent purification . Characterization of NcCyP41 reveals that it is a heat shock protein, which is active as a cyclosporin A-sensitive PPIase . Affinity chromatography using immobilized recombinant NcCyP41 yielded two major NcCyP41-binding proteins: Hsp80 (a Hsp90 ortholog from N.crassa) and CyPBP37 . CyPBP37 has not been described . In addition, this is the first record describing an interaction between a member of Cyp40-type cyclophilins and of CyPBP37-type proteins, respectively . CyPBP37 expression is repressed by thiamine and in the stationary phase in N.crassa . CyPBP37 is present in different isoforms . The expression of a CyPBP37 ortholog in yeast, Thi4p, is diminished in a mutant lacking one of the two CyP40 orthologs (Cpr7p) . In addition, the DeltaCpr7p deletion mutant shows a thiamine-dependent growth defect . We conclude that, in yeast, Cpr7p and Thi4p interact functionally. J Mol Biol, 2003 Oct 31, 333(4), 677 - 82 A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation; Lee YC et al.; In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination . Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer . To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations . The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation . Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl . It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells. FEMS Microbiol Lett, 2003 Oct 10, 227(1), 9 - 16 Two phenotypically compensating isocitrate dehydrogenases in Ralstonia eutropha; Wang ZX et al.; The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the glyoxylate bypass enzyme isocitrate lyase are involved in catabolism of isocitrate and play a key role in controlling the metabolic flux between the TCA cycle and the glyoxylate shunt . Two IDH genes icd1 and icd2 of Ralstonia eutropha HF39, encoding isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), were identified and characterized . Icd1 was functionally expressed in Escherichia coli, whereas icd2 was expressed in E . coli but no activity was obtained . Interposon-mutants of icd1 (HF39Deltaicd1) and icd2 (HF39Deltaicd2) of R . eutropha HF39 were constructed and their phenotypes were investigated . HF39Deltaicd1 retained 43% of IDH activity, which was not induced by acetate, and HF39Deltaicd2 expressed 74% of acetate-induced IDH activity . Both HF39Deltaicd1and HF39Deltaicd2 kept the same growth rate on acetate as the wild-type . These data suggested that IDH1 is induced by acetate . The interposon-mutants HF39Deltaicd1 and HF39Deltaicd2 accumulated the same amount of poly(3-hydroxybutyric acid) as the wild-type. Phytochemistry, 2003 Nov, 64(6), 1069 - 76 Cloning and regiospecificity studies of two flavonoid glucosyltransferases from Allium cepa; Kramer CM et al.; Two UDP-glucose-dependent flavonoid glucosyltransferases (EC 2.4.1.-) isolated from the epidermal layer of yellow onion (Allium cepa) were functionally expressed in Escherichia coli and their substrate specificity investigated . The two enzymes exhibited different substrate- and regio-specificity profiles . A . cepa UGT73G1 used a wide range of different flavonoid substrates including flavonoids not naturally occurring in onion . Regiospecificity was indicated for hydroxyl-groups of the C-3, C-7 and C-4' positions of the flavan backbone structure to yield flavonoid mono- and diglucosides . In contrast, A . cepa UGT73J1 showed activity only with the flavonoid mono-glucoside isoquercitrin and the isoflavone aglycone genistein, with regiospecificity for the C-7 position . The regiospecificity for both enzymes included positions that are glucosylated in flavonoids of onion bulbs, indicating their involvement in flavonoid biosynthesis in A . cepa. Biochemistry, 2003 Oct 28, 42(42), 12223 - 34 Circular dichroism and magnetic circular dichroism studies of the biferrous form of the R2 subunit of ribonucleotide reductase from mouse: comparison to the R2 from Escherichia coli and other binuclear ferrous enzymes; Strand KR et al.; Ribonucleotide reductase (RNR) catalyzes the synthesis of the four deoxyribonucleotides needed for DNA synthesis and repair in living organisms . The reduced {Fe(II)Fe(II)} form of the model mammalian enzyme, mouse RNR R2, has been studied using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD spectroscopies . Titrations of ferrous ion to the apo-enzyme have been performed and analyzed to investigate the metal binding affinity of the metal-binding site . Spectral features of individual iron sites have been analyzed to obtain detailed geometric and electronic structural information . VTVH MCD data have been collected and analyzed using two complementary models to obtain detailed ground state information including the zero-field splitting (ZFS) of both ferrous centers and the exchange coupling (J) between the two sites . These ground and excited state results provide a complete description of the biferrous site of mouse R2 . The biferrous site consists of one 4- and one 5-coordinate iron, with positive and negative ZFS values, respectively . Weak exchange coupling between the two ferrous centers is present, consistent with having carboxylate bridges . The two sites have highly cooperative and weak metal binding affinities . This may be a novel regulatory mechanism for RNR . These results are compared with those from reduced Escherichia coli R2 and reduced acyl-carrier protein Delta(9) desaturase to correlate to similarities and differences in their dioxygen reactivity. Biochemistry, 2003 Oct 28, 42(42), 12200 - 7 Drosophila TIMP is a potent inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3; Wei S et al.; The four tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the activity of matrix metalloproteinases (MMPs) and certain disintegrin and metalloproteinase (ADAM) family proteases in mammals . The protease inhibitory activity is present in the N-terminal domains of TIMPs (N-TIMPs) . In this work, the N-terminal inhibitory domain of the only TIMP produced by Drosophila (dN-TIMP) was expressed in Escherichia coli and folded in vitro . The purified recombinant protein is a potent inhibitor of human MMPs, including membrane-type 1-MMP, although it lacks a disulfide bond that is conserved in all other known N-TIMPs . Titration with the catalytic domain of human MMP-3 {MMP-3(DeltaC)} showed that dN-TIMP prepared by this method is correctly folded and fully active . dN-TIMP also inhibits, in vitro, the activity of the only two MMPs of Drosophila, dm1- and dm2-MMPs, indicating that the Drosophila TIMP is an endogenous inhibitor of the Drosophila MMPs . dN-TIMP resembles mammalian N-TIMP-3 in strongly inhibiting human tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) but is a weak inhibitor of human ADAM10 . Models of the structures of dN-TIMP and N-TIMP-3 are strikingly similar in surface charge distribution, which may explain their functional similarity . Although the gene duplication events that led to the evolutionary development of the four mammalian TIMPs might be expected to be associated with functional specialization, Timp-3 appears to have conserved most of the functions of the ancestral TIMP gene. Biochemistry, 2003 Oct 28, 42(42), 12133 - 42 Structural evidence for a 1,2-enediolate intermediate in the reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase, a member of the orotidine 5'-monophosphate decarboxylase suprafamily; Wise EL et al.; 3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5'-phosphate decarboxylase (OMPDC) are members of an enzyme suprafamily, the OMPDC suprafamily, because they are homologous enzymes that catalyze mechanistically distinct reactions using different substrates . KGPDC catalyzes the Mg(2+) ion-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate to yield L-xylulose 5-phosphate and CO(2); OMPDC catalyzes the metal ion-independent decarboxylation of OMP to UMP and CO(2) . Structural studies have shown that KGPDC and OMPDC share several strictly conserved active site residues that are used differently by each enzyme to catalyze their mechanistically distinct reactions . Although the mechanism of the KGPDC-catalyzed reaction has yet to be elucidated, it is thought to proceed via a Mg(2+) ion-stabilized 1,2-enediolate intermediate . Here we report the crystal structures of KGPDC complexed with L-gulonate 6-phosphate, L-threonohydroxamate 4-phosphate, and L-xylitol 5-phosphate, analogues of the substrate, enediolate intermediate, and product, as well as with the product, L-xylulose 5-phosphate, at 1.2, 1.8, 1.7, and 1.8 A resolution, respectively . These structures