Biochem J, 2003 Aug 1, 373(Pt 3), 987 - 92 Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta; Lim EK et al.; Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites . The compound is also an antioxidant and has potential utility as a general protectant against free radicals . Three glucosylated forms of caffeic acid are known to exist: the 3- O - and 4- O -glucosides and the glucose ester . This study describes for the first time a glucosyltransferase {UDP-glucose:glucosyltransferase (UGT)} that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid . The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro . The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs . The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter . Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides . The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3- O -glucoside . The data are discussed in the context of the utility of UGTs for natural product biotransformations. Biochemistry, 2003 May 20, 42(19), 5945 - 54 Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1; Mirshad JK et al.; The double substitution of Glu156 with Leu and Gly157 with Val in the Escherichia coli RecA protein results in a severely reduced level of recombination and constitutive coprotease behavior . Here we present our examination of the biochemical properties of this mutant protein, RecA N99, in an effort to understand its phenotype and the role of loop 1 (L1) in RecA function . We find that RecA N99 protein has reduced single-stranded DNA (ssDNA)-dependent ATP hydrolysis activity, which is not as sensitive to the presence of SSB protein as wild-type RecA protein . RecA N99 protein is also nearly unable to utilize duplex DNA as a polynucleotide cofactor for ATP hydrolysis, and it shows both a decreased rate of association with ssDNA and a diminished capacity to bind DNA in the secondary binding site . The mutant protein has a corresponding reduction in DNA strand exchange activity, which probably results in the decrease in recombination activity in vivo . The constitutive induction of the SOS response may be a consequence of the impaired ability to repair damaged DNA, resulting in unrepaired ssDNA which can act as a cofactor for the cleavage of LexA repressor . These findings point to an involvement of L1 in both the primary and secondary DNA binding sites of the RecA protein. Biochemistry, 2003 May 20, 42(19), 5937 - 44 Biochemical basis of the constitutive coprotease activity of RecA P67W protein; Mirshad JK et al.; The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes . We examined the biochemical properties of this mutant in an effort to understand these altered behaviors . We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well . This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA . An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA . As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response . The DNA strand exchange activity of RecA P67W protein is also altered . Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced . We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein. Biochemistry, 2003 May 20, 42(19), 5885 - 95 Dimerization and inter-chromophore distance of Cph1 phytochrome from Synechocystis, as monitored by fluorescence homo and hetero energy transfer; Otto H et al.; We investigated the dimerization of phytochrome Cph1 from the cyanobacterium Synechocystis by fluorescence resonance energy transfer (FRET) . As donor we used the chromophore analogue phycoerythrobilin (PEB) and as acceptor either the natural chromophore phycocyanobilin (PCB; hetero transfer) or PEB (homo transfer) . Both chromophores bind in a 1:1 stoichiometry to apo-monomers expressed in Escherichia coli . Energy transfer was characterized by time-resolved fluorescence intensity and anisotropy decay after excitation of PEB by picosecond pulses from a tunable Ti-sapphire laser system . ApoCph1 was first assembled with PEB at a low stoichiometry of 0.1 . The remaining sites were then sequentially titrated with PCB . In the course of this titration, the mean lifetime of PEB decreased from 3.33 to 1.25 ns in the P(r) form of Cph1, whereas the anisotropy decay was unaffected . In the P(fr)/P(r) photoequilibrium (about 65% P(fr)), the mean lifetime decreased significantly less, to 1.67 ns . These observations provide strong support for inter-chromophore hetero energy transfer in mixed PEB/PCB dimers . The reduced energy transfer in P(fr) may be due to a structural difference but is at least in part due to the difference in spectral overlap, which was 4.1 x 10(-13) and 1.6 x 10(-13) cm(3) M(-1) in P(r) and P(fr), respectively . From the changes in the mean lifetime, rates of hetero energy transfer of 0.68 and 0.37 ns(-1) were calculated for the P(r) form and the P(fr)/P(r) photoequilibrium, respectively . Sequential titration of apo Cph1 with PEB alone to full occupancy did not affect the intensity decay but led to a substantial increase in depolarization . This is the experimental signature of homo energy transfer . Values for the rate of energy transfer k(HT) (0.47 ns(-1)) and the angle 2theta between the transition dipole moment directions (2theta = 45 +/- 5 degrees) were determined from an analysis of the concentration dependence of the anisotropy at five different PEB/Cph1 stoichiometries . The independently determined rates of hetero and homo energy transfer are thus of comparable magnitude and consistent with the energy transfer interpretation . Using these results and exploiting the 2-fold symmetry of the dimer, the chromophore-chromophore distance R(DA) was calculated and found to be in the range 49 A < R(DA) < 63 A . Further evidence for energy transfer in Cph1 dimers was obtained from dilution experiments with PEB/PEB dimers: the lifetime was unchanged, but the anisotropy increased as the dimers dissociated with increasing dilution . These experiments allowed a rough estimate of 5 +/- 3 microM for the dimer dissociation constant . With the deletion mutant Cph1Delta2 that lacks the carboxy terminal histidine kinase domain less energy transfer was observed suggesting that in this mutant dimerization is much weaker . The carboxy terminal domain of Cph1 that is involved in intersubunit trans-phosphorylation and signal transduction thus plays a dominant role in the dimerization . The FRET method provides a sensitive assay to monitor the association of Cph1 monomers. Biochemistry, 2003 May 20, 42(19), 5867 - 76 Importance of the D and E helices of the molecular chaperone DnaK for ATP binding and substrate release; Slepenkov SV et al.; The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five alpha-helices (alphaA-alphaE) (residues 508-608) that is followed by a 30-residue disordered, flexible region (609-638) . The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region is not known . By sequentially deleting the flexible subdomain and the individual lid helices, we deduced the importance of each structural unit to creating long-lived DnaK-peptide complexes . Here we report that (i) the alphaD helix is essential for long-lived DnaK-peptide complexes . For example, ATP triggers the dissociation of a acrylodan-labeled p5 peptide (ap5, a-CLLLSAPRR) from wtDnaK and DnaK595(A-D) with k(off) equal to 7.6 and 8.9 s(-1), respectively, whereas when the D-helix is deleted, creating DnaK578(A-C), k(off) jumps to 207 s(-1) . (ii) The presence of the alphaB helix impacts the rate of the ATP-induced high-to-low affinity conformational change . For example, ATP induces this conformational change in a lidless variant, DnaK517(1/2A), with a rate constant of 442 s(-1), whereas, after adding back the B-helix (residues 518-554), ATP induces this conformational change in DnaK554(A-B) with a rate constant of 2.5 s(-1) . Our interpretation is that this large decrease occurs because the B-helix of the DnaK554(A-B) is bound in the substrate-binding site . (iii) The deletion analysis also revealed that residues 596-638, which comprise the alphaE helix and the flexible subdomain, affect ATP binding . Our results are consistent with this part of the lid producing conformational heterogeneity, perhaps by binding to the ATPase domain. Biochemistry, 2003 May 20, 42(19), 5792 - 801 Interaction with membrane lipids and heme ligand binding properties of Escherichia coli flavohemoglobin; Bonamore A et al.; Escherichia coli flavohemoglobin has been shown to be able to bind specifically unsaturated and/or cyclopropanated fatty acids with very high affinity . Unsaturated or cyclopropanated fatty acid binding results in a modification of the visible absorption spectrum of the ferric heme, corresponding to a transition from a pentacoordinated (typical of the ligand free protein) to a hexacoordinated, high spin, heme iron . In contrast, no detectable interaction has been observed with saturated fatty acid, saturated phospholipids, linear, cyclic, and aromatic hydrocarbons pointing out that the protein recognizes specifically double bonds in cis conformation within the hydrocarbon chain of the fatty acid molecule . Accordingly, as demonstrated in gel filtration experiments, flavohemoglobin is able to bind liposomes obtained from lipid extracts of E . coli membranes and eventually abstract phospholipids containing cis double bonds and/or cyclopropane rings along the acyl chains . The presence of a protein bound lipid strongly affects the thermodynamic and kinetic properties of imidazole binding to the ferric protein and brings about significant modifications in the reactivity of the ferrous protein with oxygen and carbon monoxide . The effect of the bound lipid has been accounted for by a reaction scheme that involves the presence of two sites for the lipid/ligand recognition, namely, the heme iron and a non-heme site located in a loop region above the heme pocket. Biochemistry, 2003 May 20, 42(19), 5729 - 35 Hydroperoxidase II of Escherichia coli exhibits enhanced resistance to proteolytic cleavage compared to other catalases; Chelikani P et al.; Catalase (hydroperoxidase) HPII of Escherichia coli is the largest catalase so far characterized, existing as a homotetramer of 84 kDa subunits . Each subunit has a core structure that closely resembles small subunit catalases, supplemented with an extended N-terminal sequence and compact flavodoxin-like C-terminal domain . Treatment of HPII with trypsin, chymotrypsin, or proteinase K, under conditions of limited digestion, resulted in cleavage of 72-74 residues from the N-terminus of each subunit that created a homotetramer of 76 kDa subunits with 80% of wild-type activity . Longer treatment with proteinase K removed the C-terminal domain, producing a transient 59 kDa subunit which was subsequently cleaved into two fragments, 26 and 32 kDa . The tetrameric structure was retained despite this fragmentation, with four intermediates being observed between the 336 kDa native form and the 236 kDa fully truncated form corresponding to tetramers with a decreasing complement of C-termini (4, 3, 2, and 1) . The truncated tetramers retained 80% of wild-type activity . The T(m) for loss of activity during heating was decreased from 85 to 77 degrees C by removal of the N-terminal sequence and to 59 degrees C by removal of the C-terminal domain, revealing the importance of the C-terminal domain in enzyme stability . The sites of cleavage were determined by N- and C-terminal sequencing, and two were located on the surface of the tetramer with a third being exposed by removal of the C-terminal domain. Biochemistry, 2003 May 20, 42(19), 5674 - 83 In vitro evolution of the binding specificity of neocarzinostatin, an enediyne-binding chromoprotein; Heyd B et al.; Neocarzinostatin is the most studied member of the enediyne-chromoprotein family, and is clinically used as an antitumoral agent . Neocarzinostatin could be a promising drug delivery vehicle if new binding specificities could be conferred to its protein scaffold . We used in vitro evolution methods to demonstrate that this approach is feasible . We created large libraries containing between 1.7 x 10(8) and 1.4 x 10(9) independent clones, where up to 13 side chains pointing toward the binding crevice were randomly substituted . We then used phage display to select variants that bind to a model ligand (testosterone) which is unrelated to the natural ligand of neocarzinostatin . Several different binders were selected from each library . The corresponding proteins were expressed in Escherichia coli and their affinities and specificities were characterized in detail . K(D) values of about 20 nM were obtained for streptavidin-bound testosterone . The K(D) of selected proteins for free soluble testosterone are between 7 and 