Escherichia coli

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 in the micromolar range with at least 10 times higher affinity for GDP than for GTP . IIGP1 hydrolyzes GTP to GDP, and the GTPase activity is concentration-dependent with a GTP turnover rate of 2 min-1 under saturating protein concentrations . Functional interaction between IIGP1 molecules is shown by nucleotide-dependent oligomerization in vitro . Both cooperative hydrolysis of GTP and GTP-dependent oligomerization are blocked in a mutant form of IIGP1 modified at the C terminus . IIGP1 shares micromolar nucleotide affinities and oligomerization-dependent hydrolytic activity with the 67-kDa GTPase hGBP1 (induced by type I and type II interferons), with the antiviral Mx proteins (interferon type I-induced) and with the paradigm of the self-activating large GTPases, the dynamins, with which Mx proteins show homology . The higher relative affinity for GDP and the relatively low GTPase activity distinguish IIGP1, but this study clearly adds IIGP1 and thus the p47 GTPases to the small group of cooperative GTPase families that appear to characterize the development of intracellular resistance during the interferon response to infection . The present analysis provides essential parameters to understand the molecular mechanism by which IIGP1 participates in this complex resistance program.

J Biol Chem, 2003 Jul 18, 278(29), 27105 - 11 Epub 2003 May 05.
Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide; Lybarger L et al.; Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers . These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules . In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin . Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide . These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide . In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli . SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.

J Biol Chem, 2003 Oct 10, 278(41), 40144 - 51 Epub 2003 May 05.
Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli; Gronlund H et al.; Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy . Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful . In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced . A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR . Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography . Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e . Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion . Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1 . Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients . The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1 . Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

J Microbiol Methods, 2003 Jul, 54(1), 87 - 93
Identification of EPEC and non-EPEC serotypes in the EPEC O serogroups by PCR-RFLP analysis of the fliC gene; Botelho BA et al.; Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status . The serological detection of flagellin is the basis for the H-codes typing system in E . coli . Thus, it is impossible to serotype nonmotile bacteria (i.e . to assign H-codes) . Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)) . Each motile serotype was characterized by one or two fliC specific restriction patterns . The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases . These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments . Nonmotile strains showed fliC restriction patterns identical to some known H serotypes . The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.

Curr Opin Microbiol, 2003 Apr, 6(2), 151 - 6
Control of rRNA expression in Escherichia coli; Schneider DA et al.; The control of ribosome synthesis has been a major focus in molecular biology for over 50 years . As protein synthesis is an essential, yet energetically costly, process, all cells (from bacteria to mammals) devote complex regulatory networks to fine-tune the expression of ribosomal RNA (and therefore ribosome synthesis) to the nutritional environment . In Escherichia coli, ribosomal RNA promoters are among the strongest in the cell and are regulated by trans-acting proteins (Fis and H-NS) and small molecules (guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates) . Recent work has dissected many of the molecular mechanisms responsible for the strength and regulation of rRNA promoters.

Curr Opin Microbiol, 2003 Apr, 6(2), 146 - 50
Coupling the initiation of chromosome replication to cell size in Escherichia coli; Donachie WD et al.; Bacterial cells change size dramatically with change in growth rate, but the ratio between cell volume and the number of copies of the origin of chromosome replication (oriC) is roughly constant at the time of initiation of DNA replication at almost all growth rates . Recent research on the inactivation of initiator protein (DnaA) and depletion of DnaA pools by the high-affinity DnaA-binding locus datA allows us to propose a simple model to explain the long-standing question of how Escherichia coli couples DNA replication to cell size.

Biochemistry, 2003 May 13, 42(18), 5469 - 77
Flavin redox state triggers conformational changes in the PutA protein from Escherichia coli; Zhu W et al.; The regulation of proline utilization in Escherichia coli involves the proline-dependent translocation of the PutA flavoprotein from the cytoplasm to a peripheral position on the membrane . In the cytoplasm, PutA represses transcription of the proline utilization (put) genes while membrane-bound PutA catalyzes the oxidation of L-proline to glutamate . The mechanism by which PutA switches from a DNA-binding protein to a membrane-bound enzyme involves a proline-induced conformational change that is characterized by the appearance of a 119-kDa fragment during limited proteolysis of proline-reduced PutA . To establish whether the FAD redox state is responsible for the proline-induced conformational change in PutA, we distinguished the effects that FAD reduction and proline analogue binding have on PutA conformation by limited chymotrypsin proteolysis . Controlled potentiometric proteolysis of PutA demonstrated that the formation of the 119-kDa band occurs at an E(m)(conf) value of -0.058 V (pH 7.5), which is within 20 mV of the E(m) value for FAD bound to PutA . The manipulation of the E(m)(conf) value by reconstitution of PutA with the FAD analogue, 5-deazaFAD, confirmed that the conformational change observed in the presence of proline is solely dependent on the FAD redox state . The proline analogue, L-tetrahydro-2-furoic acid (L-THFA), failed to elicit the formation of the 119-kDa fragment during chymotrypsin cleavage of PutA . Instead, a unique fragment of about 93-kDa was observed, indicating that a distinct PutA conformer is stabilized by L-THFA . Reduction of L-THFA-complexed PutA, however, regenerated the 119-kDa fragment showing that reduction of the FAD cofactor overrides conformational changes induced by L-THFA . Mapping of the protease susceptibility sites in PutA revealed that the conformational changes caused by FAD reduction and L-THFA binding are transmitted to domains outside the proline dehydrogenase active site.

Biochemistry, 2003 May 13, 42(18), 5403 - 13
Transient kinetic studies of heme reduction in Escherichia coli nitrate reductase A (NarGHI) by menaquinol; Zhao Z et al.; We have studied the transient kinetics of quinol-dependent heme reduction in Escherichia coli nitrate reductase A (NarGHI) by the menaquinol analogue menadiol using the stopped-flow method . Four kinetic phases are observed in the reduction of the hemes . A transient species, likely to be associated with a semiquinone radical anion, is observed with kinetics that correlates with one of the phases . The decay of the transient species and the formation of the second reduction phase of the hemes can be fitted to a double-exponential equation giving similar rate constants, k(1) = 9.24 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the decay of the transient species, and k(1) = 9.23 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the formation of the reduction phase . The quinol-binding-site inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and stigmatellin have significant and different inhibitory effects on the reduction kinetics . The kinetics of heme reduction in NarI expressed in the absence of the NarGH catalytic dimer (NarI(DeltaGH) exhibits only two kinetic phases, and the decay of the transient species also correlates kinetically with the second reduction phase of the hemes . We have also studied nitrate-dependent heme reoxidation following quinol-dependent heme reduction using a sequential stopped-flow method . HOQNO elicits a much stronger inhibitory effect than stigmatellin on the reoxidation of the hemes . On the basis of our results, we propose schemes for the mechanism of NarGHI reduction by menaquinol and reoxidation by nitrate.

Biochemistry, 2003 May 13, 42(18), 5387 - 94
The CXC motif: a functional mimic of protein disulfide isomerase; Woycechowsky KJ et al.; Protein disulfide isomerase (PDI) utilizes the active site sequence Cys-Gly-His-Cys (CGHC; E degrees ' = -180 mV) to effect thiol-disulfide interchange during oxidative protein folding . Here, the Cys-Gly-Cys-NH(2) (CGC) peptide is shown to have a disulfide reduction potential (E degrees ' = -167 mV) that is close to that of PDI . This peptide has a thiol acid dissociation constant (pK(a) = 8.7) that is lower than that of glutathione . These attributes endow the CGC peptide with substantial disulfide isomerization activity . Escherichia coli thioredoxin (Trx) utilizes the active site sequence Cys-Gly-Pro-Cys (CGPC; E degrees ' = -270 mV) to effect disulfide reduction . Removal of the proline residue from the Trx active site yields a CGC active site with a greatly destabilized disulfide bond (E degrees ' >or= -200 mV) . The DeltaP34 variant retains high conformational stability and remains a substrate for thioredoxin reductase . In contrast to the reduced form of the wild-type enzyme, the reduced form of DeltaP34 Trx has disulfide isomerization activity, which is 25-fold greater than that of the CGC peptide . Thus, the rational deletion of an active site residue can bestow a new and desirable function upon an enzyme . Moreover, a CXC motif, in both a peptide and a protein, provides functional mimicry of PDI.

Biochemistry, 2003 May 13, 42(18), 5333 - 40
Thermodynamic characterization of the binding of nucleotides to glycyl-tRNA synthetase; Dignam JD et al.; The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches . ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition constants, consistent with their exerting an effect by binding at the ATP site . Glycine had little effect alone but potentiated the effect of ATP in increasing the resistance to thrombin digestion, consistent with the formation of an enzyme-bound adenylate . No protection from thrombin digestion was afforded by tRNA(gly) . Binding constants were determined by isothermal titration calorimetry at 25 degrees C for ATP (2.5 x 10(5) M(-1)), AMPPNP (3.7 x 10(5) M(-1)), and AMPPCP (2.2 x 10(6) M(-1)) . The nucleotides had similar values for DeltaH (-71 kJ mol(-1)), with values for TDeltaS that accounted for the differences in the binding constants . Near-ultraviolet CD spectra of the enzyme-nucleotide complexes indicate that the nucleotides are bound in the anti configuration . A glycyl-adenylate analogue, glycine sulfamoyl adenosine (GSAd), bound with a large value for DeltaH (-187 kJ mol(-1)), which was balanced by a large TDeltaS term to give a binding constant (3.7 x 10(6) M(-1)) only slightly larger than that of AMPPCP . Glycine binding to the enzyme could not be detected calorimetrically, and its presence did not change the thermodynamic parameters for binding of AMPPCP . AMPPNP and AMPPCP were not substrates for glycyl-tRNA synthetase . Analysis of the temperature dependence of ATP binding indicated that the heat capacity change is small, whereas the binding of GSAd is accompanied by a large negative heat capacity change (-2.6 kJ K(-1) mol(-1)) . Titrations performed in buffers with different ionization enthalpies indicate that the large value for DeltaH for the adenylate analogue does not arise from a coupled protonation event . Differential scanning calorimetry indicated that glycyl-tRNA synthetase is stabilized by nucleotides . Unfolding of the protein is irreversible, and thermodynamic parameters for unfolding could therefore not be determined . The results are consistent with a significant conformational transition in glycyl-tRNA synthetase coupled to the binding of GSAd.

Biochemistry, 2003 May 13, 42(18), 5321 - 32
Allosteric activator domain of maintenance human DNA (cytosine-5) methyltransferase and its role in methylation spreading; Pradhan S et al.; The human maintenance DNA (cytosine-5) methyltransferase (hDNMT1) consists of a large N-terminal regulatory domain fused to a catalytic C-terminal domain by randomly repeated Gly-Lys dipeptides . Several N-terminal deletion mutants of hDNMT1 were made, purified, and tested for substrate specificity . Deletion mutants lacking 121, 501, 540, or 580 amino acids from the N-terminus still functioned as DNA methyltransferases, methylated CG sequences, and preferred hemimethylated to unmethylated DNA, as did the full-length hDNMT1 . Methylated DNA stimulated methylation spreading on unmethylated CpG sequences for the full-length and the 121 amino acid deletion hDNMT1 equally well but not for the mutants lacking 501, 540, or 580 amino acids, indicating the presence of an allosteric activation determinant between amino acids 121 and 501 . Peptides from the N- and C-termini bound methylated DNA independently . Point mutation analysis within the allosteric region revealed that amino acids 284-287 (KKHR) were involved in methylated DNA-mediated allosteric activation . Allosteric activation was reduced in the double point mutant enzymes D25 (K284A and K285A) and D12 (H286A and R287A) . Retinoblastoma gene product (Rb), a negative regulator of DNA methylation, bound to the allosteric site of hDNMT1 and inhibited methylation, suggesting Rb may regulate methylation spreading.

Biochemistry, 2003 May 13, 42(18), 5312 - 20
Kinetic mechanism of the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA); Van Lanen SG et al.; The bacterial enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the unprecedented transfer and isomerization of the ribosyl moiety of S-adenosylmethionine (AdoMet) to a modified tRNA nucleoside in the biosynthesis of the hypermodified nucleoside queuosine . The complexity of this reaction makes it a compelling problem in fundamental mechanistic enzymology, and as part of our mechanistic studies of the QueA-catalyzed reaction, we report here the elucidation of the steady-state kinetic mechanism . Bi-substrate kinetic analysis gave initial velocity patterns indicating a sequential mechanism, and provided the following kinetic constants: K (M)(tRNA)= 1.9 +/- 0.7 microM and K (M)(AdoMet)= 98 +/- 5.0 microM . Dead-end inhibition studies with the substrate analogues S-adenosylhomocysteine and sinefungin gave competitive inhibition patterns against AdoMet and noncompetitive patterns against preQ(1)-tRNA(Tyr), with K(i) values of 133 +/- 18 and 4.6 +/- 0.5 microM for sinefungin and S-adenosylhomocysteine, respectively . Product inhibition by adenine was noncompetitive against both substrates under conditions with a subsaturating cosubstrate concentration and uncompetitive against preQ(1)-tRNA(Tyr) when AdoMet was saturating . Inhibition by the tRNA product (oQ-tRNA(Tyr)) was competitive and noncompetitive against the substrates preQ(1)-tRNA(Tyr) and AdoMet, respectively . Inhibition by methionine was uncompetitive versus preQ(1)-tRNA(Tyr), but noncompetitive against AdoMet . However, when methionine inhibition was investigated at high AdoMet concentrations, the pattern was uncompetitive . Taken together, the data are consistent with a fully ordered sequential bi-ter kinetic mechanism in which preQ(1)-tRNA(Tyr) binds first followed by AdoMet, with product release in the order adenine, methionine, and oQ-tRNA . The chemical mechanism that we previously proposed for the QueA-catalyzed reaction {Daoud Kinzie, S., Thern, B., and Iwata-Reuyl, D . (2000) Org . Lett . 2, 1307-1310} is consistent with the constraints imposed by the kinetic mechanism determined here, and we suggest that the magnitude of the inhibition constants for the dead-end inhibitors may provide insight into the catalytic strategy employed by the enzyme.

Biochemistry, 2003 May 13, 42(18), 5301 - 11
Zinc ion effects on individual Ssp DnaE intein splicing steps: regulating pathway progression; Nichols NM et al.; Use of the naturally split, self-splicing Synechocystis sp . PCC6803 DnaE intein permits separate purification of the N- and C-terminal intein domains . Otherwise spontaneous intein-mediated reactions can therefore be controlled in vitro, allowing detailed study of intein kinetics . Incubation of the Ssp DnaE intein with ZnCl(2) inhibited trans splicing, hydrolysis-mediated N-terminal trans cleavage, and C-terminal trans cleavage reactions . Maximum inhibition of the splicing reaction was achieved at equal molar concentrations of ZnCl(2) and intein domains, suggesting a 1:1 metal ion:intein binding stoichiometry . Mutation of the (+)1 cysteine residue to valine (C(+)1V) alleviated the inhibitory effects of ZnCl(2) . Valine substitution in the absence of ZnCl(2) blocked trans splicing and decreased C-terminal cleavage kinetics in a manner similar to that of the native (+)1 cysteine in the presence of ZnCl(2) . These data are consistent with Zn(2+)-mediated inhibition of the Ssp DnaE intein via chelation of the (+)1 cysteine residue . N-Terminal trans cleavage can occur via both spontaneous hydrolysis and nucleophilic (e.g., DTT) attack . Comparative examination of N-terminal cleavage rates using amino acid substitution (C(+)1V) and Zn(2+)-mediated inhibition permitted the maximum contribution of hydrolysis to overall N-terminal cleavage kinetics to be determined . Stable intermediates consisting of the associated intein domains were detected by PAGE and provided evidence of a rapid C-terminal cleavage step . Acute control of the C-terminal reaction was achieved by the rapid reversal of Zn(2+)-mediated inhibition by EDTA . By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively.

Biochemistry, 2003 May 13, 42(18), 5292 - 300
Distal site aspartate is essential in the catalase activity of catalase-peroxidases; Jakopitsch C et al.; Structural and biochemical characterization of aspartate 152 at the distal heme side of catalase-peroxidase (KatG) from Synechocystis PCC 6803 reveals an important functional role for this residue . In the wild-type protein, the side chain carboxyl group of Asp152 is 7.8 A apart from the heme iron and is hydrogen-bonded to two water molecules and a KatG-specific large loop . We have prepared the site-specific variants Asp152Asn, Asp152Ser, Asp152Trp, and Pro151Ala . Exchange of Asp152 exhibited dramatic consequences on the bifunctional activity of this unique peroxidase . The turnover number of catalase activity of Asp152Asn is 2.7%, Asp152Ser 5.7%, and Asp152Trp is 0.6% of wild-type activity . By contrast, the peroxidase activity of the Asp152 variants was 2-7 times higher than that of wild-type KatG or Pro151Ala . The KatG-specific pH profile of the catalase activity was completely different in these variants and exchange of Asp152 made it possible to follow the transition of the ferric enzyme to the redox intermediate compound I by hydrogen peroxide spectroscopically and to determine the corresponding bimolecular rate constant to be 7.5 x 10(6) M(-1) s(-1) (pH 7 and 15 degrees C) . The reactivity of compound I toward aromatic one-electron donors was enhanced in the Asp152 variants compared with the wild-type protein, whereas the reactivity toward hydrogen peroxide was dramatically decreased . A mechanism for the hydrogen peroxide oxidation, which is different from monofunctional catalases and involves the distal residues Trp122 and Asp152, is proposed.