support a mechanism that involves the formation of a cis-1,2-enediolate intermediate . Contrary to expectations, the geometry of the intermediate does not involve bidentate coordination of both enediolate oxygen atoms to the Mg(2+) ion but rather involves only the coordination of the oxygen on C2 to the Mg(2+) ion . The oxygen atom on C1 instead forms hydrogen bonds to both Lys64 and Asp67, two strictly conserved active site residues . Lys64 also interacts with the oxygen on C2 and may serve to stabilize a cis conformation of the 1,2-enediolate . These structures also implicate His136 to be the general acid that protonates the 1,2-enediolate intermediate . This study further demonstrates that multiple unrelated enzyme functions can evolve from a single active site architecture without regard for substrate binding affinity or mechanism. Appl Microbiol Biotechnol, 2004 Apr, 64(2), 255 - 62 Epub 2003 Oct 18. SulA-independent filamentation of Escherichia coli during growth after release from high hydrostatic pressure treatment; Kawarai T et al.; To improve the efficiency of sterilization by high hydrostatic pressure treatment (HPT), it is desirable to know the biochemical process of bacteria most sensitive to the treatment . We investigated growth properties after release from HPT of exponentially growing Escherichia coli K-12 cells . We observed growth retardation after treatment (30 min at 37 degrees C) above 75 MPa . Long filamentous cells of about eight times normal cell length were observed at 90 min growth after treatment at 75 MPa . In the subsequent period the filamentous cells divided into normal-sized cells . recA and sulA mutant strains also formed filamentous cells, indicating that filamentation was SulA-independent . Nucleoids segregated normally in the filamentous cells . Only one FtsZ ring (or none) was detected at possible division sites in the elongated cells . Western blotting analysis demonstrated that the amount of FtsZ protein was not affected by the treatment . GTP-dependent in vitro polymerization of either FtsZ protein in E . coli crude extract or purified FtsZ protein, however, was sensitive to HPT . These facts suggest that HPT at 75 MPa denatures a fraction of FtsZ molecules, and that these denatured molecules interfere with the polymerization of functional FtsZ, resulting in the significantly reduced number of FtsZ rings. Gene Ther, 2003 Nov, 10(24), 2020 - 8 Creation of immune 'stealth' genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1; Ossevoort M et al.; A major obstacle in gene-therapy protocols is T-cell-mediated destruction of transgene-expressing cells . Therefore new approaches are needed to prevent rapid clearance of transduced cells . We exploited the Gly-Ala repeat (GAr) domain of the Epstein-Barr virus nuclear antigen-1, since the GAr prevents cytotoxic T-lymphocyte-epitope generation . Here we show that three different enzymes (viz . the E . coli LacZ gene encoded beta-galactosidase, firefly luciferase, and HSV1 thymidine kinase) fused with the GAr retained their function . Moreover, linking GAr with beta-galactosidase successfully prevented recognition of GAr-LacZ-expressing cells by beta-galactosidase-specific CTL . Nonetheless, vaccination with a GAr-LacZ adenovirus or with an allogeneic cell line expressing GAr-LacZ resulted in the induction of beta-gal-specific CTL . This demonstrates that the GAr domain does not inhibit cross presentation of antigens, but only affects breakdown of endogenously synthesized proteins . These data demonstrate how the GAr domain can be exploited to create immuno'stealth' genes by hiding transgene products from CTL-mediated immune attack. Nat Struct Biol, 2003 Nov, 10(11), 899 - 906 Epub 2003 Oct 19. Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy; Valle M et al.; Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP . Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of approximately 9 A, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site . In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu . This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu . From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome. Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12706 - 11 Epub 2003 Oct 17. Exploiting luminescence spectroscopy to elucidate the interaction between sugar and a tryptophan residue in the lactose permease of Escherichia coli; Vazquez-Ibar JL et al.; The crystal structure of the Escherichia coli lactose permease at 3.5 A with a bound substrate has been reported recently . The structure reveals the sugar-protein contacts, which include hydrophobic stacking between the galactopyranosyl ring of substrate and the indole side chain of Trp-151, as proposed previously . The nature of this interaction is studied here by exploiting the luminescence properties of Trp-151 in a mutant devoid of other tryptophan residues . The following phenomena are observed . (i) The fluorescence emission spectrum of Trp-151 and fluorescence-quenching experiments with water-soluble quenchers demonstrate that Trp-151 is in a hydrophilic environment . (ii) Substrate binding leads to a blue shift in the emission spectrum and reduction in accessibility to polar quenchers, indicating that Trp-151 becomes less exposed to aqueous solvent . (iii) The phosphorescence spectrum of Trp-151 is red-shifted in the presence of substrate, indicating charge separation of the triplet state due to a direct stacking interaction between the galactopyranosyl and indole rings . The spectroscopic data fully complement the x-ray structure and demonstrate the feasibility of fluorescence spectroscopy for studying sugar-protein interactions. Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12871 - 6 Epub 2003 Oct 17. Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer; Vilenchik MM et al.; This article extends our previous quantitative analysis of the relationship between the dynamics of the primary structure of DNA and mutagenesis associated with single-strand lesions to an analysis of the production and processing of endogenous double-strand breaks (EDSBs) and to their implications for oncogenesis . We estimate that in normal human cells approximately 1% of single-strand lesions are converted to approximately 50 EDSBs per cell per cell cycle . This number is similar to that for EDSBs produced by 1.5-2.0 Gy of sparsely ionizing radiation . Although EDSBs are usually repaired with high fidelity, errors in their repair contribute significantly to the rate of cancer in humans . The doubling dose for induced DSBs is similar to doubling doses for mutation and for the induction of carcinomas by ionizing radiation . We conclude that rates of production of EDSBs and of ensuing spontaneous mitotic recombination events can account for a substantial fraction of the earliest oncogenic events in human carcinomas. Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12648 - 53 Epub 2003 Oct 17. Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit; Pan H et al.; RNA pseudouridine synthase, TruB, catalyzes pseudouridine formation at U55 in tRNA . This posttranscriptional modification is almost universally conserved and occurs in the T arm of most tRNAs . We determined the crystal structure of Escherichia coli TruB apo enzyme, as well as the structure of Thermotoga maritima TruB in complex with RNA . Comparison of the RNA-free and -bound forms of TruB reveals that this enzyme undergoes significant conformational changes on binding to its substrate . These conformational changes include the ordering of the "thumb loop," which binds right into the RNA hairpin loop, and a 10 degree hinge movement of the C-terminal domain . Along with the result of docking experiments performed on apo TruB, we conclude that TruB recognizes its RNA substrate through a combination of rigid docking and induced fit, with TruB first rigidly binding to its target and then maximizing the interaction by induced fit. Int Immunol, 2003 Nov, 15(11), 1319 - 26 Thymus leukemia antigen (TL)-specific cytotoxic T lymphocytes recognize the alpha1/alpha2 domain of TL free from antigenic peptides; Tsujimura K et al.; The thymus leukemia antigens (TL) belong to the MHC class Ib family and can be recognized by CD8-dependent or -independent cytotoxic T lymphocytes (CTL) showing TL, but not H-2, restriction . We previously reported that the CTL epitope is TAP independent and in the present study we further characterize the recognition mechanism of CD8-dependent TL-specific TCRalphabeta CTL . We first prepared empty TL tetramers by way of peptide-independent folding