55 microM and therefore higher than the K(D) for streptavidin-bound testosterone . The spacer and streptavidin used during selection contributed to the high affinity of the selected binders for the target . Binding studies of 15 different steroids related to testosterone allowed us to determine that C3, 4, 5, 6, and 7 on cycles A and B and the conjugated 3 oxo group of the steroid molecule were essential for molecular recognition . Other testosterone analogues substituted on C1, 2, 9, 11, 15, and 17 were not discriminated from testosterone . These results demonstrate that the binding specificity of this protein family can be extended to compounds that are completely unrelated to the natural enediyne chromophore family . This type of highly expressed, stable proteins with tailored binding properties have a wide potential range of applications. Biochemistry, 2003 May 20, 42(19), 5632 - 9 Asymmetric binding of the high-affinity Q(H)(*)(-) ubisemiquinone in quinol oxidase (bo3) from Escherichia coli studied by multifrequency electron paramagnetic resonance spectroscopy; Grimaldi S et al.; Ubiquinone-2 (UQ-2) selectively labeled with (13)C (I =(1)/(2)) at either the position 1- or the 4-carbonyl carbon is incorporated into the ubiquinol oxidase bo(3) from Escherichia coli in which the native quinone (UQ-8) has been previously removed . The resulting stabilized anion radical in the high-affinity quinone-binding site (Q(H)(*)(-)) is investigated using multifrequency (9, 34, and 94 GHz) electron paramagnetic resonance (EPR) spectroscopy . The corresponding spectra reveal dramatic differences in (13)C hyperfine couplings indicating a strongly asymmetric spin density distribution over the quinone headgroup . By comparison with previous results on labeled ubisemiquinones in proteins as well as in organic solvents, it is concluded that Q(H)(*)(-) is most probably bound to the protein via a one-sided hydrogen bond or a strongly asymmetric hydrogen-bonding network . This observation is discussed with regard to the function of Q(H) in the enzyme and contrasted with the information available on other protein-bound semiquinone radicals. Biochemistry, 2003 May 20, 42(19), 5592 - 9 Structures along the catalytic pathway of PrmC/HemK, an N5-glutamine AdoMet-dependent methyltransferase; Schubert HL et al.; Posttranslational methylation of release factors on the glutamine residue of a conserved GGQ motif is required for efficient termination of protein synthesis . This methylation is performed by an N(5)-glutamine methyltransferase called PrmC/HemK, whose crystal structure we report here at 2.2 A resolution . The electron density at the active site appears to contain a mixture of the substrates, S-adenosyl-L-methionine (AdoMet) and glutamine, and the products, S-adenosyl-L-homocysteine (AdoHcy) and N(5)-methylglutamine . The C-terminal domain of PrmC adopts the canonical AdoMet-dependent methyltransferase fold and shares structural similarity with the nucleotide N-methyltransferases in the active site, including use of a conserved (D/N)PPY motif to select and position the glutamine substrate . Residues of the PrmC (197)NPPY(200) motif form hydrogen bonds that position the planar Gln side chain such that the lone-pair electrons on the nitrogen nucleophile are oriented toward the methyl group of AdoMet . In the product complex, the methyl group remains pointing toward the sulfur, consistent with either an sp(3)-hybridized, positively charged Gln nitrogen, or a neutral sp(2)-hybridized nitrogen in a strained conformation . Due to steric overlap within the active site, proton loss and formation of the neutral planar methylamide product are likely to occur during or after product release . These structures, therefore, represent intermediates along the catalytic pathway of PrmC and show how the (D/N)PPY motif can be used to select a wide variety substrates. Biochemistry, 2003 May 20, 42(19), 5547 - 54 Substrate-induced conformational changes in Escherichia coli taurine/alpha-ketoglutarate dioxygenase and insight into the oligomeric structure; O'Brien JR et al.; The enzymes in the alpha-ketoglutarate (alphaKG) dependent dioxygenase superfamily represent the largest class of non-heme iron oxidases and have important medical, ecological, and biotechnological roles . One such enzyme, taurine/alpha-ketoglutarate dioxygenase (TauD), catalyzes the conversion of 2-aminoethanesulfonate (taurine) to sulfite and aminoacetaldehyde while decomposing alphaKG to succinate and CO(2) . This alphaKG dependent dioxygenase is expressed in Escherichia coli under sulfur starvation conditions and allows the cell to utilize taurine, and other similar sulfonates in the environment, as an alternative sulfur source . In this work, we report the structures of the apo and holo forms of TauD to 1.9 A resolution (R(cryst) = 21.2%, R(free) = 24.9%) and 2.5 A resolution (R(cryst) = 22.5%, R(free) = 27.8%), respectively . The models reported herein provide significant new insight into the substrate orientations at the active site and the conformational changes that are induced upon taurine binding . Furthermore, analysis of our crystallographic data coupled with reanalysis of the crystallographic model (resolution = 3.0 A, R(cryst) = 28.1, R(free) = 32.0) presented by Elkins et al . (Biochemistry (2002) 41, 5185-5192) reveals an alternative oligomeric arrangement for the enzyme that is consistent with the conserved primary and secondary structure elements of other alphaKG dependent dioxygenases. Biomacromolecules, 2003 May-Jun, 4(3), 815 - 20 Synthesis and characterization of chimeric silkworm silk; Asakura T et al.; A synthetic gene encoding a chimeric silklike protein was constructed that combined a polyalanine encoding region (Ala)(18), a sequence slightly longer than the (Ala)(12-13) found in the silk fibroin from the wild silkworm Samia cynthia ricini, and a sequence encoding GVGAGYGAGAGYGVGAGYGAGVGYGAGAGY, found in the silk fibroin from the silkworm Bombyx mori . A tetramer of the chimeric repeat sequence encoding a approximately 29 kDa protein was expressed as a fusion protein in Escherichia coli . In comparison to S . c . ricini silk, the chimeric protein demonstrated improved solubility because it could be dissolved in 8 M urea . The purified protein assumed an alpha-helical structure based on solid-state (13)C CP/MAS NMR and was less prone to conformational transition to a beta-sheet, unlike native silk proteins from S . c . ricini . Model peptides representing the crystalline region of S . c . ricini silk fibroin, (Ala)(12) and (Ala)(18), formed beta-sheet structures . Therefore, the solubility and structural transitions of the chimeric protein were significantly altered through the formation of this chimeric silk . This experimental strategy to the study of silk structure and function can be used to develop an improved understanding of the contributions of protein domains in repetitive silkworm and spider silk sequences to structure development and structural transitions. Biomacromolecules, 2003 May-Jun, 4(3), 572 - 80 Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides; Trabbic-Carlson K et al.; Genetically engineered elastin-like polypeptides consisting of Val-Pro-Gly-X-Gly repeats, where X was chosen to be Lys every 7 or 17 pentapeptides (otherwise X was Val), were synthesized and expressed in E . coli, purified, and chemically cross-linked using tris-succinimidyl aminotriacetate to produce hydrogels . Swelling experiments indicate hydrogel mass decreases by 80-90% gradually over an approximate 50 degrees C temperature range . Gels ranged in stiffness from 0.24 to 3.7 kPa at 7 degrees C and from 1.6 to 15 kPa at 37 degrees C depending on protein concentration, lysine content, and molecular weight . Changes in gel stiffness and loss angle with cross-linking formulation suggest a low-temperature gel structure that is nearly completely elastic, where force is transmitted almost exclusively through fully extended polypeptide chains and chemical cross-links, and a high-temperature gel structure, where ELP chains are contracted and force is transmitted through chemical cross-links as well as frictional contact between polypeptide chains. Biomacromolecules, 2003 May-Jun, 4(3), 504 - 9 Mechanism of the polymerization reaction initiated and catalyzed by the polyhydroxybutyrate synthase of Ralstonia eutropha; Zhang S et al.; Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB . The Ralstonia eutropha PHB synthase purified from recombinant E . coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium . Several lines of evidence suggest that the homodimer is the active form of the synthase . The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site . The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified . It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site . To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C(319) mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured . The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis . Two identical thiol groups from C(319) residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions . We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase . These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme J Dairy Sci, 2003 Apr, 86(4), 1163 - 70 Characterization and proteolytic origins of specific peptides appearing during lipopolysaccharide experimental mastitis; Moussaoui F et al.; Based on the compositional change of the proteose peptone fraction, proteolysis was studied over time following lipopolysaccharide-induced experimental mastitis . Electrophoresis of the proteose peptone fraction revealed many degradation products . Five peptides were identified by amino-terminal sequencing as internal fragments of beta-, kappa-, alpha(s1)-, and alpha(s2)-casein that were generated by somatic cell proteases . Although kappa-casein is considered particularly resistant to endogenous proteolysis, a kappa-casein peptide was electrophoretically isolated in association with a beta-casein fragment . The in vitro kinetic studies of caseinate hydrolysis by elastase, one of the main polymorphonuclear neutrophil (PMN) proteases, suggested that the beta-casein peptide might be generated by elastase . In addition, elastase activity in milk PMN was higher during the inflammation of the mammary gland than prior to infusion. Parasitology, 2003 Apr, 126(Pt 4), 303 - 12 Vaccination of mice against experimental Neospora caninum infection using NcSAG1- and NcSRS2-based recombinant antigens and DNA vaccines; Cannas A et al.; NcSAG1 and NcSRS2, the two major immunodominant tachyzoite surface antigens of the apicomplexan parasite Neospora caninum, were investigated for their potential as vaccine candidates in mice . Recombinant recNcSRS2 and recNcSAG1 were expressed in Escherichia coli as poly-histidine-tagged fusion proteins . Separate groups of mice were immunized with purified recNcSAG1, recNcSRS2, or a combination of both, and were then challenged with N . caninum tachyzoites . Subsequent experiments included intramuscular vaccination of mice with the eukaryotic expression plasmid pcDNA3 containing either NcSRS2 or NcSAG1 cDNA inserts, followed by a single booster with the corresponding recombinant antigens . Immunization with a crude somatic antigen (NC1-extract) was included in the experiments . Following challenge, the presence of the parasite in the different organs was assessed by a N . caninum-specific PCR, while the parasite burden in infected brain tissue was assessed by quantitative real-time PCR . Immunization of mice employing individual recombinant antigens, or combined recNcSAG1/recNcSRS2, resulted in a lower degree of protection against cerebral infection, when compared to combined DNA/recombinant antigen vaccination . Serological analysis showed that this protective effect was associated with the occurrence of antibodies directed against native parasite antigens in those animals receiving combined DNA/recombinant antigen vaccination . Conversely, mice immunized with recombinant antigens alone generated antibodies recognizing only the recombinant antigens . Mice experiencing clinical signs such as walking disorders, rounded back, apathy and paralysis were observed only in the untreated positive control groups, but never in the vaccinated groups . Our results suggest that a combined DNA/recombinant antigen-vaccine, based on NcSAG1 and NcSRS2, respectively, exhibited a highly significant protective effect against experimentally induced cerebral neosporosis in mice. Chembiochem, 2003 May 9, 4(5), 425 - 33 Semisynthesis and characterization of the first analogues of pro-neuropeptide y; von Eggelkraut-Gottanka R et al.; Enzymatic cleavage of