Biochemistry, 2003 May 13, 42(18), 5225 - 35
The structure and specificity of Escherichia coli maltose acetyltransferase give new insight into the LacA family of acyltransferases; Lo Leggio L et al.; The crystallographic three-dimensional structure of the Escherichia coli maa gene product, previously identified as a maltose O-acetyltransferase (MAT) {Brand, B., and Boos, W . (1991) J . Biol . Chem . 266, 14113-14118} has been determined to 2.15 A resolution by the single anomalous dispersion method using data from a crystal cocrystallized with trimethyllead acetate . It is shown here that MAT acetylates glucose exclusively at the C6 position and maltose at the C6 position of the nonreducing end glucosyl moiety . Furthermore, MAT shows higher affinity toward artificial substrates containing an alkyl or hydrophobic chain as well as a glucosyl unit . The presence of a long hydrophobic patch near the acceptor site provides the structural explanation for this preference . The three-dimensional structure reveals the expected trimeric left-handed parallel beta-helix structure found in all other known hexapeptide repeat enzymes . In particular, the structure shows similarities both overall and at the putative active site to the recently determined structure of galactoside acetyltransferase (GAT), the lacA gene product {Wang, X.-G., Olsen, L . R., and Roderick, S . L . (2002) Structure 10, 581-588} . The structure, together with the new biochemical data, suggests that GAT and MAT are more closely related than previously thought and might have similar cellular functions . However, while GAT is specific for acetylation of galactosyl units, MAT is specific for glucosyl units and is able to acetylate maltooligosaccharides, an important property for biotechnological applications . Structural differences at the acceptor site reflect the differences in substrate specificity.

Biochemistry, 2003 May 13, 42(18), 5186 - 94
Protein inhibitors of serine proteinases: role of backbone structure and dynamics in controlling the hydrolysis constant; Song J et al.; Standard mechanism protein inhibitors of serine proteinases bind as substrates and are cleaved by cognate proteinases at their reactive sites . The hydrolysis constant for this cleavage reaction at the P(1)-P(1)' peptide bond (K(hyd)) is determined by the relative concentrations at equilibrium of the "intact" (uncleaved, I) and "modified" (reactive site cleaved, I*) forms of the inhibitor . The pH dependence of K(hyd) can be explained in terms of a pH-independent term, K(hyd) degrees, plus the proton dissociation constants of the newly formed amino and carboxylate groups at the cleavage site . Two protein inhibitors that differ from one another by a single residue substitution have been found to have K(hyd) degrees values that differ by a factor of 5 {Ardelt, W., and Laskowski, M., Jr . (1991) J . Mol . Biol . 220, 1041-1052}: turkey ovomucoid third domain (OMTKY3) has K(hyd) degrees = 1.0, and Indian peafowl ovomucoid third domain (OMIPF3), which differs from OMTKY3 by the substitution P(2)'-Tyr(20)His, has K(hyd) degrees = 5.15 . What mechanism is responsible for this small difference? Is it structural (enthalpic) or dynamic (entropic)? Does the mutation affect the free energy of the I state, the I* state, or both? We have addressed these questions through NMR investigations of the I and I forms of OMTKY3 and OMIPF3 . Information about structure was derived from measurements of NMR chemical shift changes and trans-hydrogen-bond J-couplings; information about dynamics was obtained through measurements of (15)N relaxation rates and (1)H-(15)N heteronuclear NOEs with model-free analysis of the results . Although the I forms of each variant are more dynamic than the corresponding I forms, the study revealed no appreciable difference in the backbone dynamics of either intact inhibitor (OMIPF3 vs OMTKY3) or modified inhibitor (OMIPF3* vs OMTKY3*) . Instead, changes in chemical shifts and trans-hydrogen-bond J-couplings suggested that the K(hyd) degrees difference arises from differential intramolecular interactions within the intact inhibitors (OMIPF3 vs OMTKY3) in a region of each protein that becomes disordered upon reactive site cleavage (to OMIPF3* and OMTKY3*).

Science, 2003 May 2, 300(5620), 801 - 5
Cooperation between RNA polymerase molecules in transcription elongation; Epshtein V et al.; Transcription elongation is responsible for rapid synthesis of RNA chains of thousands of nucleotides in vivo . In contrast, a single round of transcription performed in vitro is frequently interrupted by pauses and arrests that drastically reduce the elongation rate and the yield of the full-length transcript . Here we demonstrate that most transcriptional delays disappear if more than one RNA polymerase (RNAP) molecule initiates from the same promoter . Anti-arrest and anti-pause effects of trailing RNAP are due to forward translocation of leading (backtracked) complexes . Such cooperation between RNAP molecules links the rate of elongation to the rate of initiation and explains why elongation is still fast and processive in vivo even without anti-arrest factors.

Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5968 - 73 Epub 2003 May 01.
Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS; Abdelnoor RV et al.; The plant mitochondrial genome is retained in a multipartite structure that arises by a process of repeat-mediated homologous recombination . Low-frequency ectopic recombination also occurs, often producing sequence chimeras, aberrant ORFs, and novel subgenomic DNA molecules . This genomic plasticity may distinguish the plant mitochondrion from mammalian and fungal types . In plants, relative copy number of recombination-derived subgenomic DNA molecules within mitochondria is controlled by nuclear genes, and a genomic shifting process can result in their differential copy number suppression to nearly undetectable levels . We have cloned a nuclear gene that regulates mitochondrial substoichiometric shifting in Arabidopsis . The CHM gene was shown to encode a protein related to the MutS protein of Escherichia coli that is involved in mismatch repair and DNA recombination . We postulate that the process of substoichiometric shifting in plants may be a consequence of ectopic recombination suppression or replication stalling at ectopic recombination sites to effect molecule-specific copy number modulation.

Annu Rev Microbiol, 2003, 57, 155 - 76 Epub 2003 May 01.
Nitrogen assimilation and global regulation in Escherichia coli; Reitzer L; Nitrogen limitation in Escherichia coli controls the expression of about 100 genes of the nitrogen regulated (Ntr) response, including the ammonia-assimilating glutamine synthetase . Low intracellular glutamine controls the Ntr response through several regulators, whose activities are modulated by a variety of metabolites . Ntr proteins assimilate ammonia, scavenge nitrogen-containing compounds, and appear to integrate ammonia assimilation with other aspects of metabolism, such as polyamine metabolism and glutamate synthesis . The leucine-responsive regulatory protein (Lrp) controls the synthesis of glutamate synthase, which controls the Ntr response, presumably through its effect on intracellular glutamine . Some Ntr proteins inhibit the expression of some Lrp-activated genes . Guanosine tetraphosphate appears to control Lrp synthesis . In summary, a network of interacting global regulators that senses different aspects of metabolism integrates nitrogen assimilation with other metabolic processes.

J Biol Chem, 2003 Jul 11, 278(28), 25745 - 51 Epub 2003 May 04.
Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin; Tamarit J et al.; Grx5 is a yeast mitochondrial protein involved in iron-sulfur biogenesis that belongs to a recently described family of monothiolic glutaredoxin-like proteins . No member of this family has been biochemically characterized previously . Grx5 contains a conserved cysteine residue (Cys-60) and a non-conserved one (Cys-117) . In this work, we have purified wild type and mutant C60S and C117S proteins and characterized their biochemical properties . A redox potential of -175 mV was calculated for wild type Grx5 . The pKa values obtained by titration of mutant proteins with iodoacetamide at different pHs were 5.0 for Cys-60 and 8.2 for Cys-117 . When Grx5 was incubated with glutathione disulfide, a transient mixed disulfide was formed between glutathione and the cystein 60 of the protein because of its low pKa . Binding of glutathione to Cys-60 promoted a decrease in the Cys-117 pKa value that triggered the formation of a disulfide bond between both cysteine residues of the protein, indicating that Cys-117 plays an essential role in the catalytic mechanism of Grx5 . The disulfide bond in Grx5 could be reduced by GSH but at a rate at least 20 times slower than that observed for the reduction of glutaredoxin 1 from E . coli, a dithiolic glutaredoxin . This slow reduction rate could suggest that GSH may not be the physiologic reducing agent of Grx5 . The fact that wild type Grx5 efficiently reduced a glutathiolated protein used as a substrate indicated that Grx5 may act as a thiol reductase inside the mitochondria.

J Biol Chem, 2003 Jul 11, 278(28), 25664 - 70 Epub 2003 May 01.
Affinity purification of ribosomes with a lethal G2655C mutation in 23 S rRNA that affects the translocation; Leonov AA et al.; A method for preparation of Escherichia coli ribosomes carrying lethal mutations in 23 S rRNA was developed . The method is based on the site-directed incorporation of a streptavidin binding tag into functionally neutral sites of the 23 S rRNA and subsequent affinity chromatography . It was tested with ribosomes mutated at the 23 S rRNA position 2655 (the elongation factor (EF)-G binding site) . Ribosomes carrying the lethal G2655C mutation were purified and studied in vitro . It was found in particular that this mutation confers strong inhibition of the translocation process but only moderately affects GTPase activity and binding of EF-G.

J Biol Chem, 2003 Aug 1, 278(31), 28378 - 87 Epub 2003 Apr 30.
The GTPase activity and C-terminal cysteine of the Escherichia coli MnmE protein are essential for its tRNA modifying function; Yim L et al.; The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP . The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule . MnmE is an evolutionarily conserved protein that, in E . coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs . Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE . Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP . Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function . Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes . Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue . We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.

J Biol Chem, 2003 Jul 11, 278(28), 25657 - 63 Epub 2003 May 01.
Properties of the mouse intestinal acyl-CoA:monoacylglycerol acyltransferase, MGAT2; Cao J et al.; Acyl-CoA:monoacylglycerol acyltransferase (MGAT) plays an important role in dietary fat absorption by catalyzing a rate-limiting step in the re-synthesis of diacylglycerols in enterocytes . The present study reports further characterization of MGAT2, a newly identified intestinal MGAT (Cao, J., Lockwood, J., Burn, P., and Shi, Y . (2003) J . Biol . Chem . 278, 13860-13866) for its substrate specificity, requirement for lipid cofactors, optimum pH and Mg2+, and other intrinsic properties . MGAT2 enzyme expressed in COS-7 cells displayed a broad range of substrate specificity toward fatty acyl-CoA derivatives and monoacylglycerols, among which the highest activities were observed with oleoyl-CoA and rac-1-monolauroylglycerol, respectively . MGAT2 appeared to acylate monoacylglycerols containing unsaturated fatty acyls in preference to saturated ones . Lipid cofactors that play roles in signal transduction were shown to modulate MGAT2 activities . In contrast to oleic acid and sphingosine that exhibited inhibitory effects, phosphatidylcholine, phosphatidylserine, and phosphatidic acid stimulated MGAT2 activities . Using recombinant murine MGAT2 expressed in Escherichia coli, we demonstrated conclusively that MGAT2 also possessed an intrinsic acyl-CoA:diacylglycerol acyltransferase (DGAT) activity, which could provide an alternative pathway for triacylglycerol synthesis in the absence of DGAT . In contrast to the inhibitory effect on MGAT2 activities, nonionic and zwitterionic detergents led to a striking activation of DGAT activity of the human DGAT1 expressed in mammalian cells, which further distinguished the behaviors of the two enzymes . The elucidation of properties of MGAT2 will facilitate future development of compounds that inhibit dietary fat absorption as a means to treat obesity.

J Biol Chem, 2003 Jul 11, 278(28), 25731 - 7 Epub 2003 May 02.
The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli; Gong X et al.; An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy{3H}-6-decyl-1,4-benzoquinone ({3H}azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I) . The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of {3H}azido-Q in the dark . Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity . SDS-PAGE of the complex labeled with {3H}azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site . When the {3H}azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit . This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM . The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane . Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.

J Biol Chem, 2003 Jul 11, 278(28), 26275 - 86 Epub 2003 May 02.
Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli; Stillman TJ et al.; Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism . They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity . The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits . Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center . We report the structures of five E . coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives . Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates . Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions . The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.

J Bacteriol, 2003 May, 185(10), 3244 - 8
Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein; Camara JE et al.; Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA . Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.

J Bacteriol, 2003 May, 185(10), 3238 - 43
The yggH gene of Escherichia coli encodes a tRNA (m7G46) methyltransferase; De Bie LG et al.; We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-L-methionine-dependent formation of N(7)-methylguanosine at position 46 (m(7)G46) in tRNA . Additionally, we generated an E . coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m(7)G46) methyltransferase activity.

J Bacteriol, 2003 May, 185(10), 3139 - 46
Transcription regulation coupling of the divergent argG and metY promoters in Escherichia coli K-12; Krin E et al.; The cAMP-catabolite activator protein (CAP) complex is a pleiotropic regulator that regulates a vast number of Escherichia coli genes, including those involved in carbon metabolism . We identified two new targets of this complex: argG, which encodes the arginosuccinate synthase involved in the arginine biosynthetic pathway, and metY, which encodes one of the two methionine tRNA initiators, tRNAf2Met . The cAMP-CAP complex activates argG transcription and inhibits metY transcription from the same DNA position . We also show that ArgR, the specific repressor of the arginine biosynthetic pathway, together with its arginine cofactor, acts on the regulation of metY mediated by CAP . The regulation of the two divergent promoters is thus simultaneously controlled not only by the cAMP-CAP complex, a global regulator, but also by a specific regulator of arginine metabolism, suggesting a previously unsuspected link between carbon metabolism and translation initiation.

J Bacteriol, 2003 May, 185(10), 3101 - 10
Repair system for noncanonical purines in Escherichia coli; Burgis NE et al.; Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic . We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction . The E . coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs . moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure . Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis . Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death . Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation . This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA . We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA . The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.

J Bacteriol, 2003 May, 185(10), 3060 - 7
Characterization of the double-partitioning modules of R27: correlating plasmid stability with plasmid localization; Lawley TD et al.; Plasmid R27 contains two independent partitioning modules, designated Par1 and Par2, within transfer region 2 . Par1 is member of the type I partitioning family (Walker-type ATPase), and Par2 is a member of the type II partitioning family (actin-type ATPase) . Stability tests of cloned Par1 and Par2 and insertional disruptions of Par1 and Par2 within R27 demonstrated that Par1 is the major stability determinant whereas Par2 is the minor stability determinant . Creation of double-partitioning mutants resulted in R27 integrating into the chromosome, suggesting that at least one partitioning module is required for R27 to exist in the extrachromosomal form . Using the lacO/LacI-green fluorescent protein (GFP) system, we labeled and visualized R27 and R27 partitioning mutants (Par1(-) and Par2(-)) under different growth conditions in live Escherichia coli cells . Plasmid R27 was visualized as the discrete GFP foci present at the mid- and quarter-cell regions in >99% of the cells . Time lapse experiments demonstrated that an increase in R27 plasmid foci resulted from focus duplication in either the mid- or quarter-cell regions of E . coli . Both R27 Par(-) variants gave a high percentage of plasmidless cells, as suggested by a uniform GFP signal, and cells with GFP patterns scattered throughout the entire cell, suggesting that plasmid molecules are randomly distributed throughout the cytoplasm . Those cells that did contain R27 Par(-) with one or two discrete foci had localization patterns that were statistically different from those formed with wild-type R27 . Therefore, these results suggest that partitioning-impaired plasmids are characterized by individual and clustered plasmids that are randomly located within the host cytoplasm.

J Biotechnol, 2003 May 8, 102(3), 211 - 21
Transfection of partially purified plasmid DNA for high level transient protein expression in HEK293-EBNA cells; Wright JL et al.; One of the major constraints to performing large-scale transfections of cultured mammalian cells for the transient expression of recombinant proteins is the production of large quantities of purified plasmid DNA . In this report partially purified plasmid DNA was prepared by a method that combines alkaline lysis of E . coli with standard precipitation techniques . The efficiency of calcium phosphate-DNA co-precipitate formation with crude DNA was similar to that observed for pure DNA, but precipitate formed with crude DNA also contained RNA . The transfection of adherent and suspension-adapted HEK293-EBNA cells with partially purified pEGFPN1 resulted in levels of transient GFP expression equivalent to those achieved with pure DNA . In addition, the co-transfection of 1-200 ml cultures of suspension-adapted HEK293-EBNA cells with two different plasmids encoding the heavy and light chain genes of anti-human RhD IgG1, respectively, yielded similar IgG titers with pure and partially purified plasmid DNA . Finally, it was observed that suspension-adapted cells were more tolerant to the presence of RNA in the plasmid preparations than were adherent cells . These findings are relevant to the field of DNA transfection, including applications ranging from high-throughput screening to large-scale transient protein expression.

J Biotechnol, 2003 May 8, 102(3), 241 - 9
Activity, conformation and dynamics of cutinase adsorbed on poly(methyl methacrylate) latex particles; Baptista RP et al.; The adsorption of a recombinant cutinase from Fusarium solani pisi onto the surface of 100 nm diameter poly(methyl methacrylate) (PMMA) latex particles was evaluated . Adsorption of cutinase is a fast process since more than 70% of protein molecules are adsorbed onto PMMA at time zero of experiment, irrespective of the tested conditions . A Langmuir-type model fitted both protein and enzyme activity isotherms at 25 degrees C . Gamma(max) increased from 1.1 to 1.7 mg m(-2) and U(max) increased from 365 to 982 U m(-2) as the pH was raised from 4.5 to 9.2, respectively . A decrease (up to 50%) in specific activity retention was observed at acidic pH values (pH 4.5 and 5.2) while almost no inactivation (eta(act) congruent with 87-94%) was detected upon adsorption at pH 7.0 and 9.2 . Concomitantly, far-UV circular dichroism (CD) spectra evidenced a reduction in the alpha-helical content of adsorbed protein at acidic pH values while at neutral and alkaline pH the secondary structure of adsorbed cutinase was similar to that of native protein . Fluorescence anisotropy decays showed the release of some constraints to the local motion of the Trp69 upon protein adsorption at pH 8.0, probably due to the disruption of the tryptophan-alanine hydrogen bond when the tryptophan interacts with the PMMA surface . Structural data associated with activity measurements at pH 7.0 and 9.2 showed that cutinase adsorbs onto PMMA particles in an end-on orientation with active site exposed to solvent and full integrity of cutinase secondary structure . Hydrophobic interactions are likely the major contribution to the adsorption mechanism at neutral and alkaline pH values, and a higher amount of protein is adsorbed to PMMA particles with increasing temperature at pH 9.2 . The maximum adsorption increased from 88 to 140 mg cutinase per g PMMA with temperature raising from 25 to 50 degrees C, at pH 9.2.