with recombinant proteins produced in an Escherichia coli expression system, and showed that TL-specific CTL recognized TL without putative TL-associated peptide and/or post-translational modifications of TL by mammalian and insect cells . We next prepared transfectants expressing various chimeric TL molecules with mouse or human MHC class I as well as chimeric TL tetramers with recombinant proteins produced by insect cells, and demonstrated that chimeric TL whose alpha3 domain was replaced by that of H-2K(b), but not of HLA-A2, was sufficient for binding and activation of TL-specific CTL . These results indicate that TL-specific CTL recognize predominantly their alpha1/alpha2 domain as an epitope(s) and that the binding activity to the murine CD8 of the alpha3 domain of H-2K(b) is sufficient to induce their CTL activity, although it is known to be weaker than that of TL, but stronger than that of HLA . The results taken together indicate that CD8-dependent TL-specific TCRalphabeta CTL recognize an epitope(s) of the alpha1/alpha2 domain of TL free from antigenic molecules, and that CD8 plays an important role in stable interactions between TL and their corresponding TCR. J Interferon Cytokine Res, 2003 Sep, 23(9), 533 - 43 Microencapsulation of tumor necrosis factor oligomers: a new approach to proinflammatory cytokine inhibition; Oettinger C et al.; Antisense oligonucleotides offer great therapeutic potential provided adequate intracellular penetration can be achieved . In this study, we evaluated the effectiveness of microencapsulating antisense oligonucleotides to tumor necrosis factor (TNF) in suppressing TNF release in vitro and in vivo . Microencapsulation of TNF oligomers was performed using albumin to produce microcapsules 0.6-1.0 mum in size that target phagocytic cells . Albumin microcapsules containing fluoresceinated TNF oligomers were incubated with U-937 cells to observe uptake . Microcapsules were added to whole blood and stimulated with Escherichia coli endotoxin . Endotoxin was given intravenously (i.v.) to rats along with 100 mug microencapsulated TNF oligomers to determine TNF inhibition and animal survival . E . coli was given intraperitoneally (i.p.) along with gentamicin and microencapsulated TNF oligomers to assess TNF inhibition and animal survival . The duration of microencapsulated antisense TNF oligomers was also determined in vivo . The results demonstrated rapid uptake of the microcapsules by macrophages after 2 h and 4 h incubation . There was improvement in TNF inhibition in vitro and improved animal survival by microencapsulated antisense in both endotoxin (100% survival) and peritonitis models (70% survival) compared with free antisense oligomers in solution . Microencapsulation extended the duration of action of the oligomers to 72 h . Intracellular targeting of macrophages with antisense oligomers to TNF by microencapsules as a delivery system improves TNF inhibition using the models of whole blood endotoxin stimulation and endotoxic shock and peritonitis in rats. Biochem J, 2004 Feb 1, 377(Pt 3), 749 - 55 Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1-->6)-galactanase gene; Kotake T et al.; The nucleotide sequence depicted in Figure 1 has been submitted to the DDBJ nucleotide sequence database under the accession no . AB104898 . A gene encoding endo-beta-(1-->6)-galactanase from Trichoderma viride was cloned by reverse transcriptase-PCR and expressed in Escherichia coli . The gene contained an open reading frame consisting of 1437 bp (479 amino acids) . The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases . A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa . The gene product expressed in E . coli as a recombinant protein fused with thioredoxin and His(6) tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T . viride, i.e . recombinant enzyme endo-hydrolysed beta-(1-->6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse alpha-L-arabinofuranosidase-treated arabinogalactan protein from radish . It produced beta-(1-->6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and beta-(1-->6)-galactobiose as the major products at the final reaction stage . These results indicate that the cloned gene encodes an endo-beta-(1-->6)-galactanase . As far as we know, this is the first time an endo-beta-(1-->6)-galactanase has been cloned. Nucleosides Nucleotides Nucleic Acids, 2003 May-Aug, 22(5-8), 1539 - 43 Human cytidine deaminase: understanding the catalytic mechanism; Costanzi S et al.; In the absence of an experimentally elucidated three-dimensional structure of the human CDA, we built an homology model of this enzyme starting from the crystal structure of its E . coli homologous . Furthermore, we docked in the active site alternatively the substrate, the intermediate or the product . By means of molecular dynamics simulations, we determined the topology of the active site, identifying the amino acids involved in the catalytic mechanism, and outlining the central role played by E67. Nucleosides Nucleotides Nucleic Acids, 2003 May-Aug, 22(5-8), 1505 - 7 Cross-linking of Escherichia coli formamidopyrymidine-DNA glycosylase to DNA duplexes containing photoactivatable phenyl(trifluoromethyl)diazirine groups; Taranenko MV et al.; New reactive analogs of substrates for DNA repair enzyme E . coli Fpg protein containing the residues of 8-oxoguanine and photoactivatable phenyl(trifluoromethyl)diazirine groups were synthesized . Their substrate properties were investigated . Using photocross-linking technique, we established the presence of contacts of two nucleosides located near the oxoG with amino acids from the Fpg protein . The cross-linking efficiency achieved 10%. Nucleosides Nucleotides Nucleic Acids, 2003 May-Aug, 22(5-8), 1335 - 8 Synthesis and properties of oligonucleotide chimeras containing 5'-amino-2'-deoxy-2'-fluoroarabinonucleosides; Viazovkina E et al.; Oligonucleotide analogues comprised of 2'-deoxy-2'-fluoro-beta-D-arabinose units joined via P3'-N5' phosphoramidate linkages (2'F-ANA(5'N)) were prepared for the first time . Among the compounds prepared were a series of 2'OMe-RNA-{GAP}-2'OMe-RNA 'chimeras', whereby the "GAP" consisted of DNA, DNA(5'N), 2'F-ANA or 2'F-ANA(5'N) segments . The chimeras with the 2'F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5'-amino derivatives, i.e., 2'F-ANA > DNA > 2'F-ANA(5'N) > DNA(5'N) . Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E . coli RNase HI . In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2'F-ANA > 2'F-ANA(5'N) > DNA(5'N) . The corresponding relative rates observed with the human enzyme were: gap DNA > 2'F-ANA(5'N) > 2'F-ANA > DNA(5'N). Nucleosides Nucleotides Nucleic Acids, 2003 May-Aug, 22(5-8), 883 - 5 Thionucleotides as inhibitors of ribonucleotide reductase; Roy B et al.; Ribonucleosides and xylonucleosides bearing a disulfide function on the sugar ring were synthesized . Ribonucleosides belonging to the cytidine series were found to efficiently reduce dNTP pools in the human lymphoblastoid CEM/SS cell line. Biomed Khim, 2003 Mar-Apr, 49(2), 145 - 52 {Expression of the Mycobacterium tuberculosis cytochrome CYP51 gene in Escherichia coli}; Shavkunov AS et al.; Mycobacterium tuberculosis sterol 14 alpha-demethylase CYP51 is currently considered a promising target for a new generation of antitubercular drugs . Similarly to eucaryotic sterol 14 alpha-demethylases, candidate selective inhibitors of M . tuberculosis CYP51 belong to azoles, the N-substituted derivatives of imidazole and triazole . We have constructed a highly effective system for heterologous expression of MT CYP51 in E . coli . The recombinant protein was purified by metalloaffinity chromatography for primary in vitro screening of novel antitubercular azole compounds for their high affinity to the molecular target . Yield of the purified recombinant MT CYP51 was about 1.5 micromoles of the purified native protein per 1 l of E . coli cell culture . The recombinant protein interacted with ketoconazole with a Kd of 7.7 microM . MT CYP51 produced by our system for heterologous expression in E . coli may be used for initial testing of novel antimycobacterial azole drugs. Funct Integr Genomics, 2003 Dec, 3(4), 189 - 96 Epub 2003 Oct 16. Dynamic generation and qualitative analysis of metabolic pathways by a joint database/graph theoretical approach; Ehrentreich F et al.; The dynamic generation and qualitative analysis of metabolic networks relying on continuously growing qualified metabolic data by a joint database/graph theoretical approach is described . The procedure is applied to