prohormone neuropeptide Y (proNPY) leads to mature neuropeptide Y (NPY), a widely distributed neuropeptide with multiple functions both peripherally and centrally . A single dibasic pair of amino acids, Lys38-Arg39, represents the recognition motif for a class of hormone-processing enzymes known as prohormone convertases (PCs) . Two members of this PC family, PC1/3 and PC2, are involved in proNPY cleavage . The aim of this work was to establish an effective method for the generation of full-length 69-amino acid proNPY analogues for further studies of prohormone convertase interaction . We have chosen two ligation sites in order to perform the semisynthesis of proNPY analogues by expressed protein ligation (EPL) . By using the intein-mediated purification system (IMPACT) with improved conditions for intein splicing, we were able to isolate proNPY 1-40 and proNPY 1-54 fragments as Cterminal thioesters . Peptides bearing Nterminal cysteine instead of the naturally occurring Ser41 and Thr55 residues, respectively, were generated by solid-phase peptide synthesis . Moreover, labels (carboxyfluorescein and biotin) were inserted into the peptide sequences . The synthesis of the {C41}proNPY 41-69 fragment, which proved to be a difficult peptide sequence, could be achieved by the incorporation of two pseudo-proline derivatives . Western blot analysis revealed that all five proNPY analogues are recognized by monoclonal antibodies directed against NPY as well as against the Cflanking peptide of NPY (CPON). Chembiochem, 2003 May 9, 4(5), 406 - 12 Large-scale in vivo synthesis of the carbohydrate moieties of gangliosides GM1 and GM2 by metabolically engineered Escherichia coli; Antoine T et al.; Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc; Gal = galactose, Glc = glucose, Ac = acetyl) and GM1 (Galbeta-3GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc . The GM2 oligosaccharide-producing strain TA02 was devoid of both beta-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP = cytidine monophosphate), alpha-2,3-sialyltransferase, UDP-GlcNAc (UDP = uridine diphosphate) C4 epimerase, and beta-1,4-GalNAc transferase . When this strain was cultivated on glycerol, exogenously added lactose and sialic acid were shown to be actively internalized into the cytoplasm and converted into GM2 oligosaccharide . The in vivo synthesis of GM1 oligosaccharide was achieved by taking a similar approach but using strain TA05, which additionally overexpressed the gene for beta-1,3-galactosyltransferase . In high-cell-density cultures, the production yields for the GM2 and GM1 oligosaccharides were 1.25 g L(-1) and 0.89 g L(-1), respectively. Chembiochem, 2003 May 9, 4(5), 396 - 405 Monitoring the cellular surface display of recombinant proteins by cysteine labeling and flow cytometry; Jose J et al.; A general method is described that allows one to follow the surface display of recombinant proteins in Escherichia coli without having to use specific antibodies or enzymatic reactions . The method is based on cysteine-specific labeling through Michael addition to the double bond of maleimide and its derivatives, and takes advantage of the fact that naturally occurring surface proteins in E . coli contain no accessible cysteine residues . The method is easy to perform and could be simply applied to different analytic procedures including Western blot, spectral photometry, and flow cytometry . By using this new labeling method, single cells bearing a distinct protein at the surface could be selected by fluorescence-activated cell sorting . The data were obtained by using autodisplay, an efficient surface display system established for E . coli, but the method presented here represents rather a general solution for analyzing the surface display of recombinant proteins, independent of the cellular system used. Int Arch Allergy Immunol, 2003 Apr, 130(4), 258 - 65 Cloning and characterisation of two IgE-binding proteins, homologous to tropomyosin and alpha-tubulin, from the mite Lepidoglyphus destructor; Saarne T et al.; BACKGROUND: The dust mite Lepidoglyphus destructor is a major source of mite allergy in European rural environments, but it also causes allergy in urban populations around the world . We have previously cloned, sequenced and expressed several allergens from L . destructor (Lep d 2, Lep d 5, Lep d 7 and Lep d 13) . The aim of this study was to identify and clone additional allergens from L . destructor, and to evaluate their IgE-binding reactivities . METHODS: PCR and screening with sera from L . destructor-sensitised individuals were used to isolate new clones from a phage display L . destructor cDNA library . The complete coding sequences of the clones were determined and expressed as His(6)-tagged recombinant proteins in Escherichia coli . The recombinant proteins were analysed by SDS-PAGE, immunoblotting and mass spectrometry . RESULTS: Two new clones, showing homology to tropomyosin and alpha-tubulin in several species, were isolated from the phage display L . destructor cDNA library . Due to its homology to group 10 dust mite allergens, the tropomyosin clone was named Lep d 10 . The IgE-binding frequencies of the recombinant Lep d 10 and alpha-tubulin were 13% (18/136) and 12% (11/95), respectively, among subjects with IgE reactivity to mites and/or crustaceans . CONCLUSIONS: Two new allergens from L . destructor have been identified and can now be added to the repertoire of recombinant L . destructor allergens . In addition, both these allergens belong to highly conserved protein families and may be important for evaluation of allergenic cross-reactivity . Urol Int, 2003, 70(4), 335 - 6 Spontaneous communication between a simple renal cyst and the pyelocaliceal system with a gas-producing infection; Okubo Y et al.; We present the extremely rare case of a 44-year-old woman who presented with right flank pain and high fever, which proved to be a case of spontaneous communication between a renal cyst and the pyelocaliceal system caused by increased pressure in the renal pelvic cavity exerted by a stone leading to infection . J Atheroscler Thromb, 2003, 10(2), 99 - 108 Apoptosis of endothelial cells may be mediated by genes of peroxisome proliferator-activated receptor gamma 1 (PPARgamma 1) and PPARalpha genes; Inoue I et al.; Apoptosis in human umbilical vein endothelial cells (HUVECs) was prevented by transfection with the gene for the human full-length peroxisome proliferator-activated receptor alpha (PPARalpha), or acyl-coenzyme A synthetase (AcylCS) into HUVECs . In contrast, ligands/activators of PPARgamma 1 induced apoptosis by a cytochrome c-dependent mechanism in HUVECs transfected with human full-length PPARgamma 1, but not in hepatocytes . Co-transfection of PPARgamma 1 and PPARalpha protected the HUVEC apoptosis . The results suggest that the apoptosis of endothelial cells may be mediated by genes of PPARgamma1 and PPARalpha. J Biol Chem, 2003 Aug 1, 278(31), 29278 - 87 Epub 2003 May 11. Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B; He H et al.; The molecular machinery required for autophagy is highly conserved in all eukaryotes as seen by the high degree of conservation of proteins involved in the formation of the autophagosome membranes . Recently, both yeast Apg8p and its rat homologue Map1lc3 were identified as essential constituents of autophagosome membrane as a processed form . In addition, both the yeast and human proteins exist in two modified forms produced by a series of post-translational modifications including a critical C-terminal cleavage after a conserved Gly residue, and the smaller processed form is associated with the autophagosome membranes . Herein, we report the identification and characterization of three human orthologs of the rat Map1LC3, named MAP1LC3A, MAP1LC3B, and MAP1LC3C . We show that the three isoforms of human MAP1LC3 exhibit distinct expression patterns in different human tissues . Importantly, we found that the three isoforms of MAP1LC3 differ in their post-translation modifications . Although MAP1LC3A and MAP1LC3C are produced by the proteolytic cleavage after the conserved C-terminal Gly residue, like their rat counterpart, MAP1LC3B does not undergo C-terminal cleavage and exists in a single modified form . The essential site for the distinct post-translation modification of MAP1LC3B is Lys-122 rather than the conserved Gly-120 . Subcellular localization by cell fractionation and immunofluorescence revealed that three human isoforms are associated with membranes involved in the autophagic pathway . These results revealed different regulation of the three human isoforms of MAP1LC3 and implicate that the three isoforms may have different physiological functions. J Biol Chem, 2003 Aug 8, 278(32), 29913 - 24 Epub 2003 May 08. Mechanistic understanding of an altered fidelity simian immunodeficiency virus reverse transcriptase mutation, V148I, identified in a pig-tailed macaque; Diamond TL et al.; We have recently reported that the reverse transcriptase (RT) of SIVMNE 170 (170), which is a representative viral clone of the late symptomatic phase of infection with the parental strain, SIVMNE CL8 (CL8), has a largely increased fidelity, compared with the CL8 RT . In the present study, we analyzed the mechanistic alterations of the high fidelity 170 RT variant . First, we found that among several 170 RT mutations, only one, V148I, is solely responsible for the fidelity increase over the CL8 RT . This V148I mutation lies near the Gln-151 residue that we recently found is important to the low fidelity of RT and the binding of incoming dNTPs . Second, we compared dNTP binding affinity (Kd) and catalysis (kpol) of the CL8 RT and the CL8-V148I RT using pre-steady state kinetic analysis . In this experiment, the high fidelity CL8-V148I RT has largely decreased binding to both correct and incorrect dNTP without altering kpol . The fidelity increase imparted by the V148I mutation is likely because of the major reduction seen in RT binding to dNTPs . This parallels our findings with the Q151N mutant . Third, site-directed mutagenesis targeting amino acid residue 148 has revealed that a valine amino acid at this position is essential to RT infidelity . Based on these findings, we discuss possible structural impacts of residue 148 (and mutations at this site) on the interaction of RT with incoming dNTPs and infer how alterations in these properties may relate to viral replication and fitness. J Biol Chem, 2003 Jul 18, 278(29), 26817 - 22 Epub 2003 May 10. The C-terminally truncated NuoL subunit (ND5 homologue) of the Na+-dependent complex I from Escherichia coli transports Na+; Steuber J; The NADH:quinone oxidoreductase (complex I) from Escherichia coli acts as a primary Na+ pump . Expression of a C-terminally truncated version of the hydrophobic NuoL subunit (ND5 homologue) from E . coli complex I resulted in Na+-dependent growth inhibition of the E . coli host cells . Membrane vesicles containing the truncated NuoL subunit (NuoLN) exhibited 2-4-fold higher Na+ uptake activity than control vesicles without NuoLN . Respiratory proton transport into inverted vesicles containing NuoLN decreased upon addition of Na+, but was not affected by K+, indicating a Na+-dependent increase of proton permeability of membranes in the presence of NuoLN . The His-tagged NuoLN protein was solubilized, enriched by affinity chromatography, and reconstituted into proteoliposomes . Reconstituted His6-NuoLN facilitated the uptake of Na+ into the proteoliposomes along a concentration gradient . This Na+ uptake was prevented by EIPA (5-(N-ethyl-N-isopropyl)-amiloride), which acts as inhibitor against Na+/H+ antiporters. Biotechnol Appl Biochem, 2003 Aug, 38(Pt 1), 87 - 93 Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli; Mergulhao FJ et al.; Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ-proinsulin by Escherichia coli . The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ-proinsulin to the periplasmic space and culture medium . All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5'-untranslated regions thus making promoter strength comparisons more accurate . The highest ZZ-proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter . It was also demonstrated that the use of M9 minimal medium favours secretion. J Protein Chem, 2003 Jan, 22(1), 71 - 6 E292 is important for the aminoacylation activity of Escherichia coli leucyl-tRNA synthetase; Du X et al.; Escherichia coli leucyl-tRNA synthetase (LeuRS) has a large connecting polypeptide (CP1) inserted into its active site . It was demonstrated that the peptide bond between E292-A293 was crucial for the aminoacylation activity of E . coli LeuRS . To investigate the effect of E292 on the function of Escherichia coli LeuRS, E292 was mutated to K, F, S, D, Q and A . These mutations at 292 did not change the specific activity of the amino acid activation reaction . Though the conformational change of these mutants was not detected in CD, their aminoacylation activities were impaired to varying extents . The mutation of E to K decreased the aminoacylation activity to the largest extent . Analysis of the Km values of these mutants for the three substrates showed that the E292 was not involved in the binding of leucine and that all mutants had stronger binding with ATP. J Protein Chem, 2003 Jan, 22(1), 41 - 9 Chicken deoxyribonuclease: purification, characterization, gene cloning and gene expression; Hu CC et al.; Chicken DNase was purified to apparent homogeneity from the pancreas extract . It showed two isoforms, A and B forms, on cation-exchange chromatography . On SDS-PAGE it was a 30-kDa protein . When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882 . The enzyme was bound to concanavalin A, indicating its glycoprotein nature . The carbohydrate side chain could be removed by endoglycosidase F . Chicken DNase was activated by metal ions and for half-maximum activation, Mn2+ and Mg2+ required were 1 mM and 4 mM, respectively . The pH optimum was between 7 and 8 depending on the metal ions used . In the presence of Cu2+, it was almost completely inactivated by 0.1 M iodoacetate within 1 min . In the absence of Ca2+ at pH 8, chicken DNase resisted to the trypsin or beta-mercaptoethenol inactivation . When the purified enzyme was subjected to protein sequencing, approximately 93% of the sequence was established . Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced . The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5'-untranslated region and 166 of the 3' and, in the 5'-untranslated region, two types of sequences occurred . The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids . As compared with mammalian DNases, chicken DNase had an overall 58 +/- 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide . The cDNA was cloned into the pET15b expression vector . When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates. Chem Commun (Camb), 2003 Apr 7, (7), 822 - 3 Synthesis of protein-nucleic acid conjugates by expressed protein ligation; Lovrinovic M et al.; The synthesis of covalent conjugates of proteins and polyamide nucleic acids (PNA) is accomplished by expressed protein ligation of intein-fusion proteins and a PNA-cysteine conjugate. Neurol Res, 2003 Apr, 25(3), 263 - 7 Protective action of recombinant neurturin on dopaminergic neurons in substantia nigra in a rhesus monkey model of Parkinson's disease; Li H et al.; Parkinson's disease (PD) is a neurodegenerative disease characterized by muscular trembling palsy due to lack of dopamine (DA) in the substantia nigra-striatum (nigrostriatal) system resulting from the degeneration and necrosis of dopaminergic neurons . No effective cure has been found . Neurturin (NTN) has been demonstrated to act specifically on midbrain (mesencephalic) dopaminergic neurons with protective actions specifically . In the present study, we induced rhesus monkey model of Parkinson's disease by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) . Rhesus monkeys were randomly divided into a PD model group, NTN treatment group and normal control groups . In the NTN treatment group, 1 mg of E . coli-derived recombinant human NTN was injected into the cerebral ventricles 48 h before the injection of MPTP . Results indicated that Rhesus monkeys in the PD model group acquired PD symptoms that increasingly aggravated over time, while monkeys treated with NTN had less apparent or no symptoms . Using fluorospectrophotometry, the dopamine (DA), 5, 5-hydroxytrytamine (5-HT) and the 5-hydroxyindoleacetic acid (5-HIAA) contents of DA, 5-HT and 5-HIAA in substantia nigra, putamen and caudate nucleus in monkeys from the model group was found to be significantly lower than in the normal control group . While no significant differences were found between monkeys treated with NTN and normal control groups, the contents of DA, 5-HT and 5-HIAA in the NTN treatment group were higher than those observed in the PD model group . A dramatic loss of neurons in the substantia nigra in monkeys in the PD model group was observed by light microscopy, while no obvious loss was observed in the NTN treatment group in which the numbers of neurons were similar to those in normal controls . These results indicate that recombinant human NTN can prevent PD symptoms as well as protect dopaminergic neurons and preserve DA content in midbrain substantia nigra in rhesus monkeys exposed to MPTP. Pflugers Arch, 2004 Feb, 447(5), 760 - 2 Epub 2003 May 09. The acetyl-CoA transporter family SLC33; Hirabayashi Y et al.; The acetyl-CoA (Ac-CoA) transporter (AT-1) is a multiple transmembrane protein in the endoplasmic reticulum . Ac-CoA is transported to the lumen of the Golgi apparatus, where it serves as the substrate of acetyltransferases that modify the sialyl residues of gangliosides and glycoproteins . The AT-1 gene, originally named ACATN (acetyl-CoA transporter), was cloned from human melanoma cells . Although homologs of this family of proteins have been identified in lower organisms, such as Escherichia coli, Drosophila melanogaster, and Caenorhabditis . elegans, currently only one member of this SLC33A1 family has been identified in humans . Thus, SLC33A1 proteins should be re-named ACATN1 or AT-1 . Although acetylated gangliosides show a highly tissue-specific distribution, AT-1 is ubiquitously expressed . Phylogenetically, the AT-1 gene is highly conserved, suggesting that it is particularly significant . The precise physiological roles of this transporter protein, however, remain to be elucidated. Science, 2003 May 9, 300(5621), 976 - 80 Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump; Yu EW et al.; Multidrug efflux pumps cause serious problems in cancer chemotherapy and treatment of bacterial infections . Yet high-resolution structures of ligand transporter complexes have previously been unavailable . We obtained x-ray crystallographic structures of the trimeric AcrB pump from Escherichia coli with four structurally diverse ligands . The structures show that three molecules of ligands bind simultaneously to the extremely large central cavity of 5000 cubic angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions . Each ligand uses a slightly different subset of AcrB residues for binding . The bound ligand molecules often interact with each other, stabilizing the binding. FASEB J, 2003 Jul, 17(10), 1325 - 7 Epub 2003 May 08. Cyclooxygenase-2-deficient mice are resistant to endotoxin-induced inflammation and death; Ejima K et al.; Sepsis is a systemic inflammatory response to a blood-borne infection that is associated with an extremely high rate of morbidity and mortality . The present study investigates the role of cyclooxygenase (COX)-2 in host responses to bacterial endotoxemia . After administration of Escherichia coli lipopolysaccharide, 50% of wild-type mice die within 96 h . COX-2 deficient mice displayed a dramatic improvement in survival with reduced leukocyte infiltration into critical organs (kidneys and lungs) and a blunted and delayed induction of the cytokine inducible genes nitric oxide synthase 2 and heme oxygenase-1 . Translocation and activation of transcription factors important for signaling events during an inflammatory response, such as nuclear factor (NF)-kappaB, were also markedly reduced . While the absence of COX-2 did not alter the induction of several pro-inflammatory cytokines in tissue macrophages, induction of the anti-inflammatory cytokine IL-10 was exaggerated . Administration of IL-10 to wild-type mice reduced NF-kappaB activation . Taken together, our data suggest that COX-2 deficient mice are resistant to many of the detrimental consequences of endotoxemia . These beneficial effects occur, in part, by a compensatory increase in IL-10 that counterbalances the pro-inflammatory host response to endotoxemia. J Biol Chem, 2003 Jul 25, 278(30), 28237 - 45 Epub 2003 May 08. RNA-structural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon; Stelzl U et al.; Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence . This process presumably involves L4 interaction with the leader mRNA . Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro . Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction . Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site . Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions . Mutational analysis of the mRNA binding site is compatible with the structural model . In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how protein L4 may provoke transcription termination. J Biol Chem, 2003 Jul 18, 278(29), 26722 - 6 Epub 2003 May 08. Gain of glutaminase function in mutants of the ammonia-specific frog carbamoyl phosphate synthetase; Saeed-Kothe A et al.; Depending on their physiological role, carbamoyl phosphate synthetases (CPSs) use either glutamine or free ammonia as the nitrogen donor for carbamoyl phosphate synthesis . Sequence analysis of known CPSs indicates that, regardless of whether they are ammonia- or glutamine-specific, all CPSs contain the structural equivalent of a triad-type glutamine amidotransferase (GAT) domain . In ammonia-specific CPSs, such as those of rat or human, the catalytic inactivity of the GAT domain can be rationalized by the substitution of the Triad cysteine residue by serine (1) . The ammonia-specific CPS of Rana catesbeiana (fCPS) presents an interesting anomaly in that, despite its retention of the entire catalytic triad (2) and almost all other residues conserved in Triad GATs, it is unable to utilize glutamine as a nitrogen-donating substrate (3) . Based on our earlier work with the glutamine-utilizing E . coli CPS (eCPS), we have targeted residues Lys258 and Glu261 in the fCPS GAT domain as critical for preventing GAT function . Previously we have shown that substitution of the corresponding residues in eCPS by their fCPS counterparts (Leu --> Lys and Gln --> Glu) resulted in complete loss of GAT function in eCPS (3) . To examine the role of these residues in the fCPS GAT component, we have cloned the full-length fCPS gene from R . catesbeiana liver . Here we report the first heterologous expression of an ammonia-specific CPS and show that a single mutation of the frog enzyme, K258L, yields a gain of glutaminase function. J Biol Chem, 2003 Jul 11, 278(28), 25839 - 46 Epub 2003 May 07. Effect of protein kinase A-mediated phosphorylation on the structure and association properties of the enteropathogenic Escherichia coli Tir virulence protein; Hawrani A et al.; Enteropathogenic Escherichia coli virulence is dependent on delivery of the translocated intimin receptor protein (Tir) into host cells . Tir phosphorylation on a single tyrosine (Tyr-474) is essential in mediating cytoskeletal rearrangement correlated with disease . Tir is also phosphorylated on other residues, with cAMP-dependent kinase (PKA) modification shown to play a role in Tir function . However, the mechanism by which nontyrosine phosphorylation affects Tir function remains unclear . In this study, analytical ultracentrifugation, SDS and native gel electrophoresis revealed that both Tir and its C-terminal domain (residues 385-550 of Tir that include the PKA substrate sites) exist in an equilibrium of monomers, dimers, and in the case of Tir, higher oligomers . PKA phosphorylation (1:300, PKA/C-Tir, mol/mol) shifted the equilibrium of C-Tir, but not Tir, predominantly to the dimeric state, whereas, at 100-fold higher concentrations of PKA the phosphorylated C-Tir was largely monomeric . This monomeric state was also produced at the lower PKA concentration and physiological ionic strength . Phosphorylation-mediated effects were achieved without significant changes in secondary structure as determined by circular dichroism spectroscopy . The data presented indicate that PKA-mediated phosphorylation induces changes in the association properties of the C-terminal domain of Tir that may facilitate Tir-Tir interactions and subsequently C-Tir-host protein interactions in vivo. Blood, 2003 Sep 1, 102(5), 1753 - 63 Epub 2003 May 08. Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli; Jefford M et al.; Dendritic cells (DCs) are a family of leukocytes that initiate T- and B-cell immunity against pathogens . Migration of antigen-loaded DCs from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses . In humans, the major peripheral blood DC (PBDC) types, CD1c+ DCs and interleukin 3 receptor-positive (IL-3R+) plasmacytoid DCs, are significantly expanded in vivo with the use of Flt3 ligand (FL) . DC-like cells can also be generated from monocyte precursors (MoDCs) . A detailed comparison of the functional potential of these types of DCs (in an autologous setting) has yet to be reported . Here, we compared the functional capacity of FL-expanded CD1c+ PBDCs with autologous MoDCs in response to 3 different classes of stimuli: (1) proinflammatory mediators, (2) soluble CD40 ligand trimer (CD40L), and (3) intact bacteria (Escherichia coli) . Significant differences in functional capacities were found with respect to changes in phenotype, migratory capacity, cytokine secretion, and T-cell stimulation . MoDCs required specific stimuli for the expression of functions . They responded vigorously to CD40L or E coli, expressing cytokines known to regulate interferon-gamma (IFN-gamma) in T cells (IL-12p70, IL-18, and IL-23), but required prostaglandin E2 (PGE2) during stimulation to migrate to chemokines . In contrast, PBDCs matured in response to minimal stimulation, rapidly acquired migratory function in the absence of PGE2-containing stimuli, and were low cytokine producers . Interestingly, both types of DCs were equivalent with respect to stimulation of allogeneic T-cell proliferation and presentation of peptides to cytotoxic T lymphocyte (CTL) lines . These distinct differences are of particular importance when considering the choice of DC types for clinical applications. Clin Diagn Lab Immunol, 2003 May, 10(3), 459 - 68 Monoclonal antibodies to Escherichia coli-expressed P46 and P65 membranous proteins for specific immunodetection of Mycoplasma hyopneumoniae in lungs of infected pigs; Cheikh Saad Bouh K et al.; The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species-specific antigenic determinants . Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 bp) and N-terminally truncated P65(c) (1,200 bp) . These primers were shown to be specific to M . hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract . Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST) . Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M . hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis . Following induction by IPTG (isopropyl-beta-D-thiogalactopyranoside), both GST-P46 and GST-P65(c) recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65(c) moieties . The enriched E . coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65(c) monoclonal antibodies (MAbs) . The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain . Cross-reactivity study of the anti-P46 and anti-P65(c) MAbs towards two other M . hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes . No reactivity to other mycoplasma species tested was demonstrated . Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M . hyopneumoniae . Both anti-P46 and anti-P65(c) MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M . hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period. J Immunol Methods, 2003 May 1, 276(1-2), 175 - 83 Plaque reduction test: an alternative method to assess specific antibody response to pIII-displayed peptide of filamentous phage M13; Yang WJ et al.; Phage-displayed peptide systems have been used to identify the immunogenic epitopes and to develop the design of peptide-based or peptide-displaying phages themselves as vaccine candidates . To estimate the humoral immunity of phage-based vaccine, it is necessary to evaluate the antibody response specifically directed at the displayed peptide . Enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis are commonly used for this purpose . However, using these methods, it is not easy to distinguish the antibody response against phage coat protein or the antibody response specific to the displayed peptide . The purified anti-Mycoplasma hyopneumoniae IgG was used to screen heptapeptides displaying on the pIII coat protein of M13 phage . Four selected phage clones were chosen to immunize mice . In order to evaluate the specific antibody response that is directed against heptapeptides, advantage was taken of the natural property of M13 phage to infect Escherichia coli, which is mediated by the pIII coat protein binding with the F pili of E . coli, and plaque reduction tests were performed to assess the specificity of antibody response . By comparing the number of plaques produced by the different phages (which are the same except for the displayed peptides) neutralized by the antiserum, we could demonstrate that the specificity of antibody response is directed against the peptide displayed on pIII coat protein . The results described here indicate that plaque reduction test is a convenient and more precise method to detect the antibody against the phage-displayed peptide. Structure (Camb), 2003 May, 11(5), 591 - 603 tRNA-dependent active site assembly in a class I aminoacyl-tRNA synthetase; Sherlin LD et al.; The crystal structure of ligand-free E . coli glutaminyl-tRNA synthetase (GlnRS) at 2.4 A resolution shows that substrate binding is essential to construction of a catalytically proficient active site . tRNA binding generates structural changes throughout the enzyme, repositioning key active site peptides that bind glutamine and ATP . The structure gives insight into longstanding questions regarding the tRNA dependence of glutaminyl adenylate formation, the coupling of amino acid and tRNA selectivities, and the roles of specific pathways for transmission of tRNA binding signals to the active site . Comparative analysis of the unliganded and tRNA-bound structures shows, in detail, how flexibility is built into the enzyme architecture and suggests that the induced-fit transitions are a key underlying determinant of both amino acid and tRNA specificity. Dev Cell, 2003 May, 4(5), 610 - 1 Never fat or gaunt; James ES et al.; Bacteria stringently regulate the synthesis of their membrane phospholipids, but the responsible regulatory mechanisms are incompletely understood . In this issue of Developmental Cell, a study reports negative regulation of the transcription of several genes of fatty acid and phospholipid synthesis plus identification of the regulatory protein. Emerg Infect Dis, 2003 May, 9(5), 545 - 51 Human milk secretory antibodies against attaching and effacing Escherichia coli antigens; Noguera-Obenza M et al.; Secretory immunoglobulin A (sIgA) is a primary factor responsible for preventing attachment of enteropathogens to gut epithelium in breastfeeding infants . We compared the frequency of sIgA to major surface antigens of enterohemorrhagic Escherichia coli (EHEC) in milk of 123 women from the United States and Mexico to determine whether regional differences existed in the frequency of antibodies to these surface antigens . In both groups of women, milk commonly has sIgA against various EHEC lipopolysaccharides, EspA, EspB, intimin, and less frequently against Shiga toxin . The study suggests that persons living in the United States are exposed to attaching/effacing enteropathogens more frequently than is generally assumed . The low frequency of antibodies to Stx1 (in 12% of Mexican and in 22% of U.S . samples) suggests that the rare appearance of hemolytic uremic syndrome in adults is not due to neutralization of toxin at the gut level . Only anti-EspA is found in most milk samples from both populations of women . EspA may represent a useful target for an immunization strategy to prevent EHEC disease in humans. Proc R Soc Lond B Biol Sci, 2003 Apr 22, 270(1517), 843 - 8 The influence of cellular physiology on the initiation of mutational pathways in Escherichia coli populations; Notley-McRobb L et al.; The factors affecting the direction of evolutionary pathways and the reproducibility of adaptive responses were investigated under closely related but non-identical conditions . Replicate chemostat cultures of Escherichia coli were compared when adapting to partial or severe glucose limitation . Four independent populations used a reproducible sequence of early mutational changes under both conditions, with rpoS mutations always occurring first before mgl . However, there were interesting differences in the timing of mutational sweeps: rpoS mutations appeared in a clock-like fashion under both partial and severe glucose limitation, while mgl sweeps arose under both conditions but at different times . Interestingly, malT and mlc mutations appeared only under severe limitation . Even though the ancestors were genotypically identical, the semi-differentiated properties of bacteria growing with mild or severe glucose limitation sent the populations in characteristic directions . Mutation supply and the fitness contribution of mutations were estimated and demonstrated to be potential influences in the choice of particular adaptation pathways under severe and mild glucose limitation . Predicting all the mutations fixed in adapting populations is beyond our current understanding of evolutionary processes, but the interplay between ancestor physiology and the initiation of adaptation pathways is demonstrated and definable in bacterial populations. Biochem J, 2003 Aug 1, 373(Pt 3), 703 - 11 Dictyostelium discoideum protein phosphatase-1 catalytic subunit exhibits distinct biochemical properties; Andrioli LP et al.; Protein phosphatase-1 (PP1) is expressed ubiquitously and is involved in many eukaryotic cellular functions, although PP1 enzyme activity could not be detected in the social amoeba Dictyostelium discoideum cell extracts . In the present paper, we show that D . discoideum has a single copy gene that codes for the catalytic subunit of PP1 (DdPP1c) . DdPP1c is expressed throughout the D . discoideum life cycle with constant levels of mRNA, and its protein and amino acid sequence show a mean identity of 80% with other PP1c enzymes . However, it has a distinctive difference: the substitution of a phenylalanine residue (Phe(269) in the DdPP1c) for a highly conserved cysteine residue (Cys(273) in rabbit PP1c) in a region that was shown to have a critical role in the interaction of rabbit PP1c with toxin inhibitors . Wild-type DdPP1c and an engineered mutant form in which Phe(269) was replaced by a cysteine residue were expressed in Escherichia coli . Both recombinant activities were similarly inhibited by okadaic acid, tautomycin and microcystin . However, the Phe(269)-->Cys mutation resulted in a large increase in enzyme activity towards phosphorylase a and a higher sensitivity to calyculin A . These results, together with the molecular modelling of DdPP1c structure, indicate that the Phe(269) residue, which occurs naturally in D . discoideum, confers distinct biochemical properties on this enzyme. Cell Res, 2003 Apr, 13(2), 131 - 9 Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro; Kong XF et al.; A partial rice (Oryza sativa L.) cDNA clone, OsPI4K1c, was isolated through screening of a cDNA library constructed from tillering materials . OsPI4K1c encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa . The OsPI4K1c peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e . a lipid kinase unique (LKU) domain and a catalytic (CAT) domain . A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well . Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein . Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c . The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring . OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA) . However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4K1 in wounding responses. Cell Mol Life Sci, 2003 Mar, 60(3), 617 - 28 Design and evaluation of a diabody to improve protection against a potent scorpion neurotoxin; Aubrey N et al.; Diabodies are recombinant, dimeric, antibody-based molecules composed of two non-covalently associated single-chain antibody fragments that bind to an antigen in a divalent manner . In an attempt to develop more effective therapeutic molecules against scorpion venoms, we designed a diabody derived from monoclonal antibody 9C2, which neutralizes the toxicity of scorpion neurotoxin AahI in mammals . The recombinant diabody produced in the periplasm of Escherichia coli was purified to homogeneity in a single step by protein L-agarose affinity chromatography . It was functional, and possessed a high binding affinity to AahI (8 x 10(-11) M) . The bivalence of the diabody was confirmed by size-exclusion chromatography, isoelectrofocussing and electron microscopic observations . Finally, the diabody showed high thermal stability in serum and demonstrated protective activity when injected intraperitoneally in mice experimentally envenomed with toxin AahI . In conclusion, the diabody format gives the 9C2 molecule advantageous properties that are particularly important for potential clinical applications in the treatment of envenomations. Br Poult Sci, 2003 Mar, 44(1), 104 - 12 Interaction between genotype and dietary concentrations of methionine for immune function in commercial broilers; Rama Rao SV et al.; 1 . Growth, antibody response to sheep red blood cells (SRBC) and resistance to Escherichia coli were measured in broiler female chicks received from 4 (n = 100 in each) commercial genotypes (A, B, C and D) and fed with maize-soybean-deoiled rice bran based diets containing 4 concentrations of methionine (3.91, 4.46, 5.00 and 5.54 g/kg) . The diets were fed ad libitum from 1 to 49 d of age . 