FEBS Lett, 2003 May 8, 542(1-3), 159 - 63
A metallothionein and CPx-ATPase handle heavy-metal tolerance in the filamentous cyanobacterium Oscillatoria brevis; Liu T et al.; A metallothionein (BmtA) and a CPx-ATPase (Bxa1) have been identified and characterized from the cyanobacterium Oscillatoria brevis . Both bmtA and bxa1 expression can be markedly induced in vivo by Zn(2+) or Cd(2+) . Over-expression of bmtA or bxa1 in Escherichia coli enhances Zn(2+) and Cd(2+) tolerance in the transformant . Dynamic studies on the expression of two genes showed that the maximum expression of bxa1 induced by Zn(2+) and Cd(2+) was much quicker than that of bmtA, suggesting distinct physiological roles of metallothionein and CPx-ATPase in the handling of surplus metal.

FEBS Lett, 2003 May 8, 542(1-3), 115 - 8
Glutamate substitutions at a PKA consensus site are consistent with inactivation of calpain by phosphorylation; Smith SD et al.; Regulation of calpain by phosphorylation has often been suggested, but has proved difficult to detect . Calpains extracted from mammalian tissue are reported to contain 2-4 mol phosphate/mol of enzyme distributed over multiple sites, but phosphate groups are not detectable in the X-ray structures of recombinant calpain . Some serine and threonine residues in the large subunit of rat m-calpain were converted to aspartic or glutamic acid residues, at sites suggested by previous studies, to assess the probable effects of phosphate groups on the enzyme . Expression of the mutant calpains in Escherichia coli, and their heat stabilities, did not differ from those of the wild-type enzyme . m-Calpains with the mutations Ser50Asp, Ser50Glu, Ser67Glu, and Thr70Glu had the same specific activity and Ca(2+) requirement as the wild-type enzyme . In contrast, Ser369Asp-, Ser369Glu-, and Thr370Glu-m-calpain were inactive . This result is consistent with the recent report that phosphorylation at position 369 or 370 in vivo reduced m-calpain activation.

J Mol Biol, 2003 May 9, 328(4), 863 - 75
Evolutionary stabilization of the gene-3-protein of phage fd reveals the principles that govern the thermodynamic stability of two-domain proteins; Martin A et al.; The gene-3-protein (G3P) of filamentous phage is essential for their propagation . It consists of three domains . The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor . Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability . In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P . Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains . The method is limited by the proteolytic stability of G3P itself . We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections . Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C . The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined . In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step . N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken . The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains . These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and how mutations are optimally placed to improve the stability of such proteins.

J Mol Biol, 2003 May 16, 328(5), 1123 - 35
Expression of an RNase P ribozyme against the mRNA encoding human cytomegalovirus protease inhibits viral capsid protein processing and growth; Trang P et al.; A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein . We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro . Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably . In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant . Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked . These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly . Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined . Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.

J Mol Biol, 2003 May 16, 328(5), 1047 - 60
Energetics of lesion recognition by a DNA repair protein: thermodynamic characterization of formamidopyrimidine-glycosylase (Fpg) interactions with damaged DNA duplexes; Minetti CA et al.; As part of an overall effort to map the energetic landscape of the base excision repair pathway, we report the first thermodynamic characterization of repair enzyme binding to lesion-containing duplexes . Isothermal titration calorimetry (ITC) in conjunction with spectroscopic measurements and protease protection assays have been employed to characterize the binding of Escherichia coli formamidopyrimidine-glycosylase (Fpg), a bifunctional repair enzyme, to a series of 13-mer DNA duplexes . To resolve energetically the binding and the catalytic events, several of these duplexes are constructed with non-hydrolyzable lesion analogs that mimic the natural 8-oxo-dG substrate and the abasic-like intermediates . Specifically, one of the duplexes contains a central, non-hydrolyzable, tetrahydrofuran (THF) abasic site analog, while another duplex contains a central, carbocyclic substrate analog (carba-8-oxo-dG) . ITC-binding studies conducted between 5.0 degrees C and 15.0 degrees C reveal that Fpg association with the THF-containing duplex is characterized by binding free energies that are relatively invariant to temperature (deltaG approximately -9.5 kcalmol(-1)), in contrast to both the reaction enthalpy and entropy that are strongly temperature-dependent . Complex formation between Fpg and the THF-containing duplex at 15 degrees C exhibits an unfavorable association enthalpy (deltaH=+7.5 kcalmol(-1)) that is compensated by a favorable association entropy (TdeltaS=+17.0 kcalmol(-1)) . The entropic nature of the binding interaction, coupled with the large negative heat capacity (deltaC(p)=-0.67 kcaldeg(-1)mol(-1)), is consistent with Fpg complexation to the THF-containing duplex involving significant burial of non-polar surface areas . By contrast, under the high ionic strength buffer conditions employed herein (200 mM NaCl), no appreciable Fpg affinity for the carba-8-oxo-dG substrate analog is detected . Our results suggest that initial Fpg recognition of a damaged DNA site is predominantly electrostatic in nature, and does not involve large contact interfaces . Subsequent base excision presumably facilitates accommodation of the resulting lesion site into the binding pocket, as the enzyme interaction with the THF-containing duplex is characterized by high affinity and a large negative heat capacity change . Our data are consistent with a pathway in which Fpg glycosylase activity renders the base excision product a preferred ligand relative to the natural substrate, thereby ensuring the fidelity of removing highly reactive and potentially mutagenic abasic-like intermediates through catalytic elimination reactions.

Protein Expr Purif, 2003 May, 29(1), 123 - 31
Expression, purification, and initial characterization of a recombinant form of plant PEP-carboxylase kinase from CAM-induced Mesembryanthemum crystallinum with enhanced solubility in Escherichia coli; Ermolova NV et al.; Plant phosphoenolpyruvate-carboxylase kinase (PEPC-kinase {PpcK}) is the smallest Ser/Thr kinase identified to date, having a molecular mass of approximately 32,000 . This novel, monomeric kinase is dedicated to the phosphorylation of plant PEPC, thereby regulating this target enzyme's activity and allosteric properties . Although several recombinant, non-fusion PpcK proteins have been produced recently in Escherichia coli, these are plagued by their high degree of insolubility . Here, we report the use of the native, E . coli NusA protein and a related E . coli expression vector (pET-43a(+) {Novagen}) for enhancing the solubility of this recalcitrant Ser/Thr kinase at least 10-fold by its production as a dual 6xHis-tagged NusA/McPpcK1 fusion protein, which accounts for approximately 10% of the soluble protein fraction from induced cells . Capture of this fusion protein from the centrifuged cell extract by immobilized metal (Ni(2+)) affinity-chromatography, its "on-bead" cleavage by thrombin, and subsequent elution yielded milligram quantities of a "free," approximately 36-kDa form of PpcK for further purification by fast-protein liquid chromatography on blue dextran-agarose or preparative SDS-PAGE . Steady-state kinetic analysis of the former, active preparation revealed that this dedicated kinase discriminates against neither various isoforms of plant PEPC nor certain mutant forms of recombinant C(4) PEPC . Alternatively, the latter, electrophoretically homogeneous sample of the approximately 36-kDa polypeptide was used as antigen for polyclonal-antibody production in rabbits . The antibodies against the recombinant McPpcK1 from Mesembryanthemum crystallinum cross-reacted on Western blots with an enriched preparation of the maize-leaf kinase, but not with the parent crude extract, thus directly documenting this protein's extremely low abundance in vivo . However, these antibodies were effective in immunoprecipitating 32P-based PpcK activity from crude, desalted extracts of maize leaves and soybean root-nodules.

Protein Expr Purif, 2003 May, 29(1), 117 - 22
Characterization of factors favoring the expression of soluble protozoan tubulin proteins in Escherichia coli; MacDonald LM et al.; The alpha- and beta-tubulin genes of the parasitic protozoa Giardia duodenalis, Cryptosporidium parvum, and Encephalitozoon intestinalis have been overexpressed in soluble form using Escherichia coli-based expression systems . Several expression systems were compared in terms of the amount of soluble protein produced with different fusion partners, strains of E . coli BL21, and expression temperatures . The cleavability of the fusion partner was also assessed in terms of post-expression applications of the recombinant protein . The maltose-binding protein (MBP) and glutathione S-transferase (GST) fusion partners produced the highest expression levels for all six proteins without the formation of inclusion bodies . The expression system also provided a means of purifying the soluble protein using affinity and anion-exchange chromatography while minimizing protein losses . The yield and purity were therefore very high for both the MBP and GST systems . The tubulin monomers were demonstrated to be assembly-competent using a standard dimerization assay and also retained full antigenicity with monoclonal antibodies . This study presents several methods which are suitable for producing soluble tubulin monomers and, thus, circumventing the formation of inclusion bodies which necessitates re-folding of the tubulin.

Protein Expr Purif, 2003 May, 29(1), 85 - 93
Construction and characterization of a recombinant esterase with high activity and enantioselectivity to (S)-ketoprofen ethyl ester; Choi GS et al.; The ester-hydrolyzing enzyme families, including lipase and esterase, mediated a broad range of reactions and, thus, were able to act on a variety of ester compounds that are found naturally or exploited industrially . With the increasing demand for pharmacological use, attempts to produce an enantiomer (S)-ketoprofen from the corresponding ethyl ester have recently been proliferating, but information about the structure and function of related enzymes has not been reported to date in detail . Here, we reported the construction, expression, and one-step purification of a potential esterase in Escherichia coli with a hexahistidine tag at its N-terminus . The expression level of the enzyme was more than 20% of the total protein in E . coli, resulting in approximately 1.2mg of the purified proteins by an affinity resin, Ni-NTA, from a 0.2L of bacterial culture in a single step . As typical properties, its innate traits that revealed favorable reactions at alkaline pH and high activity to the triglycerides composed of short chain fatty acids (<C(6)) supported the enzyme to be an esterase . The enzyme was determined to be a monomer with a calculated molecular mass of 42 kDa and showed quite a high activity to rac-ketoprofen ethyl ester (27,000 U), with strict selectivity to (S)-enantiomer (>99% ee(p)) . The small-scale conversion using the recombinant enzyme strongly suggested the enzyme to be useful for enzyme-mediated chiral resolution of (S)-ketoprofen.

Protein Expr Purif, 2003 May, 29(1), 70 - 6
Chaperone-assisted expression, purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni; Stevens JM et al.; Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide . Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form . We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E . coli GroES and GroEL chaperones . The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications . In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.

Protein Expr Purif, 2003 May, 29(1), 51 - 7
Cloning, expression, and characterization of diuretic hormone Manduca diuresin from Manduca sexta in Escherichia coli; Wong WK et al.; Manduca diuresin (MD), a 30 amino acid peptide isolated from the tobacco hornworm Manduca sexta, was found to play an important role in the regulation of water and salt balance in the insect . To facilitate studies relating to the function and structure of MD, a synthetic gene encoding MD was assembled and expressed in Escherichia coli . Using an excretion vector, expression of the MD gene in an induced transformant was detected at the transcriptional and translational levels by Northern-blot and ELISA analyses, respectively . With the use of glutathione-S-transferase as the reporter protein, MD was confirmed to be expressed in E . coli . The recombinant product was resolved by reverse-phase HPLC into three peptide groups of different retention times, which were shown by mass spectrometry to be composed of MD deletants missing various lengths of the C-terminus . Despite the deletions and the absence of an amidated C-terminus, the deletants were shown to be biologically active, suggesting the importance of the N-terminus of MD for biological activity.

Protein Expr Purif, 2003 May, 29(1), 24 - 32
Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli; Riera M et al.; Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro . The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared to CK2 from human . Kinetic measurements of the recombinant maize holoenzyme (rmCK2) revealed k(cat) values for ATP and GTP of 4 and 2s(-1), respectively; whereas the recombinant maize catalytic subunit showed almost equal values for ATP and GTP, i.e., ca . 0.8s(-1) . A comparison of the k(cat)/K(m) ratio between the maize holoenzyme and the catalytic subunit from CK2 maize shows that the incorporation of the catalytic subunit into the holoenzyme leads to a 14-fold activation in the case of ATP and 8-fold activation in the case of GTP . The maize holoenzyme is about 10 times more sensitive towards CK2 inhibitor heparin, on the other hand, it is stimulated only 0% by polylysine as compared to the human counterpart . The maize holoenzyme activity is more sensitive towards NaCl concentrations higher than those of rhCK2 and treatment with urea showed that rmCK2 holoenzyme was denatured more readily than the human holoenzyme.

Protein Expr Purif, 2003 May, 29(1), 8 - 14
Overexpression and reconstitution of a Rieske iron-sulfur protein from the higher plant; Gubernator B et al.; The iron-sulfur protein subunit, known as the Rieske protein, is one of the central components of the cytochrome b(6)f complex residing in chloroplast and cyanobacterial thylakoid membranes . We have constructed plasmids for overexpression in Escherichia coli of full-length and truncated Rieske (PetC) proteins from the Spinacia oleracea fused to MalE . Overexpressed fusion proteins were predominantly found (from 55 to 70%) in cytoplasm in a soluble form . The single affinity chromatography step (amylose resine) was used to purify about 15mg of protein from 1 liter of E . coli culture . The isolated proteins were electrophoretically pure and could be used for further experiments . The NifS-like protein IscS from the cyanobacterium Synechocystis PCC 6803 mediates the incorporation of 2Fe-2S clusters into apoferredoxin and cyanobacterial Rieske apoprotein in vitro . Here, we used the recombinant IscS protein for the enzymatic reconstitution of the iron-sulfur cluster into full-length Rieske fusion and truncated Rieske fused proteins . Characterization by EPR spectroscopy of the reconstituted proteins demonstrated the presence of a 2Fe-2S cluster in both full-length and truncated Rieske fusion proteins.

Protein Expr Purif, 2003 May, 29(1), 1 - 7
Purification and characterization of a soluble form of rat liver NADPH-cytochrome P-450 reductase highly expressed in Escherichia coli; Hayashi S et al.; A recombinant cDNA of rat liver NADPH-cytochrome P-450 reductase (CPR), which lacks the N-terminal hydrophobic region, was amplified by PCR and cloned . The N-truncated cDNA named tCPR was ligated into a pBAce vector and expressed . The tCPR protein expressed in Escherichia coli was recovered into the soluble fraction of the cell lysate and purified to homogeneity by three sequential purification procedures; (I) anion-exchange chromatography on a DEAE-cellulose (DE-52) column, (II) affinity chromatography on 2('),5(')-ADP Sepharose 4B, and (III) chromatography on a hydroxyapatite column . The average yield was 47mg per liter of culture medium . The absorption spectrum of the purified tCPR protein was identical to that of a native full-length CPR purified from rat liver, indicating that tCPR also possesses one molecule each of FAD and FMN . The tCPR protein was able to reduce cytochrome c and was also able to assist heme degradation by a soluble form of rat heme oxygenase-1 . However, it failed to support the O-deethylation of 7-ethoxycoumarin by cytochrome P-450 1A1, indicating that the presence of the N-terminal hydrophobic domain is necessary for CPR to interact with cytochrome P-450 . Previously, to prepare a soluble form of CPR, full-length CPR was treated with proteinases that selectively removed the N-terminal domain . With the expression system established in this study, however, the soluble and biologically active tCPR protein can be readily prepared in large amounts . This expression system will be useful for mechanistic as well as structural studies of CPR.

Arch Biochem Biophys, 2003 May 15, 413(2), 253 - 61
Heterologous expression of Ascaris suum cytochrome b5 precursor protein: a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coli; Takamiya S et al.; The cytochrome b(5) of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b(5) . It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b(5), but possesses an NH(2)-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues {Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch . Biochem . Biophys . 328, 165-172} . The nematode cytochrome b(5) is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown . To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b(5) with a complete presequence (b5wt) and its NH(2) terminus-truncated derivatives, b5Delta18 and b5Delta28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli . As expected, all transformants, tb5wt, tb5Delta18, and tb5Delta28, produced recombinant proteins with a histidine-tagged NH(2)-terminal extension . However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall . These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E . coli.

Arch Biochem Biophys, 2003 May 15, 413(2), 243 - 52
Purification and characterization of yeast mitochondrial initiation factor 2; Garofalo C et al.; Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene . A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified . Purified ymIF2 bound both E . coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions . In addition, the isolated ymIF2 was compared to E . coli IF2 in four other assays commonly used to characterize this initiation factor . Formylated and nonformylated Met-tRNA(f)(Met) were bound to E . coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA . The GTPase activity of ymIF2 was found to be dependent on the presence of E . coli ribosomes . The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E . coli IF2 against nonenzymatic deaminoacylation . In contrast to E . coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay . In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.