analyze the connectivity of a metabolic network after enzyme removal and to subsequently perform shortest path analyses . The focus lies on the analysis of the connectivity of the metabolic network depending on model assumptions . Here we analyze the influence of the number of strongly connected components on the assignment of reversibility or irreversibility of the biochemical reactions. J Anesth, 2000, 14(3), 147 - 50 Effect of linear polarized light radiation on impaired mitochondrial oxidative phosphorylation in skeletal muscle; Kudoh A et al.; PURPOSE: The aim of this study was to investigate the effect of linear polarized light radiation (LPLR) on mitochondrial oxidative phosphorylation impaired by hemorrhagic shock or Escherichia coli lipopolysaccharide (LPS) in skeletal muscle . METHODS: We studied the effect of LPLR on mitochondrial function of skeletal muscle by using a model of mitochondria impaired by hemorrhage or LPS . The oxygen uptake in states 3 and 4, the respiratory control ratio (RCR), and the adenosine diphosphate-to-oxygen ratio (ADP/O) were measured with a Clark oxygen electrode . RESULTS: Oxygen uptake in states 3 and 4, RCR, and ADP/O were significantly decreased by hemorrhage for 4 h and by LPS treatment for 12 h . Oxygen uptake in states 3 and 4 impaired by hemorrhage increased significantly from 40.1 +/- 3.2 to 60.1 +/- 5.4 nmol O(2).min(-1).mg protein(-1) after LPLR, and oxygen uptake in state 4 decreased significantly from 22.8 +/- 2.4 to 17.7 +/- 1.5 nmol O(2).min(-1).mg protein(-1) after LPLR . RCR and ADP/O were also significantly increased from 1.8 +/- 0.3 and 0.9 +/- 0.2 to 3.4 +/- 0.3 and 1.5 +/- 0.1, respectively, by LPLR . Oxygen uptake in states 3 and 4 impaired by LPS was improved from 46.6 +/- 5.1 and 21.0 +/- 1.9 to 53.8 +/- 6.2 and 17.9 +/- 2.3 nmol O(2).min(-1).mg protein(-1), respectively following LPLR . RCR and ADP/O were also elevated significantly from 2.2 +/- 0.2 and 0.9 +/- 0.2 to 3.0 +/- 0.3 and 1.4 +/- 0.2, respectively, after LPLR . CONCLUSION: LPLR improved mitochondrial oxidative phosphorylation of skeletal muscle impaired by hemorrhagic shock or E . coli LPS. Planta, 2004 Jan, 218(3), 406 - 16 Epub 2003 Oct 15. Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate, chloride and organic-anion transporters; Roth C et al.; A protein from Arabidopsis thaliana (L.) Heynh . showing homology to animal proteins of the NaPi-1 family, involved in the transport of inorganic phosphate, chloride, glutamate and sialic acid, has been characterized . This protein, named ANTR2 (for anion transporters) was shown by chloroplast subfractionation to be localized to the plastid inner envelope in both A . thaliana and Spinacia oleracea (L.) . Immunolocalization revealed that ANTR2 was expressed in the leaf mesophyll cells as well as in the developing embryo at the upturned-U stage . Five additional homologues of ANTR2 are found in the Arabidopsis genome, of which one was shown by green fluorescent protein (GFP) fusion to be also located in the chloroplast . All ANTR proteins share homology to the animal NaPi-1 family, as well as to other organic-anion transporters that are members of the Anion:Cation Symporter (ACS) family, and share the main features of transporters from this family, including the presence of 12 putative transmembrane domains and of a 7-amino acid motif in the fourth putative transmembrane domain . ANTR2 thus represent a novel protein of the plastid inner envelope that is likely to be involved in anion transport. Mol Genet Genomics, 2003 Dec, 270(5), 378 - 86 Epub 2003 Oct 17. Isolation and characterization of a phospholipase C delta isoform from pea that is regulated by light in a tissue specific manner; Venkataraman G et al.; Phosphoinositide-specific phospholipases C (PLCs) play an important role in many cellular responses and are involved in the production of secondary messengers . We report the cloning and characterization of a cDNA encoding a PLC-delta from Pisum sativum (PsPLC) . The amino acid sequence deduced from the cDNA sequence showed 75-80% identity to other plant PLCs and contained the characteristic X, Y and C2 domains . The genomic PLC clone from pea was also characterized and found to contain eight introns . The protein was expressed in Escherichia coli, but the recombinant product did not show any phosphoinositide (PI)- or phosphatidylinositol-4, 5-bisphosphate (PIP2)-specific activity, despite having all known residues required for such activity, and in spite of the fact that its C2 domain was shown to bind calcium . Under similar in vitro assay conditions the recombinant tobacco PLC used as a control showed calcium-dependent PI- and PIP2-specific activity . Though PsPLC did not show enzyme activity in vitro, and may represent an inactive form of PLC, such as those reported in some mammalian systems, analysis of the transcription of PsPLC showed that the gene is expressed in all pea tissues, and is regulated by light in a tissue-specific manner . Roots showed higher expression of PsPLC than shoots . A putative PsPLC promoter region (792 bp) was also cloned and found to contain root-specific and light-responsive cis elements, suggesting that this form of PLC may be involved in important functions in plants. Appl Microbiol Biotechnol, 2004 Mar, 64(1), 99 - 105 Epub 2003 Oct 15. Expression of foreign proteins in Escherichia coli by fusing with an archaeal FK506 binding protein; Ideno A et al.; Improper protein-folding often results in inclusion-body formation in a protein expression system using Escherichia coli . To express such proteins in the soluble fraction of E . coli cytoplasm, we developed an expression system by fusing the target protein with an archaeal FK506 binding protein (FKBP) . It has been reported that an archaeal FKBP from a hyperthermophilic archaeon, Thermococcus sp . KS-1 (TcFKBP18), possesses not only peptidyl-prolyl cis-trans isomerase activity, but also chaperone-like activity to enhance the refolding yield of an unfolded protein by suppressing irreversible protein aggregation . To study the effect of this fusion strategy with FKBP on the expression of foreign protein in E . coli, a putative rhodanese (thiosulfate sulfurtransferase) from a hyperthermophilic archaeon and two mouse antibody fragments were used as model target proteins . When they were expressed alone in E . coli, they formed insoluble aggregates . Their genes were designed to be expressed as a fusion protein by connecting them to the C-terminal end of TcFKBP18 with an oligopeptide containing a thrombin cleavage site . By fusing TcFKBP18, the expression of the target protein in the soluble fraction was significantly increased . The percentage of the soluble form in the expressed protein reached 10-28% of the host soluble proteins . After purification and protease digestion of the expressed antibody fragment-TcFKBP18 fusion protein, the cleaved antibody fragment (single-chain Fv) showed specific binding to the antigen in ELISA . This indicated that the expressed antibody fragment properly folded to the active form. Appl Microbiol Biotechnol, 2004 Mar, 64(1), 86 - 90 Epub 2003 Oct 16. Biotransformation of glucose to 5-keto-D-gluconic acid by recombinant Gluconobacter oxydans DSM 2343; Herrmann U et al.; For the conversion of glucose to 5-keto-D-gluconate (5-KGA), a precursor of the industrially important L-(+)-tartaric acid, Gluconobacter strains were genetically engineered . In order to increase 5-KGA formation, a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreductase (gno) was overexpressed in G . oxydans strain DSM 2434 . This enzyme is involved in the nonphosphorylative ketogenic oxidation of glucose and oxidizes gluconate to 5-KGA . As the 5-KGA reductase activity depends on the cofactor NADP+, the sthA gene (encoding Escherichia coli transhydrogenase) was cloned and overexpressed in the GNO-overproducing G . oxydans strain . Growth of the sthA-carrying strains was indistinguishable from the G . oxydans wild-type strain and therefore they were chosen for the coupled overexpression of sthA and gno . G . oxydans strain DSM 2343/pRS201-gno-sthA overproducing both enzymes showed an enhanced accumulation of 5-KGA. J Bacteriol, 2003 Nov, 185(21), 6486 - 9 rRNA antitermination functions with heat shock promoters; Seoh HK et al.; Transcription antitermination in the rRNA operons of Escherichia coli requires a unique nucleic acid sequence that serves as a signal for modification of the elongating RNA polymerase, making it resistant to Rho-dependent termination . We examined the antitermination ability of RNA