2 . Body weight gain and weight gain/food intake at 2 week intervals, response of broilers to inoculation of 0.5 ml of SRBC (0.5 or 2.5%), 0.1 ml of E . coli (10(-4) dilutions) culture, and 100 microg phytohaemogglutinin-P (PHA-P) at 43 d of age were measured . The responses to SRBC and E . coli inoculation were recorded at 5 d post inoculation (PI), while the responses to PHA-P were recorded at 12 and 24 h PI . 3 . Genotype by methionine interaction was not significant for body weight gain, but significant differences in weight gain were observed among different genotypes . Variation in methionine concentration did not influence body weight gain or weight gain/food intake at 1 to 14, or 42 d of age . At 28 d of age, chicks fed on the 3.91 g methionine/kg diet weighted significantly less than those on the other methionine concentrations . Genotype by methionine interaction was observed for food efficiency at 0 to 28 d of age but not at other ages . 4 . Antibody titres against SRBC and heart and air sac lesion score to E . coli challenge were not influenced by genotype-methionine interaction . Chicks given higher concentrations of methionine had higher antibody titres and greater cutaneous basophilic hypersensitivity (CBH) response than those given low levels of methionine . Also, variation was observed in expression of CBH response to PHA-P among different genotypes . 5 . It may be concluded that, although the commercial broiler chicks do not require more than 3.91 g methionine/kg for optimum growth and food efficiency, the immunity in terms of CBH response and antibody production to SRBC increased with the concentration of methionine in the diet in the majority of genotypes, indicating a higher methionine requirement for immunity than for weight gain. Int J Mol Med, 2003 Jun, 11(6), 697 - 704 The human TruB family of pseudouridine synthase genes, including the Dyskeratosis Congenita 1 gene and the novel member TRUB1; Zucchini C et al.; A novel human gene denominated TruB pseudouridine (psi) synthase homolog 1 (E . coli) (approved symbol, TRUB1) has been identified and characterized . Spanning approximately 40 kb on chromosome 10 and including 8 exons, TRUB1 is the first described human ortholog of bacterial TruB/psi55, a gene involved in tRNA pseudouridinilation . TRUB1 gene encodes a 349-amino acid product, with a VFAVHKPKGPTSA box in positions 71-83 corresponding to motif I of the TruB family (probably involved in conserving protein structure) . The TruB domain of TRUB1 lies between W104 and I255, and contains another short motif, GGTLDS AARGVLVV, including the highly conserved D residue that characterizes motif II (involved in uridine recognition and in catalytic function of psi synthases) . Northern blot analysis revealed that TRUB1 mRNA is widely expressed in various human tissues (especially heart, skeletal muscle and liver) . Phylogenetic analysis of the TruB domain revealed another human gene (approved symbol TRUB2) encoding a conserved TruB domain, located on human chromosome 9 . Thus, the human TruB family includes at least three members: i.e . DKC1 (previously identified), TRUB1 and TRUB2 . The TRUB1 and TRUB2 products could be the hitherto unidentified human tRNA psi synthases . Although TRUB1 is not highly similar to DKC1/dyskerin (whose mutations cause X-linked dyskeratosis congenita) and putatively affects tRNA rather than rRNA modification, it is the most similar human protein to dyskerin . Study of TRUB1 (and TRUB2) should facilitate understanding of the molecular mechanisms of RNA modification and the involvement of psi synthases in human pathology, including dyskeratosis-like diseases. Protein Eng, 2003 Apr, 16(4), 295 - 301 Solubility engineering of the HhaI methyltransferase; Daujotyte D et al.; DNA methylation is involved in epigenetic control of numerous cellular processes in eukaryotes, however, many mechanistic aspects of this phenomenon are not yet understood . A bacterial prototype cytosine-C5 methyltransferase, M.HhaI, serves as a paradigm system for structural and mechanistic studies of biological DNA methylation, but further analysis of the 37 kDa protein is hampered by its insufficient solubility (0.15 mM) . To overcome this problem, three hydrophobic patches on the surface of M.HhaI that are not involved in substrate interactions were subjected to site-specific mutagenesis . Residues M51 or V213 were substituted by polar amino acids of a similar size, and/or the C-terminal tetrapeptide FKPY was replaced by a single glycine residue (Delta324G) . Two out of six mutants, delta324G and V213S/delta324G, showed improved solubility in initial analyses and were purified to homogeneity using a newly developed procedure . Biochemical studies of the engineered methyltransferases showed that the deletion mutant delta324G retained identical DNA binding, base flipping and catalytic properties as the wild-type enzyme . In contrast, the engineered enzyme showed (i) a significantly increased solubility (>0.35 mM), (ii) high-quality 2D-{(15)N,(1)H} TROSY NMR spectra, and (iii) (15)N spin relaxation times evidencing the presence of a monomeric well-folded protein in solution. Nucleic Acids Res, 2003 May 15, 31(10), 2570 - 5 Mutagenic effects of 2-hydroxy-dATP on replication in a HeLa extract: induction of substitution and deletion mutations; Satou K et al.; The mutagenicity of an oxidized form of dATP, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract . 2-OH-dATP induced mutations in a dose-dependent manner and elicited substitution and deletion mutations . Of the substitutions, a G.C-->A.T transition including a tandem (CC-->TT) mutation was mainly observed . This result agrees with our previous observation that mammalian DNA polymerase alpha misincorporates the oxidized nucleotide opposite C, but is in contrast to the finding that 2-OH-dATP elicits G.C-->T.A transversions in Escherichia coli . This type of mutation was also elicited, but to a lesser extent . Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5'-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture, and probably in living cells . These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells. Nucleic Acids Res, 2003 May 15, 31(10), 2514 - 23 Synthesis, thermal stability and resistance to enzymatic hydrolysis of the oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridines; Ito T et al.; The synthesis of oligonucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridine (D) is described, and thermal stability and resistance to enzymatic hydrolysis of the ODNs are compared with ODNs containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine (H) . The ODNs containing D and the complementary RNA demonstrated a duplex thermal stabilization of 0.4-3.9 degrees C per modification depending on the position and the number, while the ODNs containing H with the RNA showed slightly less effective thermal stabilization . Further more, the ODNs containing D were found to be more resistant to nucleolytic hydrolysis, not only by snake venom phosphodiesterase (SVPD; a 3'-exonuclease) but also by DNase I (an endonuclease) . The half-life of the 17mer containing five molecules of D against nucleolytic hydrolysis by SVPD was 240 times greater than the unmodified 17mer ODN, which is 1.8 times greater than the ODN containing 5Hs in the same sequence . Against DNase I, the same ODN containing 5Ds was 24 times greater stable than the unmodified 17mer and 15 times more stable than the ODN containing 5Hs . We also examined whether the duplexes formed by the ODNs containing D and the complementary RNAs could be a substrate of Escherichia coli RNase H . It was revealed that a minimum of five contiguous unmodified 2'-deoxyribonucleosides between Ds was required to constitute a substrate of E.coli RNase H . Thus, the ODN with Ds and at least five contiguous unmodified 2'-deoxyribonucleosides between Ds was found to be a candidate for a novel antisense molecule. J Biol Chem, 2003 Jul 18, 278(29), 26648 - 54 Epub 2003 May 07. Discrimination of ATP, ADP, and AMPPNP by chaperonin GroEL: hexokinase treatment revealed the exclusive role of ATP; Motojima F et al.; The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding) . The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding . To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results . In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase . ADP was unable to support the formation of cis ternary complex and cis folding . AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese . In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP . Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident. Am J Physiol Lung Cell Mol Physiol, 2003 Jun, 284(6), L917 - 25 Epub 2003 Jan 10. Synthesis and anti-inflammatory activity of a chimeric recombinant superoxide dismutase: SOD2/3; Gao B et al.; External surfaces of cells are normally protected by extracellular superoxide dismutase, SOD3, which binds to polyanions such as heparan sulfate . We constructed a fusion gene encoding a chimeric SOD consisting of the mature human mitochondrial SOD2 plus the COOH-terminal 26-amino acid heparin-binding "tail" from SOD3 . This tail is responsible for the enzyme's affinity for endothelial surfaces . The fusion gene was expressed in Escherichia coli, and the fully active enzyme SOD2/3 was purified . Although native SOD2 has no affinity for heparin, SOD2/3 binds to a heparin-agarose column . In a rat model of acute lung injury induced by intratracheal instillation of IL-1, SOD2/3, SOD2, and denatured SOD2/3 showed 92%, 13.8%, and 0% reduction of lung leak, respectively . Only SOD2/3 prevented neutrophil accumulation . In the carrageenan-induced foot edema model in the rat, SOD2/3 reduced edema by 62% (P < 0.003) at a dose in which native SOD2 produced no significant effect . Thus SOD2/3 appears to have properties as a therapeutic anti-inflammatory agent that are greatly superior to other available forms of the enzyme. Am J Physiol Regul Integr Comp Physiol, 2003 Jun, 284(6), R1595 - 603 Activation of central melanocortin-4 receptor suppresses lipopolysaccharide-induced fever in rats; Sinha PS et al.; Activation of central melanocortin receptors (MCR) inhibits fever, but the identity of the MCR subtype(s) mediating this antipyretic effect is unknown . To determine whether selective central melanocortin receptor-4 (MC4R) activation produces antipyretic effects, the MC4R selective agonist MRLOB-0001 (CO-His-d-Phe-Arg-Trp-Dab-NH(2)) was administered intracerebroventricularly to rats treated with Escherichia coli lipopolysaccharide (LPS, 30 microg/kg ip) . Treatment with MRLOB-0001 (150 ng icv) did not lower core body temperature (T(c)) in afebrile rats but did suppress LPS-induced increases in T(c) and associated decreases in tail skin temperature (T(sk)), an indicator of vasomotor thermoeffector function . In contrast, systemic treatment with MRLOB-0001 (150 ng iv) did not produce similar antipyretic effects . Coadministration of the selective MC4R antagonist HS014 (1 microg icv) blocked the antipyretic effects of MRLOB-0001 . HS014 alone (1 microg icv) had no significant effect on LPS-induced increases in T(c) or decreases in T(sk) and in afebrile rats had no significant effects on T(c) or T(sk) . We conclude that pharmacological activation of central MC4R suppresses febrile increases in T(c) and that inhibition of heat conservation pathways may contribute to this effect . These findings suggest that the central MC4R may mediate the long-recognized antipyretic effects of centrally administered melanocortins. BMC Microbiol . 2003 May 07;3(1):8. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria; Wunschiers R et al.; BACKGROUND: Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa . Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases . Maturation of those depends on accessory proteins encoded by hyp-genes . The last maturation step involves the cleavage of a ca . 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase . Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases . The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102), only the bidirectional (Synechocystis PCC 6803) or both NiFe-hydrogenases (Anabaena PCC 7120) prompted us to mine these genomes for hydrogenase maturation related genes . In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above . RESULTS: We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes . The genes are not part of any known hydrogenase related gene cluster . The derived amino acid sequences show only low similarity (28-41%) to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known . However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site . Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions . CONCLUSION: Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes . Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation factors that are specific . Therefore, in accordance with previous nomenclature, we propose the gene names hoxW and hupW for the bidirectional and uptake hydrogenase processing endopeptidases, respectively . Due to their constitutive expression we expect that, at least in cyanobacteria, the endopeptidases take over multiple functions. Org Lett, 2003 May 15, 5(10), 1781 - 3 Synthesis of isobutyl-C-galactoside (IBCG) as an isopropylthiogalactoside (IPTG) substitute for increased induction of protein expression; Ko KS et al.; {reaction: see text} Addition of isopropyl-beta-d-thiogalactopyranoside (IPTG) to bacterial cultures is often used to induce expression of plasmid-based genes for the production of recombinant proteins under control of the lac promoter, but a simple method to circumvent the inherent instability of this compound has not been addressed experimentally . Herein we report the first synthesis of isobutyl-C-galactoside (IBCG), the C-glycoside analogue of IPTG, and show that IBCG is superior to IPTG in inducing protein expression over long induction times. Nat Prod Rep, 2003 Apr, 20(2), 184 - 201 Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions; Jordan F; The mechanism of thiamin diphosphate-dependent enzymatic reactions is discussed, concentrating on two enzymes involved in decarboxylating pyruvic acid, the yeast pyruvate decarboxylase and the pyruvate dehydrogenase multienzyme complex from Escherichia coli . The availability of high-resolution X-ray structures for several thiamin diphosphate-dependent enzymes, the use of site-specifically substituted protein variants (resulting from site-directed mutagenesis), the development of model reactions for the various putative intermediates, and the application of new mechanistic tools in solution have all contributed to a much better understanding of the role of the protein component in catalysis . Perhaps the most important advance in our understanding of these mechanisms concerns the role of the 4'-aminopyrimidine component of the coenzyme, widely ignored prior to the publication of the X-ray results . The current view is that the two aromatic rings both contribute to catalysis, perhaps carrying out an intramolecular proton transfer to initiate the various reactions, an ability that makes this coenzyme virtually unique among coenzymes. Biol Proced Online, 1998 Jul 20, 1, 92 - 99 A Method for Assaying Deubiquitinating Enzymes; Lee JI et al.; A general method for the assay of deubiquitinating enzymes was described in detail using (125)I-labeled ubiquitin-fused alphaNH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate . Since the tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid soluble products . Using this assay protocol, we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their specific activities . Since the extracts of E . coli showed little or no activity against the substrate, the assay protocol should be useful for identification and purification of eukaryotic deubiquitinating enzymes cloned and expressed in the cells. Biol Proced Online, 2003, 5, 13 - 19 Epub 2003 Feb 17. Expression, Purification and Characterization of Ricin vectors used for exogenous antigen delivery into the MHC Class I presentation pathway; Smith DC et al.; Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation {Smith et al . J Immunol 2002; 169:99-107} . The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules . Expression in E . coli and purification by cation exchange chromatography of the fusion protein is described . Before used for delivery, the activity of the vector must be characterized in vitro, via an N-glycosidase assay, and in vivo, by a cytotoxicity assay . The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass. J Clin Microbiol, 2003 May, 41(5), 2138 - 40 Multiplex PCR for detection of three plasmid-borne genes of enteroaggregative Escherichia coli strains; Cerna JF et al.; We developed a novel multiplex PCR assay for enteroaggregative Escherichia coli (EAEC) detection, by using three plasmid-borne genes (the aggregative adherence {AA} probe, aap, and aggR) . One or more of the loci were detected in 24 (86%) of 28 patient isolates analyzed . The multiplex PCR assay is a fast, convenient, and sensitive molecular test to detect EAEC. J Clin Microbiol, 2003 May, 41(5), 2113 - 25 Rapid identification of Escherichia coli pathotypes by virulence gene detection with DNA microarrays; Bekal S et al.; One approach to the accurate determination of the pathogenic potential (pathotype) of isolated Escherichia coli strains would be through a complete assessment of each strain for the presence of all known E . coli virulence factors . To accomplish this, an E . coli virulence factor DNA microarray composed of 105 DNA PCR amplicons printed on glass slides and arranged in eight subarrays corresponding to different E . coli pathotypes was developed . Fluorescently labeled genomic DNAs from E . coli strains representing known pathotypes were initially hybridized to the virulence gene microarrays for both chip optimization and validation . Hybridization pattern analysis with clinical isolates permitted a rapid assessment of their virulence attributes and determination of the pathogenic group to which they belonged . Virulence factors belonging to two different pathotypes were detected in one human E . coli isolate (strain H87-5406) . The microarray was also tested for its ability to distinguish among phylogenetic groups of genes by using gene probes derived from the attaching-and-effacing locus (espA, espB, tir) . After hybridization with these probes, we were able to distinguish E . coli strains harboring espA, espB, and tir sequences closely related to the gene sequences of an enterohemorrhagic strain (EDL933), a human enteropathogenic strain (E2348/69), or an animal enteropathogenic strain (RDEC-1) . Our results show that the virulence factor microarray is a powerful tool for diagnosis-based studies and that the concept is useful for both gene quantitation and subtyping . Additionally, the multitude of virulence genes present on the microarray should greatly facilitate the detection of virulence genes acquired by horizontal transfer and the identification of emerging pathotypes. J Clin Microbiol, 2003 May, 41(5), 2106 - 12 Identification and characterization of a new variant of Shiga toxin 1 in Escherichia coli ONT:H19 of bovine origin; Burk C et al.; A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an Escherichia coli strain, ONT:H19, isolated from bovine feces . The complete stx(1) gene of this strain was amplified and sequenced . Nucleotide sequence homology with stx(1) from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein . Cell culture supernatant of this strain, which was negative for stx(2) by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx . PCR primers were constructed for the specific detection of the new variant . The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods. J Clin Microbiol, 2003 May, 41(5), 2033 - 9 Characterization of atypical enteropathogenic Escherichia coli strains harboring the astA gene that were associated with a waterborne outbreak of diarrhea in Japan; Yatsuyanagi J et al.; The virulence traits of the Escherichia coli strain associated with a waterborne diarrhea outbreak were examined . Forty-one of 75 students (ages 12 to 15) in Akita Prefecture, Japan, showed clinical symptoms . Seven E . coli Ouk:K-:H45 isolates were isolated from the patients as the causative agent of this outbreak . One isolate (EC-3605) showed the presence of E . coli attaching-and-effacing (eaeA) and enteroaggregative E . coli heat-stable enterotoxin-1 (astA) genes and the absence of Shiga toxin (stx1 and stx2) genes . A polymorphic enteropathogenic E . coli (EPEC) adherence factor plasmid was detected in EC-3605 with a major structural gene deletion and a regulatory gene frameshift mutation, revealing that EC-3605 represents an atypical EPEC strain harboring the astA gene . The role that atypical EPEC strains harboring the astA gene play in human disease is unclear . Our results, along with those of others, present a possibility that these strains comprise a distinct category of diarrheagenic E . coli and that astA affects the age distribution of atypical-EPEC infection. J Clin Microbiol, 2003 May, 41(5), 1957 - 62 Detection of Chlamydia pneumoniae-specific antibodies binding to the VD2 and VD3 regions of the major outer membrane protein; Klein M et al.; Although Chlamydia pneumoniae is an important human pathogen, the antigens eliciting a specific humoral immune response remain elusive . We scrutinized several recombinant chlamydial surface proteins for species-specific recognition by a panel of human sera previously tested for the presence of anti-C . pneumoniae and anti-C . trachomatis antibodies by microimmunofluorescence and enzyme-linked immunosorbent assay . The 15-kDa cysteine-rich protein (CrpA), porin-b (PorB), 9-kDa outer membrane protein (OMP3), 60-kDa outer membrane protein (OMP2), and four fragments of the major outer membrane protein (MOMP) representing each variable domain (VD) were overexpressed in Escherichia coli, affinity purified, and employed for Western blot analysis . None of the sera tested contained antibodies recognizing PorB and OMP3 of C . pneumoniae . Sera from C . pneumoniae-immune patients cross-reacted with OMP2 of C . trachomatis, and sera from C . trachomatis-immune patients cross-reacted with CrpA of C . pneumoniae, indicating that some of chlamydial surface molecules share antigenic epitopes . In contrast, the VD2, as well as the VD3, regions of the MOMP of C . pneumoniae were only recognized by C . pneumoniae-positive sera, suggesting the existence of species-specific epitopes . The identification of such epitopes of cell surface molecules provides new insights into C . pneumoniae-specific immune responses and may be of value for the improvement of C . pneumoniae-specific diagnostic assay systems based on defined recombinant antigens. J Clin Microbiol, 2003 May, 41(5), 1827 - 32 Virulence markers of enteroaggregative Escherichia coli isolated from children and adults with diarrhea in Brasília, Brazil; Piva IC et al.; Escherichia coli strains isolated from sporadic cases of acute diarrhea in children and adults and from children without diarrhea were investigated for the presence of the pAA plasmid . Strains harboring the pAA plasmid were isolated at similar frequencies from children with (19.6%) and without (10.8%) diarrhea and from adults with diarrhea (11.8%) . The genotypic and phenotypic virulence markers of these strains were further analyzed . Most of the strains were positive for EAST1 (73%), and this toxin was detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.05) . Likewise, pic sequences were detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.005) and controls (P < 0.025) . Furthermore, the association of pAA positivity (pAA(+)) and pic positivity (pic(+)) was more frequently found for strains from children with diarrhea than for strains from controls, indicating that pAA(+) pic(+) strains may represent a subset of pAA(+) strains associated with disease in children . Most of the strains (82.5%) adhered to cells presenting the typical aggregative pattern . The frequency of occurrence of enteropathogenic E . coli (EPEC) serogroups in the strains from children with diarrhea was very high (56%), while none of the strains from adults with diarrhea belonged to EPEC serogroups . Extraintestinal virulence markers were very commonly found in strains from adults with diarrhea . The frequencies of occurrence of the adhesins AFA and SFA were significantly higher in strains from adults with diarrhea than in strains from children with diarrhea . More than one extraintestinal virulence marker was found in 58% of the strains from adults with diarrhea but in only 7.7% of the strains from children with diarrhea . Our results show that pAA(+) strains isolated from children and adults with diarrhea present very different profiles when enteroaggregative E . coli virulence markers, serotypes, and extraintestinal virulence markers are considered. J Biol Chem, 2003 Jul 11, 278(28), 25947 - 51 Epub 2003 May 06. The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair; Matsuda T et al.; Damaged DNA bases are removed from mammalian genomes by base excision repair (BER) . Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase . Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold . This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner . To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes . When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are <or=0.3 to <or=2.8 x 10-4 . BER error rates are higher when excess incorrect dNTPs are included in the reaction or when wild type DNA polymerase beta is replaced by DNA polymerase beta variants that fill single nucleotide gaps with lower fidelity . Under these conditions, the base substitution fidelity of polymerase beta-dependent BER is 3-8-fold higher than is single nucleotide gap filling by polymerase beta alone . Thus other proteins in the BER reaction may enhance the base substitution fidelity of DNA polymerase beta during single nucleotide BER. J Biol Chem, 2003 Jul 11, 278(28), 25825 - 31 Epub 2003 May 06. Reconstitution of recombinant uncoupling proteins: UCP1, -2, and -3 have similar affinities for ATP and are unaffected by coenzyme Q10; Jaburek M et al.; The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP) . We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, -2, and -3 reconstituted in liposomes . We show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities . The Ki values for ATP inhibition were 50 microm (UCP1), 70 microm (UCP2), and 120 microm (UCP3) at pH 7.2 . These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (Ki = 65 microm at pH 7.2) . The Vmax values for proton transport were also similar among the UCPs, ranging from 8 to 20 micromol.min(-1).mg(-1), depending on experimental conditions . We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, -2, and -3 . We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs . We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport. Biochemistry (Mosc), 2003 Mar, 68(3), 269 - 74 Alpha-crystallin promotes assembly of a trimeric form of Mycobacterium tuberculosis Hsp16.3 in a cell free system; Abulimiti A et al.; Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis proposed to form specific trimer-of-trimers structures, acts as a molecular chaperone in vitro . The assembly mechanisms of this oligomeric protein were studied using in vitro transcription/translation systems . Analysis using a combination of non-denaturing pore gradient polyacrylamide gel electrophoresis and size-exclusion chromatography demonstrates that the predominant form of Hsp16.3 produced in the in vitro transcription/translation system is the trimer . Our result indicated that alpha-crystallin (molecular chaperone) remarkably promotes the trimer assembly of Hsp16.3, but does not convert the trimeric form to nonameric form . An "inert" Hsp16.3 dimer, which does not seem to participate in trimer assembly but may be involved in forming other forms of Hsp16.3, was also detected in the in vitro expression system . A latent phase of ~10 min in the appearance of the first detectable species indicated that Hsp16.3 assembly did not occur co-translationally. Hua Xi Yi Ke Da Xue Xue Bao, 2001 Mar, 32(1), 5 - 8 {Study on the construction of DYS385 allelic ladder and the genotype distribution in two populations}; Zhang L et al.; OBJECTIVE: We have designed a new method to produce standard DYS385 allelic ladder in order to solve the problem of the accuracy and standardization of STR-PCR typing in forensic practice . METHODS: Nine different PCR amplified DYS385 allelic fragments were isolated from the gel, eluted into the distilled water and reamplified by PCR . The purified allelic fragments were then blunt-end subcloned individually into the pUC plasmid vectors and transfected into competent E . coli DH5 alpha cells . The sequencing results confirmed that the size and the constructure of the inserts were correct . The recombinant plasmids DNA with 9 inserts were then used as templates for reamplification to generate DYS385 standard ladder . RESULTS: A large quantity of standard DYS385 allelic ladder was obtained, with which the genetic polymorphisms of DYS385 locus both in Chinese Han and German populations were studied . CONCLUSION: The STR standard ladder constructed by this method has high value in the forensic application, and the DYS385 locus is robust for forensic analysis. Anal Bioanal Chem, 2003 Apr, 375(8), 1038 - 44 Epub 2003 Feb 28. The use of thermal lensing for the determination of pyrogens; Orlova NV et al.; Based on the optimized spectrophotometric determination of pyrogens (of various classes ( p-aminophenol and endotoxins), thermal lensing was applied to the determination of these substances at the submicrogram level . The limit of detection of p-aminophenol, a pyrogenic impurity in pharmaceutical formulations of paracetamol, by reaction with resorcinol in alkaline solutions is 100 ng mL(-1) . Phloroglucinol was considered as an analog of resorcinol as a reagent in this reaction . The conditions of spectrophotometric determination of pyrogenic lipopolysaccharides (endotoxins) by ion-pair formation with methylene blue (the limit of detection is 100 ng mL(-1)), by ion-pair formation with Stains-All (1-ethyl-2-{3-(1-ethylnaphtho{1,2-d}thiazolin-2-ylidene)-2-methylpropenyl}naphtho{1,2-d}thiazolium bromide) (the limit of detection is 500 ng mL(-1)), and by reaction of 2-keto-3-deoxyoctonic acid with thiobarbituric acid (the limit of detection is 800 ng mL(-1)) were proposed . The optimized procedure for 2-keto-3-deoxyoctonic acid was applied for thermal lensing that provided a decrease in the limit of detection to 70 ng mL(-1) and was also used for lipopolysaccharide determination in the endotoxin standard from E . coli. Curr Microbiol, 2003 Apr, 46(4), 296 - 301 Biochemical and functional characterization of a eukaryotic-type protein kinase, SpkB, in the cyanobacterium, Synechocystis sp . PCC 6803; Kamei A et al.; On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp . PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily ( spkA approximately spkG) . Previously, SpkA was shown to have protein kinase activity and to be required for cell motility . Here, the role of the spkB was examined . The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni(2+) affinity chromatography . The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins . SpkB showed autophosphorylation activity in the presence of both Mg(2+) and Mn(2+), but not in Ca(2+) . Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis . These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA. Curr Microbiol, 2003 May, 46(5), 318 - 23 Generation of novel plasmids in Escherichia coli S17-1(pSUP106); Mahapatra NR et al.; When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz . S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E . coli transconjugants were isolated that contained novel plasmids and were resistant to Zn(2+) (48 m M), Cu(2+) (12 m M), Ni(2+) (12 m M), chloramphenicol (50 microg/ml), and tetracycline (25 microg/ml) . The transconjugant plasmids did not hybridize with any of the A . symbioticum KM2 plasmids . After curing of the plasmids, the transconjugants became sensitive to 12 m M Zn(2+), 12 m M Cu(2+), and 12 m M Ni(2+), but remained chloramphenicol and tetracycline resistant-the phenotypic markers that were originally present in pSUP106 . That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants . Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E . coli S17-1 and not with the chromosomal DNA of A . symbioticum KM2 or E . coli K12, suggesting their host chromosomal origin . Thus, the present study describes a unique event of genetic rearrangements in the E . coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell. J Biol Chem, 2003 Jul 11, 278(28), 26039 - 45 Epub 2003 May 05. Integrating structure, bioinformatics, and enzymology to discover function: BioH, a new carboxylesterase from Escherichia coli; Sanishvili R et al.; Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate . However, structure alone is often insufficient to deduce the specific biochemical function of a protein . Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity . BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function . The crystal structure of BioH was determined at 1.7 A resolution . An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites . This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases . Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates . The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure. J Biol Chem, 2003 Jul 25, 278(30), 27758 - 65 Epub 2003 May 05. Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA: expression in Escherichia coli confers the ability to reduce soluble Fe(III) chelates; Pitts KE et al.; Shewanella oneidensis MR-1 has the metabolic capacity to grow anaerobically using Fe(III) as a terminal electron acceptor . Growth under these conditions results in the de novo synthesis of a number of periplasmic c-type cytochromes, many of which are multiheme in nature and are thought to be involved in the Fe(III) respiratory process . To begin a biochemical study of these complex cytochromes, the mtrA gene that encodes an approximate 32-kDa periplasmic decaheme cytochrome has been heterologously expressed in Escherichia coli . Co-expression of mtrA with a plasmid that contains cytochrome c maturation genes leads to the assembly of a correctly targeted holoprotein, which covalently binds ten hemes . The recombinant MtrA protein has been characterized by magnetic circular dichroism, which shows that all ten hemes have bis-histidine axial ligation . EPR spectroscopy detected only eight of these hemes, all of which are low spin and provides evidence for a spin-coupled pair of hemes in the oxidized state . Redox titrations of MtrA have been carried out with optical- and EPR-monitored methods, and the hemes are shown to reduce over the potential range -100 to -400 mV . In intact cells of E . coli, MtrA is shown to obtain electrons from the host electron transport chain and pass these onto host oxidoreductases or a range of soluble Fe(III) species . This demonstrates the promiscuous nature of this decaheme cytochrome and its potential to serve as a soluble Fe(III) reductase in intact cells. J Biol Chem, 2003 Jul 11, 278(28), 26159 - 65 Epub 2003 May 05. Truncation by Glu180 nonsense mutation results in complete loss of slow skeletal muscle troponin T in a lethal nemaline myopathy; Jin JP et al.; A lethal form of nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), is linked to a nonsense mutation at codon Glu180 in the slow skeletal muscle troponin T (TnT) gene . We found that neither the intact nor the truncated slow TnT protein was present in the muscle of patients with ANM . The complete loss of slow TnT is consistent with the observed recessive pattern of inheritance of the disease and indicates a critical role of the COOH-terminal T2 domain in the integration of TnT into myofibrils . Expression of slow and fast isoforms of TnT is fiber-type specific . The lack of slow TnT results in selective atrophy of type 1 fibers . Slow TnT confers a higher Ca2+ sensitivity than does fast TnT in single fiber contractility assays . Despite the lack of slow TnT, individuals with ANM have normal muscle power at birth . The postnatal onset and infantile progression of ANM correspond to a down-regulation of cardiac and embryonic splice variants of fast TnT in normal developing human skeletal muscle, suggesting that the fetal TnT isoforms complement slow TnT . These results lay the foundation for understanding the molecular pathophysiology and the potential targeted therapy of ANM. J Biol Chem, 2003 Aug 1, 278(31), 29336 - 43 Epub 2003 May 05. IIGP1, an interferon-gamma-inducible 47-kDa GTPase of the mouse, showing cooperative enzymatic activity and GTP-dependent multimerization; Uthaiah RC et al.; IIGP1 belongs to a well defined family of 47-kDa GTPases whose members are present at low resting levels in mouse cells but are strongly induced transcriptionally by interferons and are implicated in cell-autonomous resistance to intracellular pathogens . Recombinant IIGP1 was expressed in Escherichia coli and purified to homogeneity . Here we present a detailed biochemical characterization of IIGP1 using various biochemical and biophysical methods . IIGP1 binds to GTP and GDP with dissociation constants