Anal Biochem, 2003 Jun 1, 317(1), 59 - 66
The effects of Ca(2+) binding on the conformation of calbindin D(28K): a nuclear magnetic resonance and microelectrospray mass spectrometry study; Venters RA et al.; Calbindin D(28K) is a six-EF-hand calcium-binding protein found in the brain, peripheral nervous system, kidney, and intestine . There is a paucity of information on the effects of calcium binding on calbindin D(28K) structure . To further examine the mechanism and structural consequences of calcium binding to calbindin D(28K) we performed detailed complementary heteronuclear NMR and microelectrospray mass spectrometry investigations of the calcium-induced conformational changes of calbindin D(28K) . The combined use of these two powerful analytical techniques clearly and very rapidly demonstrates the following: (i) . apo-calbindin D(28K) has an ordered structure which changes to a notably different ordered conformation upon Ca(2+) loading, (ii) . calcium binding is a sequential process and not a simultaneous event, and (iii) . EF-hands 1, 3, 4, and 5 take up Ca(2+), whereas EF-hands 2 and 6 do not . Our results support the opinion that calbindin D(28K) has characteristics of both a calcium sensor and a buffer.

Biosci Biotechnol Biochem, 2003 Feb, 67(2), 368 - 71
Periplasmic secretion of native ovalbumin without signal cleavage in Escherichia coli; Arii Y et al.; In Escherichia coli cells carrying wild-type ovalbumin cDNA, some of the recombinant protein was secreted into the periplasmic space . In contrast, a signal-region mutant form of ovalbumin (deletion, Gly1 to Ala39) was not detected in the periplasm despite being synthesized at the same level as the wild-type protein . Chemical and spectroscopic analyses showed that periplasmic ovalbumin assumes a conformation indistinguishable from that of native egg white ovalbumin . We concluded that a process resembling the secretion of ovalbumin process in the oviduct occurs also in bacteria.

Biosci Biotechnol Biochem, 2003 Feb, 67(2), 347 - 53
Novel gene encoding a Ca2+-binding protein and under hexokinase-dependent sugar regulation; Otsuki S et al.; A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis . Here we report that this cDNA encodes a novel Ca2+-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1) . The recombinant OsSUR1 protein expressed in Escherichia coli had 45Ca2+-binding activity . Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination . Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2+ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase . These results suggested that Os-SUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.

Biosci Biotechnol Biochem, 2003 Feb, 67(2), 341 - 6
Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs; Suzukawa K et al.; We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E . coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity . Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate . Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity . Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251 . Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena . The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

J Plant Res, 2003 Jun, 116(3), 259 - 63 Epub 2003 May 01.
A new method for enzymatic preparation of isopentenyladenine-type and trans-zeatin-type cytokinins with radioisotope-labeling; Takei K et al.; We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides . The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli . The A . thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-( E)-butenyl diphosphate as isoprenoid donors . The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates . Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used . These labeled compounds will be useful tools for cytokinin research, such as receptor-ligand assays and cell metabolism studies.

Farmaco, 2003 Apr, 58(4), 329 - 36
Synthesis of new P3CS derivatives and their mitogenic activity on in vitro mice splenocytes; Pappalardo M et al.; Vaccination against tumors represents a relevant issue in current human cancer therapy . The N-terminal part of the lipoprotein from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-Cys-Ser (P(3)CS) and analogs with longer aminoacidic sequence are polyclonal activators for B-lymphocytes . Previous study reported that their N-2,2,2-trichloroethoxycarbonyl (Troc) derivatives increase immunocyte mitogenic activity . Therefore, in order to obtain compounds of greater activity and to investigate relationships between molecular structure of S-glyceryl skeleton and biological activity, we synthesized new Troc derivatives of P(3)CS . The mitogenicity of compounds was determined in vitro, by measuring in vitro {3H}-thymidine incorporation into splenocytes from Balb/c mice . Concentrations of compounds ranged from 0 to 64 micro g/ml . In particular, S-{2,3-bis(trichloroethoxycarbonyloxy)}-N-trichloroethoxycarbonyl dipeptide derivative exhibited significant mitogenic activity endowed with high pharmacological potency . These new series of compounds could be used as potent immunoadjuvants for the development of novel synthetic vaccines for tumor immunotherapy.

FEMS Immunol Med Microbiol, 2003 May 15, 36(1-2), 1 - 7
Endotoxic and immunobiological activities of a chemically synthesized lipid A of Helicobacter pylori strain 206-1; Ogawa T et al.; A synthetic lipid A of Helicobacter pylori strain 206-1 (compound HP206-1), which is similar to its natural lipid A, exhibited no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506) . Furthermore, compound HP206-1 as well as its natural lipid A demonstrated no or very low mitogenic responses in murine spleen cell . On the other hand, compound HP206-1 showed a weaker but significant production of interleukin-8 in a gastric cancer cell line, MKN-1, in comparison with compound 506 . Furthermore, compound HP206-1 exhibited induction of tumor necrosis factor-alpha production in human peripheral blood mononuclear cells and the cytokine production was clearly inhibited by mouse anti-human Toll-like receptor (TLR) 4 monoclonal antibody HTA125 . Our findings indicate that the chemically synthesized lipid A, mimicking the natural lipid A portion of lipopolysaccharide from H . pylori strain 206-1, has a low endotoxic potency and immunobiological activities, and is recognized by TLR4.

Gene, 2003 Apr 24, 309(1), 49 - 56
Hypomutable regions of yeast TFIIB in a unigenic evolution test represent structural domains; Zeng X et al.; As genome sequences of many organisms - including humans - are being decoded, there is a great need for genetic tools to analyze newly discovered genes/proteins . A 'unigenic evolution' approach has been previously proposed for dissecting protein domains, which is based on the assumption that functionally important regions of a protein may tolerate missense mutations less well than other regions . We describe a unigenic evolution analysis of general transcription factor IIB (TFIIB) - a protein that is well characterized both structurally and functionally - to better understand the molecular basis of this genetic approach . The overall distribution profile of hypomutable regions within yeast TFIIB correlates extremely well with the known compact structural domains, suggesting that the unigenic evolution approach can help reveal structural properties of a protein . We further show that a small region located immediately carboxyl-terminal to the zinc ribbon motif is functionally important despite its strong hypermutability . Our study further demonstrates the usefulness of the unigenic evolution approach in dissecting protein domains, but suggests that the mutability of different regions of a protein in such a test is determined primarily by their structural properties.

Biophys Chem, 2003 Mar 25, 103(3), 223 - 37
Inter-subunit recognition and manifestation of segmental mobility in Escherichia coli RNA polymerase: a case study with omega-beta' interaction; Ghosh P et al.; Omega (omega), consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region . Our earlier experiments have shown a chaperone-like function of omega in which it helps to maintain beta' in a correct conformation and recruit it to the alpha(2)beta subassembly to form a functional core enzyme (alpha(2)betabeta'omega) . The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch onto the N-terminal and C-terminal ends of the beta'-subunit . In the present study we have monitored the conformational changes in beta' as the denatured protein is refolded in the presence and absence of omega using tryptophan fluorescence emission of beta' as well as acrylamide quenching of Trp fluorescence . Results indicate that the presence of stoichiometric amounts of omega is helpful in beta' refolding . We have also monitored the behavior of the C-terminal tail of omega by engineering three cysteine residues at three different sites in omega and subsequently labeling them with a sulphydryl-specific fluorescent probe . Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of omega is mobile in the free protein and gets restrained in the presence of beta' . Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the beta' subunit whereas the end of the C-terminal of omega, although it is restrained in the presence of beta', has no interacting partner within a 4-A radius.

Water Res, 2003 May, 37(10), 2505 - 11
Bioaccumulation of nickel from aqueous solutions by genetically engineered Escherichia coli; Deng X et al.; This study constructed a genetically engineered Escherichia coli JM109 which simultaneously expressed nickel transport system and metallothionein to remove and recover Ni(2+) from aqueous solution . Bioaccumulation process was rapid and followed linearized Langmuir isotherm . A more than six-fold increase of Ni(2+) binding capacity was obtained by genetically engineered E . coli cells compared with original host E . coli cells . A pH assay showed genetically engineered E . coli cells accumulated Ni(2+) effectively over a broad range of pH (4-10) . The presence of 1000 mg/L Na(+) and Ca(2+), or 50mg/L Cd(2+) or Pb(2+) did not have a significant effect on Ni(2+) bioaccumulation, while Mg(2+), Hg(2+) and Cu(2+) posed a severe adverse influence on Ni(2+) uptake by genetically engineered E . coli . Furthermore, genetically engineered E . coli cells did not require extra nutrients for Ni(2+) bioaccumulation.

Biochem Biophys Res Commun, 2003 May 16, 304(4), 625 - 31
Engineering nuclear localization signals in modular protein vehicles for gene therapy; Aris A et al.; Amino acids from 126 to 135 of the SV40 virus T antigen act as efficient nuclear localization signal during infection but also when fused to recombinant proteins . This peptide has been inserted into two alternative acceptor sites of a modified Escherichia coli beta-galactosidase which also displays a DNA-binding domain, a cell-binding motif for integrin alpha(v)beta(3) targeting and cell internalization, and a cryptic nuclear targeting signal naturally present in the bacterial enzyme . In cultured cells, the presence of the SV40 peptide enhances the expression of a delivered DNA up to 30-fold . However, the DNA expression levels are largely depending on the chosen insertion site for the SV40 segment concomitant to the structural impact of peptide accommodation on the protein vehicle . The structural stability of the hybrid protein, apparently critical for efficient gene transfer, is discussed in the context of modular protein engineering to develop non-viral vectors for gene therapy.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 257 - 62
Maximization of recombinant Helicobacter pylori neutrophil activating protein production in Escherichia coli: improvement of a chemically defined medium using response surface methodology; Niccolai A et al.; Medium optimization for the production of constitutive recombinant Helicobacter pylori neutrophil activating protein (NAP) in Escherichia coli was investigated by using response surface methodology . Carbon to nitrogen ratio, concentrations of sodium polyphosphate and magnesium sulfate were considered as independent variables . The optimized medium was a chemically defined medium with a carbon to nitrogen ratio of 14.4 and with concentrations of sodium polyphosphate and magnesium sulfate about 7.1 g l(-1) and 3.04 g l(-1) respectively . The maximum recombinant NAP production level (1184.6 mg l(-1)) was 29.96% higher than that in control medium.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 237 - 42
RNase H overproduction allows the expression of stress-induced genes in the absence of topoisomerase I; Cheng B et al.; Induction of stress proteins in response to heat shock was found to be reduced significantly in Escherichia coli with DeltatopA mutation . RNase H overexpression in the DeltatopA mutant partially restored the sigma(32)-dependent induction of stress genes in response to high temperature and ethanol . The presence of overexpressed RNase H also improved the survival rate of the DeltatopA mutant after high temperature and oxidative challenges . Topoisomerase I is likely required during stress response for preventing accumulation of transcription-driven hypernegative supercoiling and R-loop formation at induced stress genes loci.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 161 - 5
The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity; Nolan LM et al.; The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide . The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile . The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen . The recombinant enzyme shows highest nitrilase activity with benzonitrile . Site-directed mutagenesis of the F . lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity . This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same . This is the first evidence of cyanide hydratase having nitrilase activity.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 155 - 9
Carbon supply and 2-oxoglutarate effects on expression of nitrate reductase and nitrogen-regulated genes in Synechococcus sp . strain PCC 7942; Vazquez-Bermudez MF et al.; Synthesis of nitrate reductase in the unicellular cyanobacterium Synechococcus sp . strain PCC 7942 took place at a slow rate when the cells were incubated without a supply of inorganic carbon, but addition to these cells of CO(2)/bicarbonate or, in a Synechococcus strain transformed with a gene encoding a 2-oxoglutarate permease, 2-oxoglutarate stimulated expression of the enzyme . Induction by 2-oxoglutarate was also observed at the mRNA level for two nitrogen-regulated genes, nir and amt1, but not for the photosystem II D1 protein-encoding gene psbA1 . Our results are consistent with a role of 2-oxoglutarate in nitrogen control in cyanobacteria.

Chem Biol, 2003 Apr, 10(4), 301 - 11
The biosynthetic gene cluster for the beta-lactam carbapenem thienamycin in Streptomyces cattleya; Nunez LE et al.; beta-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic beta-lactam antibiotics . This involves the participation of a beta-lactam synthetase . Using available information from beta-lactam synthetases, we generated a probe for the isolation of the thienamycin cluster from Streptomyces cattleya . Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified . They would participate in early steps of thienamycin biosynthesis leading to the formation of the beta-lactam ring . Other genes necessary for the biosynthesis of thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system . Involvement of the cluster in thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating thienamycin nonproducing mutants.

Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5801 - 6 Epub 2003 Apr 30.
A conserved function of YidC in the biogenesis of respiratory chain complexes; van der Laan M et al.; The Escherichia coli inner membrane protein (IMP) YidC is involved in the membrane integration of IMPs both in concert with and independently from the Sec translocase . YidC seems to be dispensable for the assembly of Sec-dependent IMPs, and so far it has been shown to be essential only for the proper Sec-independent integration of some phage coat proteins . Here, we studied the physiological consequences of YidC depletion in an effort to understand the essential function of YidC . The loss of YidC rapidly and specifically induced the Psp stress response, which is accompanied by a reduction of the proton-motive force . This reduction is due to defects in the functional assembly of cytochrome o oxidase and the F(1)F(o) ATPase complex, which is reminiscent of the effects of mutations in the yidC homologue OXA1 in the yeast mitochondrial inner membrane . The integration of CyoA (subunit II of the cytochrome o oxidase) and F(o)c (membrane subunit of the F(1)F(o) ATPase) appeared exceptionally sensitive to depletion of YidC, suggesting that these IMPs are natural substrates of a membrane integration and assembly pathway in which YidC plays an exclusive or at least a pivotal role.

Mol Cell Biol, 2003 May, 23(10), 3392 - 404
Tax recruitment of CBP/p300, via the KIX domain, reveals a potent requirement for acetyltransferase activity that is chromatin dependent and histone tail independent; Georges SA et al.; Robust transcription of human T-cell leukemia virus type 1 (HTLV-1) genome requires the viral transactivator Tax . Although Tax has been previously shown to interact with the KIX domain of CBP/p300 in vitro, the precise functional relevance of this interaction remains unclear . Using two distinct approaches to interrupt the physical interaction between Tax and KIX, we find that Tax transactivation from chromatin templates is strongly dependent on CBP/p300 recruitment via the KIX domain . Additionally, we find that the primary functional contribution of CBP/p300 to Tax transactivation resides in the intrinsic acetyltransferase activity of the coactivators . These studies unexpectedly uncover a specific requirement for CBP/p300 acetyltransferase activity on chromatin templates assembled with nucleosomes lacking their amino-terminal tails . Together, these data indicate that the KIX domain of CBP/p300 is essential for targeting the acetyltransferase activity of the coactivator to the Tax-CREB (Tax/CREB) complex . Significantly, these observations reveal the presence of one or more CBP/p300 acetyltransferase targets that function specifically on chromatin templates, are independent of the histone tails, and are critical to Tax transactivation.

Microbiology, 2003 May, 149(Pt 5), 1297 - 310
Detection and analysis of transpositionally active head-to-tail dimers in three additional Escherichia coli IS elements; Szeverenyi I et al.; This study demonstrates that Escherichia coli insertion elements IS3, IS150 and IS186 are able to form transpositionally active head-to-tail dimers which show similar structure and transpositional activity to the dimers of IS2, IS21 and IS30 . These structures arise by joining of the left and right inverted repeats (IRs) of two elements . The resulting junction includes a spacer region (SR) of a few base pairs derived from the flanking sequence of one of the reacting IRs . Head-to-tail dimers of IS3, IS150 and IS186 are unstable due to their transpositional activity . They can be resolved in two ways that seem to form a general rule for those elements reported to form dimers . One way is a site-specific process (dimer dissolution) which is accompanied by the loss of one IS copy along with the SR . The other is 'classical' transposition where the joined ends integrate into the target DNA . In intramolecular transposition this often gives rise to deletion formation, whereas in intermolecular transposition it gives rise to replicon fusion . The results presented for IS3, IS150 and IS186 are in accordance with the IS dimer model, which is in turn consistent with models based on covalently closed minicircles.

Microbiology, 2003 May, 149(Pt 5), 1265 - 73
n-Hexane sensitivity of Escherichia coli due to low expression of imp/ostA encoding an 87 kDa minor protein associated with the outer membrane; Abe S et al.; Most Escherichia coli strains are resistant to n-hexane . E . coli OST4251 is a n-hexane-sensitive strain that was constructed by transferring the n-hexane-sensitive phenotype from a n-hexane-sensitive strain by P1 transduction . OST4251 is resistant to diphenyl ether, which is less harmful than n-hexane to micro-organisms . The genetic determinant responsible for this subtle difference in the solvent resistance is mapped at 1.2 min on the E . coli chromosome . Nucleotide sequence analysis showed that IS2 and IS5 had integrated upstream of the imp/ostA structural gene in OST4251 . The integration of IS2 decreased the activity of the imp/ostA promoter . A product of the gene was identified immunologically as an 87 kDa minor protein associated with the outer membrane . Upon transformation with plasmids containing the imp/ostA gene, OST4251 produced a high level of the gene product in the membrane and acquired n-hexane resistance . Thus, the low level of promoter activity resulted in low Imp production and the n-hexane-sensitivity phenotype . It is likely that the gene product contributes to n-hexane resistance by reducing the influx of n-hexane.