polymerase elongation complexes that had initiated at three different heat shock promoters, dnaK, groE, and clpB, and then transcribed the antitermination sequence to read through a Rho-dependent terminator . Terminator bypass comparable to that seen with sigma(70) promoters was obtained . Lack of or inversion of the sequence abolished terminator readthrough . We conclude that RNA polymerase that uses sigma(32) to initiate transcription can adopt a conformation similar to that of sigma(70)-containing RNA polymerase, enabling it to interact with auxiliary modifying proteins and bypass Rho-dependent terminators. J Bacteriol, 2003 Nov, 185(21), 6477 - 80 Thymine at -5 is crucial for cpc promoter activity of Synechocystis sp . strain PCC 6714; Imashimizu M et al.; The levels of transcripts of the cpc operon were highly reduced in a PD-1 mutant of cyanobacterium Synechocystis sp . strain PCC 6714 . This was due to a substitution of C for T that occurred at 5 bp upstream of the transcription initiation site of the cpc operon . Any substitution for T at the -5 position drastically reduced both in vivo and in vitro promoter activity in cyanobacterium Synechococcus sp . strain PCC 7942 but not the in vivo activity in Escherichia coli . This suggests that the requirement of -5T appears to be specific for a cyanobacterial RNA polymerase-promoter combination. J Bacteriol, 2003 Nov, 185(21), 6472 - 6 A transcriptional pause synchronizes translation with transcription in the tryptophanase operon leader region; Gong F et al.; Regulation of transcription of the tryptophanase operon requires that translation of its leader peptide coding region, tnaC, be coupled with its transcription . We show in vitro that a transcription pause site exists at the end of the tnaC coding region and that translation of tnaC releases the paused transcription complex, coupling transcription with translation. J Bacteriol, 2003 Nov, 185(21), 6448 - 55 The gene yjcG, cotranscribed with the gene acs, encodes an acetate permease in Escherichia coli; Gimenez R et al.; We isolated an Escherichia coli mutant strain that suppresses the glycolate-negative phenotype of a strain deficient in both GlcA and LldP transporters of this compound . This suppressing phenotype was assigned to yjcG, a gene whose function was previously unknown, which was found to encode a membrane protein able to transport glycolate . On the basis of sequence similarity, the yjcG gene product was classified as a member of the sodium:solute symporter family . Northern experiments revealed that yjcG is cotranscribed with its neighbor, acs, encoding acetyl coenzyme A synthetase, which is involved in the scavenging acetate . The fortuitous presence of an IS2 element in acs, which impaired yjcG expression by polarity in our parental strain, allowed us to conclude that the alternative glycolate carrier became active after precise excision of IS2 in the suppressed strain . The finding that yjcG encodes a putative membrane carrier for glycolate and the cotranscription of yjcG with acs suggested that the primary function of the yjcG gene product (proposed gene name, actP) could be acetate transport and allowed us to define an operon involved in acetate metabolism . The time course of {1,2-(14)C}acetate uptake and the results of a concentration kinetics analysis performed with cells expressing ActP or cells deficient in ActP supported the the hypothesis that this carrier is an acetate transporter and suggested that there may be another transport system for this monocarboxylate. J Bacteriol, 2003 Nov, 185(21), 6400 - 8 Description and interpretation of adaptive evolution of Escherichia coli K-12 MG1655 by using a genome-scale in silico metabolic model; Fong SS et al.; Genome-scale in silico metabolic networks of Escherichia coli have been reconstructed . By using a constraint-based in silico model of a reconstructed network, the range of phenotypes exhibited by E . coli under different growth conditions can be computed, and optimal growth phenotypes can be predicted . We hypothesized that the end point of adaptive evolution of E . coli could be accurately described a priori by our in silico model since adaptive evolution should lead to an optimal phenotype . Adaptive evolution of E . coli during prolonged exponential growth was performed with M9 minimal medium supplemented with 2 g of alpha-ketoglutarate per liter, 2 g of lactate per liter, or 2 g of pyruvate per liter at both 30 and 37 degrees C, which produced seven distinct strains . The growth rates, substrate uptake rates, oxygen uptake rates, by-product secretion patterns, and growth rates on alternative substrates were measured for each strain as a function of evolutionary time . Three major conclusions were drawn from the experimental results . First, adaptive evolution leads to a phenotype characterized by maximized growth rates that may not correspond to the highest biomass yield . Second, metabolic phenotypes resulting from adaptive evolution can be described and predicted computationally . Third, adaptive evolution on a single substrate leads to changes in growth characteristics on other substrates that could signify parallel or opposing growth objectives . Together, the results show that genome-scale in silico metabolic models can describe the end point of adaptive evolution a priori and can be used to gain insight into the adaptive evolutionary process for E . coli. J Bacteriol, 2003 Nov, 185(21), 6340 - 7 Biochemical properties and regulated gene expression of the superoxide dismutase from the facultatively aerobic hyperthermophile Pyrobaculum calidifontis; Amo T et al.; Superoxide dismutase (SOD) was purified from a facultatively aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis VA1 . The purified native protein from aerobically grown cells exhibited 1,960 U of SOD activity/mg and contained 0.86 +/- 0.04 manganese and <0.01 iron atoms per subunit . The gene encoding SOD was cloned and expressed in Escherichia coli . Although the recombinant protein was soluble, little activity was observed due to the lack of metal incorporation . Reconstitution of the enzyme by heat treatment with either Mn or Fe yielded a highly active protein with specific activities of 1,970 and 434 U/mg, respectively . This indicated that the SOD from P . calidifontis was a cambialistic SOD with a preference toward Mn in terms of activity . Interestingly, reconstitution experiments in vitro indicated a higher tendency of the enzyme to incorporate Fe than Mn . When P . calidifontis was grown under anaerobic conditions, a majority of the native SOD was incorporated with Fe, indicating the cambialistic property of this enzyme in vivo . We further examined the expression levels of SOD and a previously characterized Mn catalase from this strain in the presence or absence of oxygen . Northern blot, Western blot, and activity measurement analyses revealed that both genes are expressed at much higher levels under aerobic conditions . We also detected a rapid response in the biosynthesis of these enzymes once the cells were exposed to oxygen. J Bacteriol, 2003 Nov, 185(21), 6225 - 32 Putative interhelical interactions within the PheP protein revealed by second-site suppressor analysis; Dogovski C et al.; Highly conserved glycine residues within span I and span II of the phenylalanine and tyrosine transporter PheP were shown to be important for the function of the wild-type protein . Replacement by amino acids with increasing side chain volume led to progressive loss of transport activity . Second-site suppression studies performed with a number of the primary mutants revealed a tight packing arrangement between spans I and II that is important for function and an additional interaction between spans I and III . We also postulate that a third motif, GXXIG, present in span I and highly conserved within different members of the amino acid-polyamine-organocation family, may function as a dimerization motif . Surprisingly, other highly conserved residues, such as Y60 and L41, could be replaced by various residues with no apparent loss of activity. J Biol Chem, 2004 Jan 23, 279(4), 2350 - 9 Epub 2003 Oct 16. Evidence for a second interaction between the regulatory amino-terminal and central output domains of the response regulator NtrC (nitrogen regulator I) in Escherichia coli; Harrod AC et al.; Nitrogen limitation in Escherichia coli activates about 100 genes . Their expression requires the response regulator NtrC (also called nitrogen regulator I or NR(I)) . Phosphorylation of the amino-terminal domain (NTD) of NtrC activates the neighboring central domain and leads to transcriptional activation from promoters that require sigma(54)-containing RNA polymerase . The NTD has five beta strands alternating with five alpha helices . Phosphorylation of aspartate 54 has been shown to reposition alpha helix 3 to beta strand 5 (the "3445 face") within the NTD . To further study the interactions between the amino-terminal and central domains, we isolated strains with alterations in the NTD that were able to grow on a poor nitrogen source in the absence of phosphorylation by the cognate sensor kinase . We identified strains with alterations located in the 3445 face and alpha helix 5 . Both types of alterations stimulated central domain activities . The alpha helix 5 alterations differed from those in the 3445 face . They did not cause a large scale conformational change in the NTD, which is not necessary for transcriptional activation in these mutants . Yeast two-hybrid analysis indicated that substitutions in both alpha helix 5 and the 3445 face diminish the interaction between the NTD and the central domain . Our results suggest that alpha helix 5 of the NTD, in addition to the 3445 face, interacts with the central domain . We present a model of interdomain signal transduction that proposes different functions for alpha helix 5 and the 3445 face. J Biol Chem, 2003 Dec 26, 278(52), 52865 - 72 Epub 2003 Oct 15. Tom1, a VHS domain-containing protein, interacts with tollip, ubiquitin, and clathrin; Yamakami M et al.; The gene for Tom1 was initially identified as a specific target of the oncogene v-myb . The Tom1 protein belongs to the VHS domain-containing protein family, and it has a GAT domain in a central part as well as an N-terminal VHS domain . VHS domain-containing proteins, including Hrs/Vps27, STAM, and GGA proteins, have been implicated in intracellular trafficking and sorting, but the role of Tom1 has not yet been elucidated . In this study, we found that Tom1 binds directly with ubiquitin chains and Tollip, which was initially isolated as a mediator of interleukin-1 signaling and has a capacity to bind ubiquitin chains . Gel filtration and subsequent Western blot analysis showed that endogenous Tom1 associates with Tollip to form a complex . In addition, Tom1 was found to be capable of binding to clathrin heavy chain through a typical clathrin-binding motif . Fluorescence microscopic analysis revealed that green fluorescent protein-Tom1 was localized predominantly in the cytoplasm, whereas its mutant with deletion of the clathrin-binding motif had a diffuse localization throughout the cell . Thus, we propose that a Tom1-Tollip complex functions as a factor that links polyubiquitinated proteins to clathrin. Mol Biochem Parasitol, 2003 Nov, 132(1), 47 - 53 Cloning and characterisation of the UDP-glucose 4'-epimerase of Trypanosoma cruzi; Roper JR et al.; Trypanosoma cruzi incorporates galactose into many of its cell-surface glycoconjugates but it is unable to transport this sugar through its hexose transporter . Epimerisation of UDP-glucose to UDP-galactose by UDP-glucose 4'-epimerase may be the only way that the parasites can obtain galactose . Here, we describe cloning the T . cruzi UDP-Glc 4'-epimerase (TcGALE) gene and show that it is functional by complementing an Escherichia coli epimerase-deficient strain . The T . cruzi GALE gene encodes a 42.4 kDa protein and the recombinant protein expressed in E . coli is a homodimer in solution with a specific activity of 3.8 U mg(-1) and K(m) for UDP-Gal of 114 microM . Unlike the human epimerase, T . cruzi UDP-Glc 4'-epimerase is unable to inter-convert UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine . This may explain why T . cruzi initiates O-glycosylation of its abundant GPI-anchored surface mucins via GlcNAcalpha1-O-Thr/Ser rather than the GalNAcalpha1-O-Thr/Ser linkage that is common for mucins from many other eukaryotes. Hepatol Res, 2003 Oct, 27(2), 136 - 142 Early development of cavernomatous vasculatures in portal venous thrombosis: morphometric kinetics in rabbit model; Ide T et al.; We tried to establish an animal model of portal venous thrombosis in order to analyze the ensuing pathological changes of the liver . An emulsion with Escherichia coli endotoxin and Lipiodol-Ultra-Fluide was injected into the portal vein of the left anterior lobe of the rabbit liver . The target lobe and portal vein were time-sequentially examined . In the experimental groups, it was found that fibrin thrombi were formed in the portal vein within 48 h after the injection and thrombi persisted for over 7 days . In an association with thrombus formation, numerous tortuously dilated vasculatures developed in the portal tract (cavernomatous vasculature) within 48 h . Both the number and the total area of the cavernomatous vasculatures increased from 2- to 3-fold more than in the control group at 72 h . The majority of proliferated vasculatures were positive for alpha-smooth muscle actin, thus suggesting that they derived from the portal venous branches . In conclusion, portal endotoxemia may be one of the pathogenetic factors of portal venous thrombosis and, in this model, the cavernomatous vasculatures rapidly developed from the portal venous branches. Biochim Biophys Acta, 2003 Oct 20, 1634(1-2), 52 - 60 Synthetic capacity of Arabidopsis phosphatidylinositol synthase 1 expressed in Escherichia coli; Justin AM et al.; Phosphatidylinositol (PtdIns) synthase 1 from the plant Arabidopsis thaliana has been expressed in Escherichia coli in order to study the synthetic capacities of the enzyme . Analysis of the total fatty acid content of the bacteria shows that PtdIns synthase activity does not have a profound effect on the proportions of the different fatty acids produced, even if the presence of an extra acidic phospholipid leads to a global reduction of the lipid content . A closer analysis carried out on individual phospholipids reveals a global fatty acid composition almost unchanged in the two major bacterial lipids phosphatidylethanolamine (PtdEtn) and phosphatidylglycerol (PtdGro) . Phosphatidylinositol has a very unusual composition that shows the ability of the plant enzyme to use CDP-diacylglycerol molecular species absent from plants . We identified the various PtdIns molecular species . They represent a pool of the major molecular species of PtdEtn and PtdGro . These results, together with the determination of the apparent affinity constants of AtPIS1 for myo-inositol and CDP-diacylglycerol, allow us to discuss some of the constraints of PtdIns synthesis in plants in terms of specificity, which will depend on the subcellular localization of the protein. Insect Biochem Mol Biol, 2003 Nov, 33(11), 1075 - 84 Distinct LPS-induced signals regulate LPS uptake and morphological changes in medfly hemocytes; Soldatos AN et al.; Recently we demonstrated that lipopolysaccharide (LPS) promotes activation of the Ras/ERK cascade in medfly hemocytes and that phagocytosis of Escherichia coli by insect hemocytes is mediated by an integrin-dependent process via the activation of FAK/Src complex (J Biol Chem 273 (1998) 14813; FEBS Letters 496 (2001) 55) . In the current study we wanted to further elucidate the effects of LPS on medfly hemocytes, in order to better understand the regulation of the evolutionary conserved signaling mechanisms between insects and mammals . We initially observed that different stimuli, including LPS, E . coli, RGD, fibronectin and heat shock activate hemocyte ERK . The response of hemocytes to these stimuli denoted that hemocyte ERK is evidently stimulated by at least an LPS receptor and via an integrin-mediated process . The medfly hemocytes respond to LPS by changing their morphology, inducing the activation of several signaling pathways, including Ras/MEK/ERK, PI-3K/ERK and Rho pathways and contributing to LPS uptake . Experiments based on inhibitors of specific signaling pathways, such as manumycin A, toxin A, U0126, PD98059 and wortmannin revealed that Ras, MEK and PI-3K are involved in the activation of ERK . Whether PI-3K is an intermediate of Ras/MEK/ERK pathway or activates ERK via other signaling pathway it remains to be elucidated . ERK is not activated via Rho pathway, denoting that Rho may not be an upstream effector molecule of ERK pathway . Regarding the role(s) that these kinases play in hemocytes, it can be suggested that PI-3K and Rho GTPases can modulate hemocyte shape changes, whereas ERK, Ras and MEK cannot . In addition, PI-3K as well as Ras and MEK through ERK activation participate in LPS endocytosis . Therefore, PI-3K