J Biol Chem, 2003 Jul 18, 278(29), 27068 - 71 Epub 2003 Apr 30.
Direct interaction of subunits a and b of the F0 complex of Escherichia coli ATP synthase by forming an ab2 subcomplex; Stalz WD et al.; The addition of a His6 tag to the N terminus of subunit a of the F0 complex of the Escherichia coli ATP synthase allowed the purification of an ab2 subcomplex after solubilization of membranes with n-dodecyl-beta-d-maltoside and subsequent nickel-nitrilotriacetic acid affinity chromatography . After co-reconstitution of the ab2 subcomplex with purified subunit c, passive proton translocation rates as well as coupled ATPase activities after binding of F1 were measured that were comparable with those of wild type F0 . The interaction between subunits a and b, which has been shown to be stoichiometric and functional, is not triggered by any cross-linking reagent and therefore reflects subunit interactions occurring within the F0 complex in vivo.

J Pharm Pharmacol, 2003 Mar, 55(3), 301 - 6
The effect of protective agents on the stability of plasmid DNA by the process of spray-drying; Kuo JH; The effect of several protective agents was assessed on the stability of spray-dried plasmid DNA . The spray-drying process had adverse effects on the tertiary structure of plasmid DNA with the protective agents of sucrose, glycine and agarose . With the protection of these noncondensing agents, a band corresponding to the linear form of plasmid DNA was observed in the gel electrophoresis between the supercoiled circular (SC) form and the open circular (OC) form . On the contrary, spray-dried plasmid DNA maintained some degree of structural integrity under the protection of condensing agents . For the protection by neutral condensing polymers, such as polyethylene glycol 1000 and 4000, no linear form between the SC form and the OC form of plasmid DNA was revealed in the gel electrophoresis . Also, excess cationic condensing polymer, polyethyleneimine, had the ability to provide the plasmid DNA with protection from degradation as indicated by the preservation in SC and OC forms of plasmid DNA on the agarose gel electrophoresis . Moreover, DNA topology was unchanged after six-month storage at 4 degrees C by the protection of these neutral and cationic condensing agents . Accordingly, DNA condensation induced by condensing agents may provide a way to minimize damage to plasmid DNA by the process of spray drying.

Biosci Biotechnol Biochem, 2003 Mar, 67(3), 584 - 91
Cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol; Sugiyama M et al.; Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621 . The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0 . Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli . The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa . The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family . Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G . oxydans that had up to 11-fold greater XDH activity than the wild-type strain . When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol . These results demonstrated that increasing XDH activity in G . oxydans improved xylitol productivity.

Genome, 2003 Apr, 46(2), 177 - 81
An expression cDNA library for suppression cloning in yeast mutants, complementation of a yeast his4 mutant, and EST analysis from the symbiotic basidiomycete Hebeloma cylindrosporum; Wipf D et al.; An oriented expression library was constructed from the mycelia of the symbiotic model fungus Hebeloma cylindrosporum in the high-level yeast expression vector pDR196 . DNA sequencing of approximately 500 expressed sequence tags (ESTs) showed that 15% correspond to known genes, two thirds contain sequences with unknown function, andthe remaining 20% showed no significant similarity to any known genes . The ESTs had a GC content between 44 and 56%, with most of them having a GC content of 52-54%, which could be correlated with GC contents of fungal genes . The library was successfully used to identify the Hebeloma HIS4 gene by functional complementation of a yeast his4 mutant . Thus, the library may serve as a powerful tool for identification and characterization of mycorrhizal genes by EST analysis and for the identification of ectomycorrhizal genes by means of suppression cloning.

Genetika, 2003 Mar, 39(3), 326 - 35
{Determination of functional role of nucleotide composition in the transcription start region of the Escherichia coli udp gene}; Ovcharova IV et al.; The promoter of the Escherichia coli udp gene contains the poly-T (5'-TTTTT-3') motif in the transcription start region located at the distance of 3 nucleotides with respect to the Pribnow box . By means of site-directed mutagenesis, mutations in the +1, -1, and +3 positions of this region were isolated and their functional role in transcription initiation was determined . It was shown that in addition to the thymine nucleotide earlier identified at position 4 (with respect to the 5' end of the poly-T motif), the third thymine nucleotide may serve as an efficient transcription site . The functional significance of the presence of the poly-T motif in the transcription initiation region is discussed . Analysis of the isolated mutant promoters revealed the ability of the guanine nucleotide at the +3 position to stabilize the formation of a productive transcription complex and to serve as an additional transcription start.

Genetika, 2003 Mar, 39(3), 318 - 25
{Cloning and expression of the Mycoplasma hominis ftsZ for a cell division protein}; Momynaliev KT et al.; A Mycoplasma hominis chromosomal fragment containing the full-length ftsZ gene was cloned and sequenced . Natural expression of this gene was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) with total RNA . The M . hominis FtsZ protein was shown to differ substantially from its counterparts of two other Mycoplasma species, M . genitalium and M . pneumoniae . The possibility of M . hominis ftsZ expression in Escherichia coli was demonstrated with several bacterial strains . The M . hominis FtsZ protein was isolated from E . coli cells transformed with recombinant plasmids carrying the M . hominis ftsZ gene . Complementation between the E . coli and M . hominis FtsZ proteins was observed in transformants.

Phytother Res, 2003 Apr, 17(4), 372 - 5
Butanolides from Machilus thunbergii and their inhibitory activity on nitric oxide synthesis in activated macrophages; Kim NY et al.; In activated macrophages the inducible form of nitric oxide synthase (iNOS) generates high amounts of the toxic mediator, nitric oxide (NO), that contributes to the circulatory failure associated with septic shock . Three butanolides were isolated from Machilus thunbergii as active principles which inhibit the production of NO in lipopolysaccharide-activated RAW 264.7 cells, and their structures were identified as litsenolide A2 (1), B1 (2) and B2 (3) . They showed dose-dependent inhibition of NO syntheses and the IC(50)s were 3.36, 3.70 and 6.19 micro m, respectively . These new inhibitors of iNOS may have potential in the treatment of endotoxaemia and inflammation accompanied by the overproduction of NO .

Planta, 2003 May, 217(1), 66 - 74 Epub 2003 Feb 20.
Allelic variation and differential expression of methionine-rich delta-zeins in maize inbred lines B73 and W23a1; Kim WS et al.; The sulfur-amino-acid-rich delta-zeins of maize ( Zea mays L.) are represented by 18-kDa and 10-kDa proteins . We have cloned a novel 11-kDa methionine-rich delta-zein from developing endosperm of the inbred line W23a1 . The nucleotide sequence of this new delta-zein is identical to the published 10-kDa delta-zein, except for an insertion of 18 nucleotides between +316 and +333 bp from the translation start site . Antibodies raised against the recombinant 18-kDa delta-zein recognized both the 18-kDa and 10-kDa delta-zein from total seed protein extracts of different maize inbred lines . Western blot analysis revealed differences in the levels of the delta-zeins in different inbred lines and some of the inbred lines lacked either the 10-kDa or the 18-kDa delta-zeins . Northern blot analysis revealed temporal differences in the RNA transcript levels of the 11-kDa and 18-kDa delta-zeins between B73 and W23a1 . Such differences were not evident on Western blot analysis where similar protein accumulation profiles were seen for both lines . Immunostaining of paraffin sections of developing maize endosperm with the 18-kDa delta-zein antibodies revealed specific labeling of protein bodies found in the first few starchy layers from the aleurone layer . Electron-microscopic observation of thin-sections of B73 and W23a1 endosperm cells confirmed the presence of recently discovered novel, vacuole-like structures in these inbred lines . Immunogold labeling studies revealed that the delta-zeins were localized in the endoplasmic-reticulum-derived protein bodies and showed no preferential gold particle labeling over either the light or electron-dense material found in these protein bodies.

Mol Biotechnol, 2003 May, 24(1), 1 - 10
Efficient display of two enzymes on filamentous phage using an improved signal sequence; Strobel H et al.; Directed protein-evolution strategies generally make use of a link between a protein and the encoding DNA . In phage-display technology, this link is provided by fusion of the protein with a coat protein that is incorporated into the phage particle containing the DNA . Optimization of this link can be achieved by adjusting the signal sequence of the fusion . In a previous study, directed evolution of signal sequences for optimal display of the Taq DNA polymerase I Stoffel fragment on phage yielded signal peptides with a 50- fold higher incorporation of fusion proteins in phage particles . In this article, we show that for one of the selected signal sequences, improved display on phage can be generalized to other proteins, such as adenylate cyclases from Escherichia coli and Bordetella pertussis, and that this is highly dependent on short sequences at the C-terminus of the signal peptide . Further, the display of two enzymes on phage has been achieved and may provide a strategy for directing coevolution of the two proteins . These findings should be useful for display of large and cytoplasmic proteins on filamentous phage.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 481 - 91
Permeation associated with three-phase-partitioning method on release of green fluorescent protein; Penna TC et al.; Transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were subjected to two methods of extraction: (1) freezing/thawing/sonication (FTS) cycles prior to the three-phase partitioning (TPP) method, or (2) directly to TPP extraction . The amount of GFPuv released by the FTS plus TPP method varied: 374 microg/mL (first cycle), 93-442 microg/mL (second cycle), 32-359 microg/mL (third cycle), 18-115 microg/mL (fourth cycle) . The GFPuv yields by the second method (TPP only) were, 23-54 microg/mL for the first extract and 33-91 microg/mL for the second . The FTS plus TPP method released similar amounts of GFPuv to that extracted by TPP; and provided a better mixture elution through the hydrophobic interaction column: 13-63 microg/mL for FTS plus TPP methods, and 2.5-13 microg/mL for TPP . The results showed that although selective permeation is a more laborious methodology, it was more efficient for obtaining of GFPuv in relation to the direct extraction of the cells for TPP.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 775 - 85
CelF of Orpinomyces PC-2 has an intron and encodes a cellulase (CelF) containing a carbohydrate-binding module; Chen H et al.; A cDNA, designated celF, encoding a cellulase (CelF) was isolated from the anaerobic fungus Orpinomyces PC-2 . The open reading frame contains regions coding for a signal peptide, a carbohydrate-binding module (CBM), a linker, and a catalytic domain . The catalytic domain was homologous to those of CelA and CelC of the same fungus and to that of the Neocallimastix patriciarum CELA, but CelF lacks a docking domain, characteristic for enzymes of cellulosomes . It was also homologous to the cellobiohydrolase IIs and endoglucanases of aerobic organisms . The gene has a 111-bp intron, located within the CBM-coding region . Some biochemical properties of the purified recombinant enzyme are described.

Biol Trace Elem Res, 2003 Apr, 92(1), 61 - 70
Study of the thermokinetic properties of copper(II) on Escherichia coli growth; Jun Y et al.; By using an LKB2277 Bioactivity Monitor, stop-flow mode, the power-time curves of Escherichia coli at 37 degrees C affected by Cu(II) were determined . Some parameters, such as growth rate constants k, inhibitory ratio I, the heat output Qlog in the log phase, and the generation times G were obtained . According to these parameters, we found that a low concentration of Cu(II) (0-20 microg/mL) had an promoting action on the growth of E . coli, but a high concentration of Cu(II) (40-100 microg/mL) had an inhibitory action . The toxicity of Cu(II) can also be expressed as the half-inhibitory concentration IC50; the value is 69.7 microg/mL . The assay is quantitative, inexpensive, and versatile.

Plant Cell Physiol, 2003 Apr, 44(4), 395 - 403
Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential expression in tissues and by stress compounds; Yamazaki Y et al.; Camptothecin derivatives are clinically used anti-tumor compounds that biogenetically belong to a group of monoterpenoid indole alkaloids (TIA) . We have already established a hairy root culture of Ophiorrhiza pumila (Rubiaceae) that produces camptothecin . The present study describes the cloning and characterization of cDNAs encoding strictosidine synthase (OpSTR; EC 4.3.3.2) and tryptophan decarboxylase (OpTDC; EC 4.1.1.28), two key enzymes in the biosynthesis of TIA from hairy roots of O . pumila . We also isolated the cDNA coding for NADPH:cytochrome P450 reductase (OpCPR; EC 1.6.2.4) that is presumed to be indirectly involved in camptothecin synthesis . The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli . The tissue-specific and stress-inducible expression patterns of OpSTR and OpTDC were quite similar, unlike those of OpCPR . The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis . Plant stress compounds like salicylic acid repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.

Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5670 - 5 Epub 2003 Apr 29.
Catalytic and structural role of the metal ion in dUTP pyrophosphatase; Mustafi D et al.; The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods . Values of k(cat) and k(cat)K(m) were 4.5 +/- 0.1 s(-1) and 0.49 +/- 0.1 x 10(6) M(-1).s(-1) in the absence of divalent metal ions, 14.7 +/- 2.2 s(-1) and 25.1 +/- 7.4 x 10(6) M(-1).s(-1) in the presence of Mg(2+) or Mn(2+), and 24.2 +/- 3.6 s(-1) and 2.4 +/- 0.7 x 10(6) M(-1).s(-1) when supported by VO(2+) or bis(acetylacetonato)oxovanadium(IV) . Binding of VO(2+) to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg(2+) . Electron paramagnetic resonance spectra of the ternary enzyme-VO(2+)-chelate-dUDP complex revealed a pattern of (31)P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO(2+) ion . Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced . By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO(2+) . We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO(2+) and the VO(2+)-chelate in dUTP hydrolysis . In this location the metal ion could promote an ordered conformation of the C-terminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.

J Pharmacol Exp Ther, 2003 Aug, 306(2), 744 - 51 Epub 2003 Apr 29.
Threonine-205 in the F helix of p450 2B1 contributes to androgen 16 beta-hydroxylation activity and mechanism-based inactivation; Lin HL et al.; Four mutants of Thr-205 in cytochrome p450 2B1 were constructed and expressed in Escherichia coli . The Ser-, Ala-, and Val-mutants displayed stable reduced CO difference spectra and were able to metabolize 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, androstenedione, and benzphetamine . The Arg-mutant displayed an unstable reduced CO difference spectrum at 450 nm, was concomitantly converted to a denatured form with a peak at 422 nm, and showed no catalytic activity with any of the four substrates tested . The Ser-mutant displayed activity and metabolite profiles for testosterone and androstenedione similar to those of the wild-type p450 2B1 (WT) . Substitution of Thr-205 with Ala or Val markedly suppressed the 16 beta-hydroxylation activity but exhibited little effect on the 16 alpha-hydroxylation activity for testosterone and androstenedione . Because 16 beta-hydroxylation activity of androgens is a specific p450 2B subfamily marker and residue 205 is located in the F helix, which forms the ceiling of the active site, we postulate that the gamma-hydroxyl side chain of Thr may play an important role in directing the 16 beta-face of testosterone and androstenedione toward the active site . Surprisingly, the Val-mutant retained full activity for benzphetamine demethylation . When mechanism-based inactivators for p450 2B1 were used to evaluate the susceptibility to inactivation, the Val-mutant was resistant to inactivation by 17 alpha-ethynylestradiol and less sensitive to inactivation by 2-ethynylnaphthalene compared with the WT enzyme . Our results demonstrate the importance of Thr-205 in determining substrate specificity and product formation as well as in influencing the susceptibility of p450 2B1 to mechanism-based inactivators.

Biochem J, 2003 Jul 15, 373(Pt 2), 559 - 69
Biochemical and genetic characterization of a murine class Kappa glutathione S-transferase; Jowsey IR et al.; The class Kappa family of glutathione S-transferases (GSTs) currently comprises a single rat subunit (rGSTK1), originally isolated from the matrix of liver mitochondria {Harris, Meyer, Coles and Ketterer (1991) Biochem . J . 278, 137-141; Pemble, Wardle and Taylor (1996) Biochem . J . 319, 749-754} . In the present study, an expressed sequence tag (EST) clone has been identified which encodes a mouse class Kappa GST (designated mGSTK1) . The EST clone contains an open reading frame of 678 bp, encoding a protein composed of 226 amino acid residues with 86% sequence identity with the rGSTK1 polypeptide . The mGSTK1 and rGSTK1 proteins have been heterologously expressed in Escherichia coli and purified by affinity chromatography . Both mouse and rat transferases were found to exhibit GSH-conjugating and GSH-peroxidase activities towards model substrates . Analysis of expression levels in a range of mouse and rat tissues revealed that the mRNA encoding these enzymes is expressed predominantly in heart, kidney, liver and skeletal muscle . Although other soluble GST isoenzymes are believed to reside primarily within the cytosol, subcellular fractionation of mouse liver demonstrates that this novel murine class Kappa GST is associated with mitochondrial fractions . Through the use of bioinformatics, the genes encoding the mouse and rat class Kappa GSTs have been identified . Both genes comprise eight exons, the protein coding region of which spans approx . 4.3 kb and 4.1 kb of DNA for mGSTK1 and rGSTK1 respectively . This conservation in primary structure, catalytic properties, tissue-specific expression, subcellular localization and gene structure between mouse and rat class Kappa GSTs indicates that they perform similar physiological functions . Furthermore, the association of these enzymes with mitochondrial fractions is consistent with them performing a specific conserved biological role within this organelle.

J Am Chem Soc, 2003 May 7, 125(18), 5298 - 307
An unnatural hydrophobic base pair with shape complementarity between pyrrole-2-carbaldehyde and 9-methylimidazo{(4,5)-b}pyridine; Mitsui T et al.; An unnatural hydrophobic base, pyrrole-2-carbaldehyde (denoted as Pa), was developed as a specific pairing partner of 9-methylimidazo{(4,5)-b}pyridine (Q) . The Q base is known to pair with 2,4-difluorotoluene (F) as an isostere of the A-T pair, and F also pairs with A efficiently in replication . In contrast, the Q-Pa pair showed specific selectivity in replication, and the five-membered-ring base Pa paired efficiently with Q but paired poorly with A . In addition, the interaction of Pa with DNA polymerases was superior, in comparison to that of F . The aldehyde group of Pa was recognized well by the Klenow fragment of Escherichia coli DNA polymerase I and the reverse transcriptase of Avian myeloblastosis virus . The structural features of the Q-Pa pair in a DNA duplex were analyzed by NMR, showing the shape complementarity of the Pa fitting with Q . The structurally unique base Pa provides valuable information for the development of unnatural base pairs toward the expansion of the genetic alphabet.

J Agric Food Chem, 2003 May 7, 51(10), 3049 - 55
Phytoene desaturase inhibition by O-(2-phenoxy)ethyl-N-aralkylcarbamates; Ohki S et al.; O-{1-Ethyl-2-(3-trifluoromethylphenoxy)}ethyl-N-benzylcarbamate exhibits a marked inhibition of carotenoid biosynthesis . Forty-one analogues were synthesized and assayed for plant-type phytoene desaturase (PDS) and zeta-carotene desaturase (ZDS) inhibition in a cell-free system using recombinant enzymes obtained from Escherichia coli transformants . The target enzyme of all carbamates synthesized in this study is PDS and not ZDS; no inhibition of ZDS was observed using a 10(-4) M inhibitor concentration . Four compounds, O-{1-ethyl-2-(3-trifluoromethylphenoxy)}ethyl-N-(2-phenylethyl)carbamate (23), O-{1-ethyl-2-(3-trifluoromethylphenoxy)}ethyl-N-(2-chlorobenzyl)carbamate (25), O-{1-ethyl-2-(3-trifluoromethylphenoxy)}ethyl-N-(2-chlorobenzyl)carbamate (26), and O-{1-methyl-2-(3-trifluoromethylphenoxy)}ethyl-N-benzylcarbamate (30), were the most potent PDS inhibitors . Their pI(50) values, the negative logarithms of the molar concentration that produces a 50% inhibition, were 7.5, representing the same inhibitory activity as norflurazon . With respect to a structure-activity relationship the oxygen atom of the phenoxy group and a carbamate structure in O-(1-ethyl-2-phenoxy)ethyl-N-aralkylcarbamates studied were found to be essential for strong PDS inhibitors . Also, introduction of an ethyl group at the alpha-position of the ethylene bridge between the phenoxy group and the carbamate was important for a strong PDS inhibitor . Substituents at the 2- and/or 3-position of the phenoxybenzene ring were found to be favorable to a strong PDS inhibition of the analogues.

Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 186 - 90 Epub 2003 Apr 26.
Cloning and characterization of the gene coding for the aerobic azoreductase from Pigmentiphaga kullae K24; Blumel S et al.; The gene coding for an aerobic azoreductase was cloned from Pigmentiphaga kullae K24, which is able to grow with the carboxylated azo compound 1-(4'-carboxyphenylazo)-4-naphthol (carboxy-Orange I) as sole source of carbon and energy . The gene encoded a protein with a molecular weight of 20,557 Da, with a conserved putative NAD(P)H-binding site in the amino-terminal region . The deduced amino acid sequence showed no further significant sequence homologies to previously studied aerobic azoreductases . The azoreductase was heterologously expressed in Escherichia coli and shown to convert the sulfonated azo dye Orange I and furthermore Magneson II {4-(4-nitrophenylazo)-1-naphthol}.

Cancer Gene Ther, 2003 May, 10(5), 396 - 402
Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy; Barresi V et al.; Since neural progenitor cells can engraft stably into brain tumors and differentiate along the neuronal and glial line, we tested the hypothesis that transplanted cytosine deaminase (CD)-expressing ST14A cells (an immortalized neural progenitor cell line) can convert locally 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) and produce a regression of glioma tumors . ST14A, retrovirally transduced with the E . coli CD gene, showed a strong bystander effect on glioma cells as assessed by in vitro assay . Intracerebral injection of C6 glioma cells generated a rapidly growing tumoral mass . DiI prelabeled ST14A, coinjected into the rat brain with C6 glioma cells, survived in the tumoral mass up to 10 days and their number was not affected by in vivo 5-FC treatment . In contrast, a significant decrease of the glioma tumoral mass (-50%) was observed in 5-FC-treated rats . 5-FC had no effect on the tumor in the absence of CD-expressing ST14A cells . Our results support the feasibility of systems based on intratumoral transplantation of prodrug-converting cells for brain tumor therapy.

J Biol Chem, 2003 Jul 11, 278(28), 25302 - 7 Epub 2003 Apr 28.
Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA; Guse A et al.; Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes . MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein . Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product . To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family . Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA . They were also characterized by x-ray structural analysis . Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme . Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.

Biophys J, 2003 May, 84(5), 2990 - 8
On translocation through a membrane channel via an internal binding site: kinetics and voltage dependence; Schwarz G et al.; Here we present a model for maltodextrin translocation through maltoporin channels . In a first step, our theoretical analysis does consider the case of a single binding site for a given substrate in a structurally unaffected channel with a possibly different entrance barrier on either side . It is shown how by means of conventional electrical conductance measurements (including current noise analysis) the basic equilibrium and rate constants can be determined as functions of the applied voltage . Then also the net translocation rate of the substrate becomes accessible quantitatively . This most simple model mechanism has been extended to include a voltage-dependent fast conformational change of the channel that prevents the binding process . The so developed approach has been tested with experimental data for a single maltoporin trimer being reconstituted in black lipid membranes when studied in the presence of maltohexaose as the substrate . The experimental results turned out to be clearly incompatible with binding alone . They are, however, very satisfactorily fitted by pertinent theoretical curves if also inhibition of binding by a conformational transition is taken into account . Accordingly, quantitative evaluations of the underlying parameters and eventually of the translocation rate have been carried out successfully . Our analysis reveals a set of parameters necessary for an optimal translocation that nicely corresponds to natural conditions.

Biophys J, 2003 May, 84(5), 2841 - 51
Feedback regulation in the lactose operon: a mathematical modeling study and comparison with experimental data; Yildirim N et al.; A mathematical model for the regulation of induction in the lac operon in Escherichia coli is presented . This model takes into account the dynamics of the permease facilitating the internalization of external lactose; internal lactose; beta-galactosidase, which is involved in the conversion of lactose to allolactose, glucose and galactose; the allolactose interactions with the lac repressor; and mRNA . The final model consists of five nonlinear differential delay equations with delays due to the transcription and translation process . We have paid particular attention to the estimation of the parameters in the model . We have tested our model against two sets of beta-galactosidase activity versus time data, as well as a set of data on beta-galactosidase activity during periodic phosphate feeding . In all three cases we find excellent agreement between the data and the model predictions . Analytical and numerical studies also indicate that for physiologically realistic values of the external lactose and the bacterial growth rate, a regime exists where there may be bistable steady-state behavior, and that this corresponds to a cusp bifurcation in the model dynamics.

J Struct Biol, 2003 Apr, 142(1), 133 - 43
Protein production in Escherichia coli for structural studies by X-ray crystallography; Goulding CW et al.; The arrival of genomic sequences to the database has provided a seemingly unlimited supply of targets for protein structure determination and the possibility of solving the structure of an entire proteome . Based on our experience with the proteomes of Pyrobaculum aerophilum and Mycobacterium tuberculosis, we have developed a simple strategy for the production of proteins for structural studies by X-ray crystallography . Our scheme demonstrates a strong protein target commitment and includes the expression of genes from these organisms in Escherichia coli . These proteins are expressed with affinity tags and purified for characterization and crystallization . We have identified protein solubility and crystallization as the two major bottlenecks in the process toward the determination of protein structures by X-ray diffraction . Strategies to overcome these bottlenecks are discussed.

Mol Cell, 2003 Apr, 11(4), 1067 - 78
Crystal structure of Escherichia coli sigmaE with the cytoplasmic domain of its anti-sigma RseA; Campbell EA et al.; The sigma factors are the key regulators of bacterial transcription . ECF (extracytoplasmic function) sigma's are the largest and most divergent group of sigma(70) family members . ECF sigma's are normally sequestered in an inactive complex by their specific anti-sigma factor, which often spans the inner membrane . Here, we determined the 2 A resolution crystal structure of the Escherichia coli ECF sigma factor sigma(E) in an inhibitory complex with the cytoplasmic domain of its anti-sigma, RseA . Despite extensive sequence variability, the two major domains of sigma(E) are virtually identical in structure to the corresponding domains of other sigma(70) family members . In combination with a model of the sigma(E) holoenzyme and biochemical data, the structure reveals that RseA functions by sterically occluding the two primary binding determinants on sigma(E) for core RNA polymerase.

Mol Cell, 2003 Apr, 11(4), 875 - 84
Crystal structure of the MazE/MazF complex: molecular bases of antidote-toxin recognition; Kamada K et al.; A structure of the Escherichia coli chromosomal MazE/MazF addiction module has been determined at 1.7 A resolution . Addiction modules consist of stable toxin and unstable antidote proteins that govern bacterial cell death . MazE (antidote) and MazF (toxin) form a linear heterohexamer composed of alternating toxin and antidote homodimers (MazF(2)-MazE(2)-MazF(2)) . The MazE homodimer contains a beta barrel from which two extended C termini project, making interactions with flanking MazF homodimers that resemble the plasmid-encoded toxins CcdB and Kid . The MazE/MazF heterohexamer structure documents that the mechanism of antidote-toxin recognition is common to both chromosomal and plasmid-borne addiction modules, and provides general molecular insights into toxin function, antidote degradation in the absence of toxin, and promoter DNA binding by antidote/toxin complexes.

BMC Mol Biol . 2003 Apr 28;4(1):5.
Accumulation of large non-circular forms of the chromosome in recombination-defective mutants of Escherichia coli; Handa N et al.; BACKGROUND: Double-strand breakage of chromosomal DNA is obviously a serious threat to cells because various activities of the chromosome depend on its integrity . However, recent experiments suggest that such breakage may occur frequently during "normal" growth in various organisms - from bacteria through vertebrates, possibly through arrest of a replication fork at some endogenous DNA damage . RESULTS: In order to learn how the recombination processes contribute to generation and processing of the breakage, large (> 2000 kb) linear forms of Escherichia coli chromosome were detected by pulsed-field gel electrophoresis in various recombination-defective mutants . The mutants were analyzed in a rich medium, in which the wild-type strain showed fewer of these huge broken chromosomes than in a synthetic medium, and the following results were obtained: (i) Several recB and recC null mutants (in an otherwise rec+ background) accumulated these huge linear forms, but several non-null recBCD mutants (recD, recC1001, recC1002, recC1003, recC1004, recC2145, recB2154, and recB2155) did not . (ii) In a recBC sbcA background, in which RecE-mediated recombination is active, recA, recJ, recQ, recE, recT, recF, recO, and recR mutations led to their accumulation . The recJ mutant accumulated many linear forms, but this effect was suppressed by a recQ mutation . (iii) The recA, recJ, recQ, recF and recR mutations led to their accumulation in a recBC sbcBC background . The recJ mutation showed the largest amount of these forms . (iv) No accumulation was detected in mutants affecting resolution of Holliday intermediates, recG, ruvAB and ruvC, in any of these backgrounds . CONCLUSION: These results are discussed in terms of stepwise processing of chromosomal double-strand breaks.

Biochem J, 2003 Aug 1, 373(Pt 3), 957 - 63
Heterologous expression of four glutathione transferase genes genetically linked to a major insecticide-resistance locus from the malaria vector Anopheles gambiae; Ortelli F et al.; A cluster of eight genes encoding glutathione transferases (GSTs) are located on division 33B of polytene chromosome arm 3R of the African malaria mosquito, Anopheles gambiae . This region of the genome contains a major 1,1,1-trichloro-2,2-bis-( p -chlorophenyl)ethane (DDT)-resistance locus, rtd1 . These GSTs belong to the insect-specific Epsilon class and share between 22.6 and 65.2% identity at the amino acid level . Two distinct allelic variants of the Epsilon GST, GSTe1, differing at 12 out of 224 amino acid residues, are present in laboratory and field populations of A . gambiae . To investigate the possible role of these GSTs in conferring resistance to the insecticide DDT, both GSTe1 alleles, plus three additional members of this gene cluster, were expressed in Escherichia coli and the recombinant proteins biochemically characterized . The five putative glutathione transferases encoded catalytically active subunits with variable biochemical properties . For example, the two allelic variants of GSTE1-1 encoded proteins with over 100-fold variation in peroxidase activity, while the three remaining GSTs had no detectable peroxidase activity . Only GSTE2-2 was able to metabolize DDT . Western blots using antibodies raised against these GSTs indicated that the expression of GSTE2-2 is elevated in a DDT-resistant strain of A . gambiae.

Biochemistry, 2003 May 6, 42(17), 5159 - 67
Peptidyl aldehydes as reversible covalent inhibitors of SRC homology 2 domains; Park J et al.; Src homology 2 (SH2) domains are phosphotyrosine- (pY-) binding modules found in a variety of signal-transducing proteins and constitute an important class of drug targets for the treatment of signaling related diseases/conditions . To date, a large number of peptidic as well as nonpeptidic SH2 domain inhibitors have been reported . However, all of these inhibitors contain a negatively charged pY mimetic as the core structure and generally have poor membrane permeability . We report here that peptidyl cinnamaldehydes function as reversible, slow-binding inhibitors toward the SH2 domains of protein tyrosine phosphatase SHP-1 . Specific interactions between the SH2 domains and the aldehydes were assessed by their ability to relieve the autoinhibitory effect of the N-terminal SH2 domain on SHP-1 catalytic activity and the surface plasmon resonance technique . The most potent inhibitor (Cinn-GEE) displayed a K(D) value of 1.3 microM against the N-terminal SH2 domain of SHP-1 . The mechanism of inhibition was investigated by site-directed mutagenesis and by using Cinn-GEE specifically labeled with (13)C at the aldehyde carbon and (1)H-(13)C heteronuclear single-quantum coherence spectroscopy . The proposed mechanism involves the formation of an initial noncovalent E.I complex, which is slowly converted into a covalent imine/enamine adduct (E.I) between the aldehyde group of the inhibitor and the guanidine group of Arg betaB5 in the pY-binding pocket of the SH2 domains . These aldehydes should provide a general, neutral pharmacophore for the further development of potent, specific, and membrane-permeable SH2 domain inhibitors.

Biochemistry, 2003 May 6, 42(17), 5025 - 34
Mutational analysis of an RNA internal loop as a reactivity epitope for Escherichia coli ribonuclease III substrates; Calin-Jageman I et al.; The enzymatic cleavage of double-stranded (ds) RNA is an obligatory step in the maturation and decay of many cellular and viral RNAs . The primary agents of dsRNA processing are members of the ribonuclease III (RNase III) superfamily, which are highly conserved in eukaryotic and bacterial cells . Escherichia coli RNase III participates in the maturation of the ribosomal RNAs and in the maturation and decay of cellular and phage mRNAs . E . coli RNase III-dependent cleavage events can regulate gene expression by controlling mRNA stability and translational activity . RNase III recognizes its substrates and selects the scissile phosphodiester(s) by recognizing specific RNA sequence and structural elements, termed reactivity epitopes . Some E . coli RNase III substrates contain an internal loop, in which is located the single scissile phosphodiester . The specific features of the internal loop that establish the pattern of single-strand cleavage are not known . A mutational analysis of the asymmetric {4 nt/5 nt} internal loop of the phage T7 R1.1 substrate reveals that cleavage reactivity is largely independent of internal loop sequence . Instead, the {4/5} asymmetry per se is the primary determinant of cleavage of a single bond within the 5 nt strand of the internal loop . The T7 R1.1 internal loop lacks elements of local tertiary structure, as revealed by sensitivity to cleavage by terbium ion and by the ability of the internal loop to destabilize a small model duplex . The internal loop functions as a discrete structural element in that the pattern of cleavage can be controlled by the specific type of asymmetry . The implications of these findings are discussed in light of RNase III substrate function as a gene regulatory element.

Biochemistry, 2003 May 6, 42(17), 4954 - 61
Thermodynamic and kinetic analysis of a peptide-class I MHC interaction highlights the noncovalent nature and conformational dynamics of the class I heterotrimer; Binz AK et al.; The class I major histocompatibility (MHC) molecule is a heterotrimer composed of a heavy chain, the small subunit beta(2)-microglobulin (beta(2)m), and a peptide . Fluorescence anisotropy has been used to assay the interaction of a labeled peptide with a recombinant, soluble form of the class I MHC HLA-A2 . Consistent with earlier work, peptide binding is shown to be a two-step process limited by a conformational rearrangement in the heavy chain/beta(2)m heterodimer . However, we identify two pathways for peptide dissociation from the heterotrimer: (1) initial peptide dissociation leaving a heavy chain/beta(2)m heterodimer and (2) initial dissociation of beta(2)m, followed by peptide dissociation from the heavy chain . Eyring analyses of rate constants measured as a function of temperature permit for the first time a complete thermodynamic characterization of peptide binding . We find that in this case peptide binding is mostly entropically driven, likely reflecting the hydrophobic character of the peptide binding groove and the peptide anchor residues . Thermodynamic and kinetic analyses of peptide-MHC interactions as performed here may be of practical use in the engineering of peptides with desired binding properties and will aid in the interpretation of the effects of MHC and peptide substitutions on peptide binding and T cell reactivity . Finally, our data suggest a role for beta(2)m in dampening conformational dynamics in the heavy chain . Remaining conformational variability in the heavy chain once beta(2)m has bound may be a mechanism to promote promiscuity in peptide binding.