shares a dual role; it is involved both in cell shape changes and in LPS endocytosis . Since ERK activation appears to be independent of the integrity of actin filaments, as cytochalasin D and latrunculin A did not block ERK activation, it can be concluded that LPS endocytosis is independent of actin cytoskeleton remodeling as is the case in mammalian systems. J Eukaryot Microbiol, 2003 Sep-Oct, 50(5), 317 - 23 Isolation and characterization of glutathione reductase from Physarum polycephalum and stage-specific expression of the enzyme in life-cycle stages with different oxidation-reduction levels; Minami Y et al.; Physarum polycephalum has a life cycle with several distinct phases that have different oxidation-reduction requirements . To investigate the relationship between the life cycle and the oxidation-reduction state, we isolated glutathione reductase (GR; EC 1.6.4.2) from Physarum microplasmodia . The enzyme was found to be a homodimer with a subunit M(r) of 49,000, and K(m) values for oxidized glutathione and NADPH of 40 and 28.6 microM, respectively . We then constructed a cDNA library from microplasmodium mRNA and cloned GR cDNA from the library . The isolated cDNA consisted of 1,475 bp encoding a polypeptide of 452 amino acids . The amino acid sequence similarity was about 50% with GRs of other organisms, and several conserved sequence motifs thought to be necessary for activity are evident in the Physarum enzyme . Escherichia coli transformed with an expression vector containing the cDNA synthesized the active GR . Genomic Southern blot analysis indicated that the GR gene is present as a single copy in the Physarum genome . Immunoblot analysis and RT-PCR analysis detected GR mRNA expression in the microplasmodium, plasmodium, and sclerotium, but not in the spore or flagellate . GR activity was low in the spore and flagellate . These results suggest that the glutathione oxidation-reduction system relates to the Physarum life cycle. Avian Dis, 2003 Jul-Sep, 47(3), 672 - 80 Comparison of the intravenous chicken challenge method with the embryo lethality assay for studies in avian colibacillosis; Gibbs PS et al.; In previous studies, the embryo lethality assay (ELA) was able to discriminate between virulent and avirulent avian Escherichia coli isolates and to predict percent mortality of the embryos resulting from an isolate based on three traits . The abilities to resist host complement, presence of Colicin V activity, and presence of the increased serum survival gene cluster (iss), were used together in a logistic regression analysis to predict the percentage of embryo deaths resulting from each of 20 avian E . coli isolates used in the ELA . In the present study, the same 20 isolates are used in an intravenous chicken challenge model in an effort to determine whether the ELA could be used to replace chicken challenge studies . Correlations between the mortality and a combination of mortality and morbidity (the survivors at trial termination with lesions suggestive of colibacillosis) and the previous ELA results and with selected isolate traits were performed . Additionally, resulting body weights in surviving chickens were compared between groups . The highest positive correlations were observed between the ELA and the combined mortality/morbidity of the intravenous challenge (r = 0.861, P < 0.0001 for the first ELA challenge, and r = 0.830, P < 0.0001 for the second ELA challenge) . The IV challenge combined mortality/morbidity results had the highest correlation coefficients with the presence of iss (r = 0.864, P < 0.0001) and the expression of ColV (r = 0.878, P < 0.0001) . The presence of tsh was slightly correlated with mortality (r = 0.465, P = .0389) but demonstrated a higher correlation with the combined mortality and morbidity of the IV challenge (r = 0.558, P = 0.0106) . Even though the ELA results in a higher number of nonspecific deaths, the two challenge methods exhibit similar results and a high correlation with each other . Interestingly, some of the isolates showed differences in their ability to cause mortality between the ELA and the IV challenge model . Furthermore, some isolates reflected significant differences in body weights of surviving birds at IV trial termination. World J Gastroenterol, 2003 Oct, 9(10), 2164 - 8 Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli; Ning JY et al.; AIM: To clone and express the antigen of monoclonal antibody (MAb) PD4 for further investigation of its function . METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen . After failed in the library screening, immunoprecipitation and SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis . The antigen coming from Mycoplasma hyorhinis (M . hyorhinis) was further confirmed with Western blot analysis by infecting M . hyorhinis -free HeLa cells and eliminating the M . hyorhinis from MGC803 cells . The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations . Immunofluorescence assay was used to demonstrate if p37 protein could directly bind to gastric tumor cell AGS . RESULTS: The cDNA library constructed with MGC803 cells was screened by MAb PD4 as probes . Unfortunately, the positive clones identified with MAb PD4 were also reacted with unrelated antibodies . Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasma hyorhinis (M . hyorhinis) by sequencing of N-terminal amino acid residues . The membrane protein was intensively verified with Western blot by eliminating M . hyorhinis from MGC803 cells and by infecting M . hyorhinis-free HeLa cells . The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations . Immunofluorescence demonstrated that p37 protein could directly bind to gastric tumor cell AGS . CONCLUSION: The antigen recognized by MAb PD4 is from M . hyorhinis, which suggests the actions involved in MAb PD4 is possibly mediated by p37 protein or M . hyorhinis . As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation. Chemistry, 2003 Oct 17, 9(20), 4887 - 99 Stereoselective aldol additions catalyzed by dihydroxyacetone phosphate-dependent aldolases in emulsion systems: preparation and structural characterization of linear and cyclic iminopolyols from aminoaldehydes; Espelt L et al.; The potential of dihydroxyacetone phosphate (DHAP)-dependent aldolases to catalyze stereoselective aldol additions is, in many instances, limited by the solubility of the acceptor aldehyde in aqueous/co-solvent mixtures . Herein, we demonstrate the efficiency of emulsion systems as reaction media for the class I fructose-1,6-bisphosphate aldolase (RAMA) and class II recombinant rhamnulose-1-phosphate aldolase from E . coli (RhuA)-catalyzed aldol addition between DHAP and N-benzyloxycarbonyl (N-Cbz) aminoaldehydes . The use of emulsions improved the RAMA-catalyzed aldol conversions by three to tenfold relative to those in conventional DMF/water mixtures . RhuA was more reactive than RAMA towards the N-Cbz aminoaldehydes regardless of the reaction medium . With (S)- or (R)-Cbz-alaninal, RAMA exhibited preference for the R enantiomer, while RhuA had no enantiomeric discrimination . The linear N-Cbz aminopolyols thus obtained were submitted to catalytic intramolecular reductive amination to afford the corresponding iminocyclitols . This reaction was diastereoselective in all cases examined; the face selectivity was controlled by the stereochemistry of the newly formed hydroxyl group originating from the aldehyde . Characterization of the resulting iminocyclitols allowed the assessment of the diastereoselectivity of the enzymatic aldol reactions with respect to the N-protected aminoaldehyde . RAMA formed single diastereoisomers from N-Cbz-glycinal and from both enantiomers of N-Cbz-alaninal, while 14 % of the epimeric product was observed from N-Cbz-3-aminopropanal . Diastereoselectivity from RhuA was lower than that observed from RAMA . Interestingly, a single diastereoisomer was formed from (S)-Cbz-alaninal, whereas only a 34 % diastereomeric excess was observed from its enantiomer (i.e., (R)-Cbz-alaninal). Nature, 2003 Oct 16, 425(6959), 717 - 20 RNA molecules stimulate prion protein conversion; Deleault NR et al.; Much evidence supports the hypothesis that the infectious agents of prion diseases are devoid of nucleic acid, and instead are composed of a specific infectious protein . This protein, PrP(Sc), seems to be generated by template-induced conformational change of a normally expressed glycoprotein, PrP(C) (ref . 2) . Although numerous studies have established the conversion of PrP(C) to PrP(Sc) as the central pathogenic event of prion disease, it is unknown whether cellular factors other than PrP(C) might be required to stimulate efficient PrP(Sc) production . We investigated the biochemical amplification of protease-resistant PrP(Sc)-like protein (PrPres) using a modified version of the protein-misfolding cyclic amplification method . Here we report that stoichiometric transformation of PrP(C) to PrPres in vitro requires specific RNA molecules . Notably, whereas mammalian RNA preparations stimulate in vitro amplification of PrPres, RNA preparations from invertebrate species do not . Our findings suggest that host-encoded stimulatory RNA molecules may have a role in the pathogenesis of prion disease . They also provide a practical approach to improve the sensitivity of diagnostic techniques based on PrPres amplification. Oncogene, 2003 Oct 16, 22(46), 7101 - 7 The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form Lys63-linked polyubiquitin chains; Bothos J et al.; We recently described a novel checkpoint pathway that functions early in mitosis to delay chromosome condensation in response to microtubule poisons . The only gene implicated so far in this checkpoint pathway is chfr, whose protein product contains a RING domain and has ubiquitin ligase activity in vitro . The significance of this activity in vivo is unclear . A recent report suggested that the Chfr protein targets itself for proteasome-dependent degradation in mitotic cells through autoubiquitination . However, we observe that in mitosis Chfr exhibits a phosphorylation-dependent electrophoretic mobility shift with no change in overall protein levels . Further analysis of its ubiquitin ligase activity revealed that Chfr can catalyse the formation of noncanonical Lys63-linked polyubiquitin chains with Ubc13-Mms2 acting as the ubiquitin-conjugating enzyme . Ubc13-Mms2 and Lys63-polyubiquitin chains are not associated with targeting proteins to the proteasome, but rather with signaling cellular stress . We propose that Chfr may have a role in signaling the presence of mitotic stress induced by microtubule poisons. Br J Cancer, 2003 Oct 20, 89(8), 1517 - 23 O6-methylguanine-DNA-methyltransferase expression and gene polymorphisms in relation to chemotherapeutic response in metastatic melanoma; Ma S et al.; In a retrospective study, O(6)-methylguanine-DNA-methyltransferase (MGMT) expression was analysed by immunohistochemistry using monoclonal human anti-MGMT antibody in melanoma metastases in patients receiving dacarbazine (DTIC) as single-drug therapy or as part of combination chemotherapy with DTIC-vindesine or DTIC-vindesine-cisplatin . The correlation of MGMT expression levels with clinical response to chemotherapy was investigated in 79 patients with metastatic melanoma . There was an inverse relationship between MGMT expression and clinical response to DTIC-based chemotherapy (P=0.05) . Polymorphisms in the coding region of the MGMT gene were also investigated in tumours from 52 melanoma patients by PCR/SSCP and nucleotide sequence analyses . Single-nucleotide polymorphisms (SNPs) in exon 3 (L53L and L84F) and in exon 5 (I143V/K178R) were identified . There were no differences in the frequencies of these polymorphisms between these melanoma patients and patients with familial melanoma or healthy Swedish individuals . Functional analysis of variants MGMT-I143V and -I143V/K178R was performed by in vitro mutagenesis in Escherichia coli . There was no evidence that these variants decreased the MGMT DNA repair activity compared to the wild-type protein . All melanoma patients with the MGMT 53/84 polymorphism except one had tumours with high MGMT expression . There was no significant correlation between any of the MGMT polymorphisms and clinical response to chemotherapy, although an indication of a lower response rate in patients with SNPs in exon 5 was obtained . Thus, MGMT expression appears to be more related to response to chemotherapy than MGMT polymorphisms in patients with metastatic melanoma. RNA, 2003 Nov, 9(11), 1308 - 14 Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs; Moll I et al.; The Escherichia coli RNA chaperone Hfq was discovered originally as an accessory factor of the phage Qbeta replicase . More recent work suggested a role of Hfq in cellular physiology through its interaction with ompA mRNA and small RNAs (sRNAs), some of which are involved in translational regulation . Despite their stability under certain conditions, E . coli sRNAs contain putative RNase E recognition sites, that is, A/U-rich sequences and adjacent stem-loop structures . We show herein that an RNase E cleavage site coincides with the Hfq-binding site in the 5'-untranslated region of E . coli ompA mRNA as well as with that in the sRNA, DsrA . Likewise, Hfq protects RyhB RNA from in vitro cleavage by RNase E . These in vitro data are supported by the increased abundance of DsrA and RyhB sRNAs in an RNase E mutant strain as well as by their decreased stability in a hfq(-) strain . It is commonly believed that the RNA chaperone Hfq facilitates or promotes the interaction between sRNAs and their mRNA targets . This study reveals another role for Hfq, that is, protection of sRNAs from endonucleolytic attack. J Biol Chem, 2004 Jan 16, 279(3), 1637 - 42 Epub 2003 Oct 15. Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase; Fan K et al.; The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS . In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase . Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays . The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase . By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency . The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1' positions react slower . The P2 position of the substrates seems to favor large hydrophobic residues . Secondary structure studies for the peptide substrates revealed that substrates with more beta-sheetlike structure tend to react fast . This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus. J Biol Chem, 2004 Jan 9, 279(2), 1533 - 40 Epub 2003 Oct 15. The mitochondrial citrate transport protein: probing the secondary structure of transmembrane domain III, identification of residues that likely comprise a portion of the citrate transport pathway, and development of a model for the putative TMDIII-TMDIII' interface; Ma C et al.; The mitochondrial citrate transport protein (CTP) has been investigated by mutating 28 consecutive residues within transmembrane domain III (TMDIII), one at a time, to cysteine . A cysteine-less CTP that retains wild-type functional properties, served as the starting template . The single Cys CTP mutants were abundantly expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system . The accessibility of each single Cys mutant to two methanethiosulfonate reagents was evaluated by determining the rate constants for inhibition of CTP function . These rate constants varied by over five orders of magnitude . With two independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of 4 was observed from residues 123-137 . Based on the pattern of accessibility we conclude that residues 123-137 exist as an alpha-helix . Although less certain, a combination of the rate constant data and the specific activity data with the single Cys mutants suggests that the alpha-helical secondary structure may extend to residue 113 . Furthermore, the rate constant data define water-accessible and water-inaccessible faces of the helix . We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer . Finally, based on a combination of the CTP inhibition rate constant data and the existence of significant sequence identity with a transmembrane segment within glycophorin A that forms a portion of its dimer interface, a model for a putative CTP TMDIII-TMDIII' dimer interface has been developed. J Biochem (Tokyo), 2003 Sep, 134(3), 403 - 13 High-level expression of porcine liver cytochrome P-450 reductase catalytic domain in Escherichia coli by modulating the predicted local secondary structure of mRNA; Kimura S et al.; A direct expression system for the solubilized catalytic domains of NADPH-cytochrome P-450 reductase (sCPR) from rat (RsCPR) and porcine (PsCPR) in Escherichia coli cells was constructed using the expression plasmid pCWori(+) . PsCPR was minimally expressed, whereas RsCPR was highly expressed . Replacement of the nucleotides encoding Thr(60)Ser(61)Ser(62) in PsCPR with those for Ala(60)Pro(61)Pro(62) in RsCPR markedly increased the expression level of the protein . The local secondary structures of the mRNAs, which were predicted with the prediction p