Biochemistry, 2003 May 6, 42(17), 4937 - 44
The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70; Wittung-Stafshede P et al.; The Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK . ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis . However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis . The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide . In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide . However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides . Binding of a fusion protein that contains a defective J-domain is insensitive to ATP . According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C) . The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range . In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture . The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity . The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.

Biochemistry, 2003 May 6, 42(17), 4926 - 36
Structure and energetics of an allele-specific genetic interaction between dnaJ and dnaK: correlation of nuclear magnetic resonance chemical shift perturbations in the J-domain of Hsp40/DnaJ with binding affinity for the ATPase domain of Hsp70/DnaK; Landry SJ; The molecular chaperone machine composed of Escherichia coli Hsp70/DnaK and Hsp40/DnaJ binds and releases client proteins in cycles of ATP-dependent protein folding, membrane translocation, disassembly, and degradation . The J-domain of DnaJ simultaneously stimulates ATP hydrolysis in the ATPase domain and capture of the client protein in the peptide-binding domain of DnaK . ATP-dependent binding of DnaJ to DnaK mimics DnaJ-dependent capture of a client protein . The dnaJ mutation that replaces aspartate-35 with asparagine (D35N) in the J-domain causes a defect in binding of DnaJ to DnaK . The dnaK mutation that replaces arginine-167 with alanine (R167A) in the ATPase domain of DnaK(R167A) restores binding of DnaJ(D35N) . This genetic interaction was said to be allele-specific because wild-type DnaJ does not bind to DnaK(R167A) . The J-domain of DnaJ binds to the ATPase domain of DnaK in its capacity as modulator of DnaK ATPase activity and conformational behavior . Surprisingly, the mutations affect the domainwise interaction in an almost opposite manner . D35N increases the affinity of the J-domain for the ATPase domain . R167A has no affect on the affinity of the ATPase domain for the D35N mutant J-domain, but it reduces the affinity for the wild-type J-domain . Previous amide ((1)H, (15)N) NMR chemical shift perturbation mapping in the J-domain suggested that the ATPase domain binds to J-domain helix II and the flanking loops . In the D35N mutant J-domain, chemical shift perturbations include additional effects at amides in the flexible loop II-III and helix III, which have been proposed to undergo an induced fit conformational change upon binding to DnaK . The integrated magnitudes of chemical shift perturbations for the various J-domain and ATPase domain pairs correlate with the free energies of binding . Thus, the J-domain structure can be described as a dynamic ensemble of conformations that is constrained by binding to the ATPase domain . J-domain helix II bends upon binding to the ATPase domain . D35N increases helix II bending, but less so in combination with R167A in the ATPase domain . Taken together, the results suggest that D35N overstabilizes an induced fit conformational change in loop II-III and helix III that is necessary for the J-domain to couple ATP hydrolysis with a conformational change in DnaK, and R167A destabilizes the induced conformation . Conclusions from this work have implications for understanding mechanisms of protein-protein interaction that are involved in allosteric regulation and genetic suppression.

Biochemistry, 2003 May 6, 42(17), 4918 - 25
RAR antagonists diminish the level of DNA binding by the RAR/RXR heterodimer; Poujol N et al.; A purified RAR/RXR-DeltaAB heterodimer was obtained by production of His-tagged RAR and untagged RXR in Escherichia coli, followed by combined purification on a Ni(2+) affinity column using excess RXR extract, and finally a gel filtration chromatography step to isolate a pure heterodimer . The purified heterodimer preparation bound 9-cisRA at a level of 0.85-0.95 mol of binding sites per mole of protein monomer . Titration of a 26 kDa fluorescent labeled fragment of the SRC-1 coactivator protein with the purified heterodimer in the presence of the agonist 9-cisRA yielded a binding affinity near 300 nM, whereas no binding was observed in the absence of agonist . Binding of the purified heterodimer to a DR5 target was identical in the absence of ligand and in the presence of 9-cisRA . Competition by unlabeled specific and nonspecific DNA allowed us to demonstrate that the binding curve was bimodal . The first phase of binding was highly specific and of high affinity . This phase also exhibited a high degree of cooperativity in the binding profile . Nonspecific DNA efficiently competes for the second phase . Thus, the first phase of binding likely corresponds to the formation of the specific heterodimer complex in which heterodimerization is energetically coupled to DNA binding . While agonist binding had no effect on the apparent affinity of the heterodimer for DR5, a series of antagonists significantly destabilized the heterodimer-DR5 complex, either through a direct decrease in the affinity of the protein for the DNA or through destabilization of the heterodimer itself . Impeding the interaction between the heterodimer and DNA appears as an additional mechanism of antagonist action of varying efficiency, depending upon the chemical structure of the antagonist.

Biochemistry, 2003 May 6, 42(17), 4904 - 8
Site-directed sulfhydryl labeling of helix IX in the lactose permease of Escherichia coli; Zhang W et al.; Site-directed sulfhydryl modification of transmembrane helix IX in the lactose permease of Escherichia coli was studied in right-side-out membrane vesicles with the thiol-specific reagents N-{(14)C}ethylmaleimide (NEM) and methanethiosulfonate ethylsulfonate (MTSES) which are permeant and impermeant, respectively . Out of approximately 20 mutants with a single Cys residue at each position in the helix, only five mutants label with NEM . (i) Cys residues at positions 291, 308, and 310 label at 25 degrees C, and binding of substrate has no effect . (ii) Cys residues at positions 295 and 298 label only in the presence of substrate . NEM labeling at 0 degrees C indicates that alkylation of Cys residues at positions 295 and 308 is dependent on the thermal motion of the protein . In contrast, temperature has little effect on labeling of Cys residues at positions 291, 298, and 310 . Interestingly, pretreatment with MTSES blocks NEM labeling of all the mutants . The findings demonstrate that the face of helix IX on which Arg302 is located is involved in ligand-induced conformational changes and accessible to water from the periplasmic surface of the membrane . Since Arg302 facilitates deprotonation of Glu325 (helix X) during turnover {Sahin-Toth, M., and Kaback, H . R . (2001) Proc . Natl . Acad . Sci . U.S.A . 98, 6068-6073}, the findings are consistent with the idea that this face of helix IX may comprise part of the H(+) translocation pathway.

Biochemistry, 2003 May 6, 42(17), 4874 - 82
High-resolution X-ray structure of isoaspartyl dipeptidase from Escherichia coli; Thoden JB et al.; Isoaspartyl dipeptidase from Escherichia coli functions in protein degradation by catalyzing the hydrolysis of beta-L-isoaspartyl linkages in dipeptides . The best substrate for the enzyme reported thus far is iso-Asp-Leu . Here we report the X-ray analysis of the enzyme in its resting state and complexed with aspartate to 1.65 and 2.1 A resolution, respectively . The quaternary structure of the enzyme is octameric and can be aptly described as a tetramer of dimers . Each subunit folds into two distinct domains: the N-terminal region containing eight strands of mixed beta-sheet and the C-terminal motif that is dominated by a (beta,alpha)(8)-barrel . A binuclear zinc center is located in each subunit at the C-terminal end of the (beta,alpha)(8)-barrel . Ligands to the binuclear metal center include His 68, His 70, His 201, His 230, and Asp 285 . The two zincs are bridged by a carboxylated lysine residue (Lys 162) and a solvent molecule, most likely a hydroxide ion . The product of the reaction, aspartate, binds to the enzyme by displacing the bridging solvent with its side chain functional group . From this investigation it is proposed that the reaction mechanism of the enzyme proceeds through a tetrahedral intermediate and that the bridging solvent attacks the re face of the carbonyl carbon of the scissile peptide bond . This structural analysis confirms the placement of isoaspartyl dipeptidase into the urease-related amidohydrolase superfamily.

Biochemistry, 2003 May 6, 42(17), 4864 - 73
Temperature range of thermodynamic stability for the native state of reversible two-state proteins; Kumar S et al.; The difference between the heat (T(G)) and the cold (T(G)') denaturation temperatures defines the temperature range (T(Range)) over which the native state of a reversible two-state protein is thermodynamically stable . We have performed a correlation analysis for thermodynamic parameters in a selected data set of structurally nonhomologous single-domain reversible two-state proteins . We find that the temperature range is negatively correlated with the protein size and with the heat capacity change (DeltaC(p)) but is positively correlated with the maximal protein stability {DeltaG(T(S))} . The correlation between the temperature range and maximal protein stability becomes highly significant upon normalization of the maximal protein stability with protein size . The melting temperature (T(G)) also shows a negative correlation with protein size . Consistently, T(G) and T(G)' show opposite correlations with DeltaC(p), indicating a dependence of the T(Range) on the curvature of the protein stability curve . Substitution of proteins in our data set with their homologues and arbitrary addition or removal of a protein in the data set do not affect the outcome of our analysis . Simulations of the thermodynamic data further indicate that T(Range) is more sensitive to variations in curvature than to the slope of the protein stability curve . The hydrophobic effect in single domains is the principal reason for these observations . Our results imply that larger proteins may be stable over narrower temperature ranges and that smaller proteins may have higher melting temperatures, suggesting why protein structures often differentiate into multiple substructures with different hydrophobic cores . Our results have interesting implications for protein thermostability.

Biochemistry, 2003 May 6, 42(17), 4843 - 54
Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues; Howe DL et al.; Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P) . However, E . coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate . The mechanism of this abortive utilization of PEP was investigated using {2,3-(13)C(2)}-PEP and {3-F}-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA) . The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule . In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and {2,3-(13)C(2)}-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E . coli KDO8-P synthase.

Biochemistry, 2003 May 6, 42(17), 4809 - 18
Determinants of DNA bending in the DNA-cyclic AMP receptor protein complexes in Escherichia coli; Lin SH et al.; The activated Escherichia coli cAMP receptor protein, CRP, is capable of regulating the expression of more than 20 genes by binding to specific DNA sites . DNA bending is an important structural feature that has been observed in the regulatory mechanism of gene expression by CRP . On the basis of the results of the fluorescence energy transfer study of the gal P1 promoter, gal bends asymmetrically upon binding to CRP, although DNA bends symmetrically in the CRP-lac complex . The flanking sequence proximal to the TGTGA motif is involved in a sharper bend than the other side with an overall bending angle of approximately 90-125 degrees, without wrapping around the CRP molecule . To understand the factors that control the symmetry in DNA bending, a series of DNA sequences was tested to dissect the contribution of half-sites and flanking sequences, using the natural gal P1 and lac P1 sequences as initial targets . The extent of DNA bending induced by CRP was monitored by the difference in fluorescence anisotropy between free DNA and the DNA-CRP complex . The extent of bending was sequence-dependent, and most importantly, the symmetry of bending was a function of the symmetry of the DNA sequence . For example, in the lac promoter the two binding half-sites (TGTGA and TCACT) were almost symmetric as an inverted repeat . The recognition F-helices of the two CRP subunits would bind to these half-sites with a 2-fold symmetry . The flanking sequences (ATAAA and CATTA) were almost identical mirror images . Thus, they are expected to bend in a similar manner . Finally, the sequence symmetry properties of a series of natural CRP promoters were analyzed . A strong tendency for symmetry sequence was encoded in class I promoter sites but not in class II promoter sites . Results from this analysis support the conclusion that the geometry of the CRP-DNA complex plays a major role in determining the molecular mechanism in gene transcription.

Biochemistry, 2003 May 6, 42(17), 4800 - 8
Mutagenesis of subunit N of the Escherichia coli complex I . Identification of the initiation codon and the sensitivity of mutants to decylubiquinone; Amarneh B et al.; The last gene in the nuo operon of Escherichia coli, nuoN, encodes a membrane-bound subunit of Complex I (NADH:ubiquinone oxidoreductase) . In this report, the gene for subunit N was disrupted by a 163 bp deletion in the chromosome, resulting in the loss of Complex I function, as measured by deamino-NADH oxidase activity . This activity could be recovered after transformation of the mutant strain by a plasmid that contains the previously identified nuoN gene and the upstream intergenic region between nuoM and nuoN . Mutagenesis of the first ATG downstream of nuoM led to a loss of function, indicating that this is the likely initiation codon for nuoN, and predicting a protein of 485 amino acids and 52 044 Da . Thirty site-specific mutations in nuoN at 19 different positions were constructed in a vector that expresses the full-length subunit N with both an octahistidine tag and an HA epitope tag at the carboxyl terminus . Highly conserved charged and aromatic residues were selected for mutagenesis, as well as a substitution that occurs as a secondary mutation in Leber's hereditary optic neuropathy (LHON) . Membranes from the mutant strains were tested for production of subunit N by immunoblots and for NADH-linked activities . Mutants with substitutions at six different positions (K158, K217, H224, K247, Y300, and K395) had rates of deamino-NADH oxidase activity that were no more than 50% of that of the wild type and had reduced rates of proton translocation . These mutants also showed enhanced inhibition by decylubiquinone, indicating that subunit N interacts with quinones . The mutation associated with LHON, G391S, had little effect on these functions.

Biochemistry, 2003 May 6, 42(17), 4780 - 6
Binding of NAP-22, a calmodulin-binding neuronal protein, to raft-like domains in model membranes; Khan TK et al.; The cholesterol-binding protein NAP-22 is a major component of the detergent-insoluble low-density fraction of rat brain . In this study, we found, using fluorescence microscopy, that native NAP-22, but not a demyristoylated form, binds to cholesterol-rich raft-like domains in planar-supported monolayers and remains bound after nonionic detergent extraction . NAP-22 also protects the cholesterol-rich domains during extraction by methyl-beta-cyclodextrin . The lateral mobility of this protein is much lower than that of other raft components in model membranes, suggesting that both cholesterol binding and inter-NAP-22 interactions markedly reduce its lateral diffusion . This study suggests that NAP-22 binding may be employed to image cholesterol-rich regions, such as caveolae/rafts, on the plasma membrane of cells, and preliminary efforts in that direction are presented.

Can J Microbiol, 2003 Feb, 49(2), 110 - 9
Melanin biosynthesis in the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus); Lanisnik Rizner T et al.; Curvularia lunata (teleomorph: Cochliobolus lunatus) is a known plant and human pathogen . Tricyclazole, a specific inhibitor of pentaketide melanin biosynthesis, blocked the biosynthesis of melanin in Curvularia lunata and caused the accumulation of the melanin metabolites flaviolin and 2-hydroxyjuglone . This showed that melanin in Curvularia lunata is produced by a pentaketide pathway from 1,8-dihydroxynaphthalene . The 1,3,8-trihydroxynaphthalene reductase (3HNR) gene, associated with the melanin pathway of Curvularia lunata, was identified and characterized . An alignment of 3HNR sequences enabled the design of primers covering conserved regions . A PCR-amplified fragment of Curvularia lunata genomic DNA was used for screening the cDNA library . Three independent cDNA clones revealed an 801-bp open reading frame encoding a 267 amino acid protein . The protein was expressed in Escherichia coli and purified to homogeneity . The predicted amino acid sequence of the 28.6-kDa protein demonstrated homology to other fungal 3HNR and other members of the short-chain dehydrogenase super family . Northern analyses revealed that 3HNR from Curvularia lunata is expressed synchronously with melanization after 3 days of Curvularia lunata growth in malt extract medium . No 3HNR reductase gene expression nor melanization was observed when Curvularia lunata was grown in yeast nitrogen base medium.

World J Gastroenterol, 2003 May, 9(5), 1077 - 81
Repression of allo-cell transplant rejection through CIITA ribonuclease P+ hepatocyte; Guo R et al.; AIM: Allo-cell transplant rejection and autoimmune responses were associated with the presence of class II major histocompatibility complex (MHC II) molecules on cells . This paper studied the effect of Ribonuclease P (RNase P) against CIITA, which was a major regulator of MHCII molecules, on repressing the expression of MHCII molecules on hepatocyte . METHODS: M1-RNA is the catalytic RNA subunit of RNase P from Escherichia coli . It were constructed that M1-RNA with guide sequences (GS) recognizing the 452, 3408 site of CIITA by PCR from pTK117 plasmid, then were cloned into the EcoRI/BglII or EcoR//SalIsite of vector psNAV (psNAV-M1-452-GS, psNAV-M1-3408-GS) respectively . The target mould plate (3176-3560) of CIITA was obtained from Raji cell by RT-PCR, and then inserted into the XhoI/EcoRIof pGEM-7zf(+) plasmid (pGEM-3176) . These recombinant plasmids were screened out by sequence analysis . psNAV-M1-452-GS, psNAV-M1-3408-GS and its target RNA pGEM-3176 were transcribed and then mixed up and incubated in vitro . It showed that M1-3408-GS could exclusively cleave target RNA that formed a base pair with the GS . Stable transfectants of hepatocyte cell line with psNAV-M1-3408-GS were tested for expression of class II MHC through FCM, for mRNA abundance of MHCII, Ii and CIITA by RT-PCR., for the level of IL-2 mRNA on T cell by mixed lymphocyte reaction . RESULTS: When induced with recombinant human interferon-gamma (IFN-gamma), the expression of HLA-DR, -DP, -DQ on psNAV-M1-3408-GS(+) hepatocyte was reduced 83.27 %, 88.93 %, 58.82 % respectively, the mRNA contents of CIITA, HLA-DR, -DP, -DQ and Ii decreased significantly . While T cell expressed less IL-2 mRNA in the case of psNAV-M1-3408-GS(+) hepatocyte . CONCLUSION: The Ribonuclease P against CIITA-M1-3408-GS could effectively induce antigen-specific tolerance through cleaving CIITA . These results provided insight into the future application of M1-3408-GS as a new nucleic acid drug against allo-transplantation rejection and autoimmune diseases.

World J Gastroenterol, 2003 May, 9(5), 1003 - 7
Expression of hepatitis C virus hypervariable region 1 and its clinical significance; Zhang XX et al.; AIM: To explore the properties of hypervariable region 1 (HVR1) in the envelope 2 gene of hepatitis C virus by analyzing the reactivity of HVR1 fusion proteins from different Chinese HCV strains with sera of patients with chronic hepatitis C and by comparing their reactivity between interferon therapy responders and non-responders . METHODS: Gene fragments of HVR1 of four HCV strains (three genotype 1b and one genotype 2a) were amplified from pGEMT-E2 plasmids and sub-cloned into pQE40 vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli strain TG1 . The purified DHFR- HVR1 proteins were then used to detect the anti-HVR1 antibodies in 70 serum samples of patients with chronic hepatitis C . RESULTS: Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320-800 ug fusion proteins per 100 ml culture) . Each fusion protein (SH1b, BJ1b, SD1b and SD2a) reacted with 72.8 % (51/70), 60 % (42/70), 48.6 % (34/70), and 58.6 % (41/30) of the anti-HCV positive patients' sera respectively by ELISA . 57 % (4/7) of non responders reacted with all four HVR1 fusion proteins, while only 15.3 % (2/13) of responders reacted with all of them . The O.D . values of sera from IFN therapy responders were significantly higher than those of non responders (P<0.05) . CONCLUSION: The selected HVR1 fusion proteins expressed in E . coli can broadly react with HCV-infected patients' sera . The intensity and/or quality of the immune response against HCV may be a critical factor determining the response to interferon treatment . With the evolution of virus strains, anti-HVR1 antibodies can not neutralize all the quasispecies . A polyvalent and high immunogenic vaccine comprising a mixture of several HVR1 sequences that cover the reactivity of most HCV isolates may be useful.

Environ Mol Mutagen, 2003, 41(4), 253 - 9
Novel transgenic rat for in vivo genotoxicity assays using 6-thioguanine and Spi- selection; Hayashi H et al.; Transgenic rodents are valuable models for investigating the genotoxicity of chemicals in vivo . Here, we report the establishment of a novel transgenic rat for genotoxicity analysis . In this model, about 10 copies of lambdaEG10 DNA carrying the gpt gene of E . coli and the red/gam genes of lambda phage are integrated per haploid genome of Sprague-Dawley rats at position 4q24-q31 . After recovery of lambdaEG10 phage, point mutations in the gpt gene and deletions in the red/gam genes are identified by 6-thioguanine and Spi(-) selection, respectively . To examine the suitability of these rats for performing in vivo mutagenicity assays, rats were treated with single intraperitoneal injections of ethylnitrosourea (ENU; 100 mg/kg) or benzo{a}pyrene (B{a}P; 62.5 and 125 mg/kg), and the mutant frequencies (MFs) in the liver were determined 7 days after the treatment . ENU enhanced the gpt MF about 7-fold over the control while it did not significantly increase the Spi(-) MF . B{a}P increased both the gpt and Spi(-) MFs several-fold in a dose-dependent manner . To examine the kinetics of MF, ENU was administered (50 mg/kg/day for 5 successive days) and gpt MFs in the liver were determined 7, 21, 35, and 70 days after the last injection . The MF increased to 8-fold and 13-fold over the control at 7 and 35 days, respectively, after the last injection and then slightly declined at 70 days . These kinetics are similar to those reported for ENU-treated lacZ transgenic mice . This novel transgenic rat could be useful for investigating species differences between rats and mice in their response to genotoxic agents .

J Infect Dis, 2003 May 1, 187(9), 1442 - 51 Epub 2003 Apr 15.
Overexpression of interleukin-15 protects against Escherichia coli-induced shock accompanied by inhibition of tumor necrosis factor-alpha-induced apoptosis; Hiromatsu T et al.; Interleukin (IL)-15, a potent inhibitor of tumor necrosis factor (TNF)-alpha-mediated apoptosis, causes multiple organ failure during endotoxic shock . We investigated the potential role of IL-15 in protection against Escherichia coli-induced shock by using IL-15 transgenic (Tg) mice . These mice were resistant to an otherwise lethal challenge with E . coli, although bacterial burden and serum levels of TNF-alpha were similar in non-Tg mice . Apoptosis in cells of the peritoneal cavity, liver, spleen, or lung was significantly suppressed in IL-15 Tg mice after E . coli infection . Peritoneal cells from naive IL-15 Tg mice were also resistant to TNF-alpha-induced apoptosis in vitro, and neutralization of endogenous IL-15 significantly aggravated TNF-alpha-induced apoptosis . Exogenous IL-15 prevented TNF-alpha-induced apoptosis in normal mice in vitro and improved the survival rate after E . coli challenge . These results suggest that IL-15 overexpression can prevent TNF-alpha-induced apoptosis and protect against E . coli-induced shock, indicating a possible therapeutic application of IL-15 for septic shock.

EMBO Rep, 2003 May, 4(5), 499 - 504
Maintenance of the correct open reading frame by the ribosome; Hansen TM et al.; During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein . The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis . Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear . In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon-anticodon interactions at the P site . The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage . We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3 . An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.

EMBO Rep, 2003 May, 4(5), 479 - 83
A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides; Ishibashi T et al.; MutT-related proteins, including the Escherichia coli MutT and human MutT homologue 1 (MTH1) proteins, degrade 8-oxo- 7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to a monophosphate, thereby preventing mutations caused by the misincorporation of 8-oxoguanine into DNA . Here, we report that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication . The human Nudix type 5 (NUDT5) protein hydrolyses 8-oxo-dGDP to monophosphate with a K(m) of 0.77 microM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5 . NUDT5 hydrolyses 8-oxo-dGTP only at very low levels, but is able to substitute for MutT when it is defective . When NUDT5 is expressed in E . coli mutT(-) cells, the increased frequency of spontaneous mutations is decreased to normal levels . Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8-oxoguanine in human cells.

Protein Sci, 2003 May, 12(5), 1087 - 96
Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles; Wang G et al.; The N-terminal domain of enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system confers amphitropism to the protein, allowing IIA(Glc) to shuttle between the cytoplasm and the membrane . To further understand this amphitropic protein, we have elucidated, by NMR spectroscopy, the solution structure of a synthetic peptide corresponding to the N-terminal domain of IIA(Glc) . In water, this peptide is predominantly disordered, consistent with previous data obtained in the absence of membranes . In detergent micelles of dihexanoylphosphatidylglycerol (DHPG) or sodium dodecylsulfate (SDS), however, residues Phe 3-Val 10 of the peptide adopt a helical conformation in the ensemble of structures calculated on the basis of NOE-derived distance restraints . The root mean square deviations for superimposing the backbone atoms of the helical region are 0.18 A in DHPG and 0.22 A in SDS . The structure, chemical shifts, and spin-spin coupling constants all indicate that, of the four lysines in the N-terminal domain of IIA(Glc), only Lys 5 and Lys 7 in the amphipathic helical region interact with DHPG . In addition, the peptide-detergent interactions were investigated using intermolecular NOESY experiments . The aliphatic chains of anionic detergents DHPG, SDS, and 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) all showed intermolecular NOE cross-peaks to the peptide, providing direct evidence for the putative membrane anchor of IIA(Glc) in binding to the membrane-mimicking micelles.

Protein Sci, 2003 May, 12(5), 893 - 902
Energy-dependent degradation: Linkage between ClpX-catalyzed nucleotide hydrolysis and protein-substrate processing; Burton RE et al.; ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation . The steady-state parameters for hydrolysis of ATP and ATPgammaS by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides . ClpX hydrolyzed ATPgammaS to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP . K(M) and k(cat) for hydrolysis of ATP and ATPgammaS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect k(cat) rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of approximately 15 micro M on K(D) for both nucleotides . Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPgammaS ( approximately 2 micro M) and ADP ( approximately 3 micro M) under the reaction conditions used for steady-state kinetics . In the absence of Mg(2+), where hydrolysis does not occur, the inhibition constant for ATP ( approximately 55 micro M) was weaker but very similar to the value for ATPgammaS ( approximately 45 micro M) . Compared with ATP, ATPgammaS supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA . These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis.

J Biol Chem, 2003 Jul 11, 278(28), 25977 - 81 Epub 2003 Apr 25.
PfSPATR, a Plasmodium falciparum protein containing an altered thrombospondin type I repeat domain is expressed at several stages of the parasite life cycle and is the target of inhibitory antibodies; Chattopadhyay R et al.; The annotated sequence of chromosome 2 of Plasmodium falciparum was examined for genes encoding proteins that may be of interest for vaccine development . We describe here the characterization of a protein with an altered thrombospondin Type I repeat domain (PfSPATR) that is expressed in the sporozoite, asexual, and sexual erythrocytic stages of the parasite life cycle . Immunoelectron microscopy indicated that this protein was expressed on the surface of the sporozoites and around the rhoptries in the asexual erythrocytic stage . An Escherichia coli-produced recombinant form of the protein bound to HepG2 cells in a dose-dependent manner and antibodies raised against this protein blocked the invasion of sporozoites into a transformed hepatoma cell line . Sera from Ghanaian adults and from a volunteer who had been immunized with radiation-attenuated P . falciparum sporozoites specifically recognized the expression of this protein on transfected COS-7 cells . These data support the evaluation of this protein as a vaccine candidate.

J Biol Chem, 2003 Jul 11, 278(28), 26065 - 70 Epub 2003 Apr 25.
The rate of peptidyl-tRNA dissociation from the ribosome during minigene expression depends on the nature of the last decoding interaction; Cruz-Vera LR et al.; The expression of some very short open reading frames (ORFs) in Escherichia coli results in peptidyl-tRNA accumulation that is lethal to cells defective in peptidyl-tRNA hydrolase activity . In an attempt to understand the factors that affect this phenotype, we have surveyed the toxicity of a complete set of two-codon ORFs cloned as minigenes in inducible expression vectors . The minigenes were tested in hydrolase-defective hosts and classified according to their degree of toxicity . In general, minigenes harboring codons belonging to the same box in the standard table of the genetic code mediated similar degrees of toxicity . Moreover, the levels of peptidyl-tRNA accumulation for synonymous minigenes decoded by the same tRNA were comparable . However, two exceptions were observed: (i) expression of minigenes harboring the Arg codons CGA, CGU, and CGC, resulted in the accumulation of different levels of the unique peptidyl-tRNAArg-2 and (ii) the toxicity of minigenes containing CUG and UCU codons, each recognized by two different tRNAs, depended on peptidyl-tRNA accumulation of only one of them . Non-toxic, or partly toxic, minigenes prompted higher accumulation levels of peptidyl-tRNA upon deprivation of active RF1, implying that translation termination occurred efficiently . Our data indicate that the nature of the last decoding tRNA is crucial in the rate of peptidyl-tRNA release from the ribosome.

J Biol Chem, 2003 Jul 4, 278(27), 25191 - 206 Epub 2003 Apr 25.
Solution structure of the phosphoryl transfer complex between the signal-transducing protein IIAGlucose and the cytoplasmic domain of the glucose transporter IICBGlucose of the Escherichia coli glucose phosphotransferase system; Cai M et al.; The solution structure of the final phosphoryl transfer complex in the glucose-specific arm of the Escherichia coli phosphotransferase system, between enzyme IIAGlucose (IIAGlc) and the cytoplasmic B domain (IIBGlc) of the glucose transporter IICBGlc, has been solved by NMR . The interface (approximately 1200-A2 buried surface) is formed by the interaction of a concave depression on IIAGlc with a convex protrusion on IIBGlc . The phosphoryl donor and acceptor residues, His-90 of IIAGlc and Cys-35 of IIBGlc (residues of IIBGlc are denoted in italics) are in close proximity and buried at the center of the interface . Cys-35 is primed for nucleophilic attack on the phosphorus atom by stabilization of the thiolate anion (pKa approximately 6.5) through intramolecular hydrogen bonding interactions with several adjacent backbone amide groups . Hydrophobic intermolecular contacts are supplemented by peripheral electrostatic interactions involving an alternating distribution of positively and negatively charged residues on the interaction surfaces of both proteins . Salt bridges between the Asp-38/Asp-94 pair of IIAGlc and the Arg-38/Arg-40 pair of IIBGlc neutralize the accumulation of negative charge in the vicinity of both the Sgamma atom of Cys-35 and the phosphoryl group in the complex . A pentacoordinate phosphoryl transition state is readily accommodated without any change in backbone conformation, and the structure of the complex accounts for the preferred directionality of phosphoryl transfer between IIAGlc and IIBGlc . The structures of IIAGlc.IIBGlc and the two upstream complexes of the glucose phosphotransferase system (EI.HPr and IIAGlc.HPr) reveal a cascade in which highly overlapping binding sites on HPr and IIAGlc recognize structurally diverse proteins.

J Biol Chem, 2003 Jul 18, 278(29), 26505 - 10 Epub 2003 Apr 27.
A backbone-reversed form of an all-beta alpha-crystallin domain from a small heat-shock protein (retro-HSP12.6) folds and assembles into structured multimers; Shukla A et al.; The structural consequences of polypeptide backbone reversal ("retro" modification) remain largely unexplored, in particular, for the retro forms of globular all-beta-sheet proteins . To examine whether the backbone-reversed form of a model all-beta-sheet protein can fold and adopt secondary and tertiary structure, we created and examined the recombinant retro form of a 110-residue-long polypeptide, an alpha-crystallin-like small heat-shock protein, HSP12.6, from C . elegans . Following intracellular overexpression in fusion with a histidine affinity tag in Escherichia coli, purification under denaturing conditions, and removal of denaturant through dialysis, retro-HSP12.6 was found to fold to a soluble state . The folded protein was examined using fluorescence and CD spectroscopy, gel filtration chromatography, non-denaturing electrophoresis, differential scanning calorimetry, and electron microscopy and confirmed to have adopted secondary structure and assembled into a multimer . Interestingly, like its parent polypeptide, retro-HSP12.6 did not aggregate upon heating; rather, heating led to a dramatic increase in structural content and the adoption of what would appear to be a very well folded state at high temperatures . However, this was essentially reversed upon cooling with some hysteresis being observed resulting in greater structural content in the heated-cooled protein than in the unheated protein . The heated-cooled samples displayed CD spectra indicative of structural content comparable to that of any naturally occurring globular protein . Attempts are being made to refine crystallization conditions for the folded protein.

Am J Clin Nutr, 2003 May, 77(5), 1287 - 95
Lack of effect of foods enriched with plant- or marine-derived n-3 fatty acids on human immune function; Kew S et al.; BACKGROUND: Greatly increasing dietary flaxseed oil {rich in the n-3 polyunsaturated fatty acid (PUFA) alpha-linolenic acid (ALA)} or fish oil {rich in the long-chain n-3 PUFAs eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids} can reduce markers of immune cell function . The effects of more modest doses are unclear, and it is not known whether ALA has the same effects as its long-chain derivatives . OBJECTIVE: The objective was to determine the effects of enriching the diet with ALA or EPA+DHA on immune outcomes representing key functions of human neutrophils, monocytes, and lymphocytes . DESIGN: In a placebo-controlled, double-blind, parallel study, 150 healthy men and women aged 25-72 y were randomly assigned to 1 of 5 interventions: placebo (no additional n-3 PUFAs), 4.5 or 9.5 g ALA/d, and 0.77 or 1.7 g EPA+DHA/d for 6 mo . The n-3 PUFAs were provided in 25 g fat spread plus 3 oil capsules . Blood samples were taken at 0, 3, and 6 mo . RESULTS: The fatty acid composition of peripheral blood mononuclear cell phospholipids was significantly different in the groups with higher intakes of ALA or EPA+DHA . The interventions did not alter the percentages of neutrophils or monocytes engaged in phagocytosis of Escherichia coli or in phagocytic activity, the percentages of neutrophils or monocytes undergoing oxidative burst in response to E . coli or phorbol ester, the proliferation of lymphocytes in response to a T cell mitogen, the production of numerous cytokines by monocytes and lymphocytes, or the in vivo delayed-type hypersensitivity response . CONCLUSION: An intake of <or= 9.5 g ALA/d or <or= 1.7 g EPA+DHA/d does not alter the functional activity of neutrophils, monocytes, or lymphocytes, but it changes the fatty acid composition of mononuclear cells.

Am J Physiol Lung Cell Mol Physiol, 2004 Mar, 286(3), L494 - 501 Epub 2003 Apr 25.
Eosinophils and monocytes produce pulmonary and activation-regulated chemokine, which activates cultured monocytes/macrophages; Schraufstatter I et al.; Pulmonary and activation-regulated chemokine (PARC/CCL18) belongs to the family of CC chemokines and shares 61% sequence identity with monocyte inflammatory protein (MIP)-1alpha . Produced by dendritic cells and macrophages primarily in the lung, PARC is known to be chemotactic for T cells . Because PARC's biological function is largely unknown, we screened various leukocyte populations for PARC expression and for response to PARC, with the idea that the cellular source may link PARC to disease states in which it may be involved . Here we report that eosinophils obtained from individuals with mild eosinophilia express PARC as assessed by RT-PCR on eosinophil RNA . The eosinophil preparations were free of monocytes, a known source of PARC, and no RT-PCR product was obtained from neutrophils . Furthermore, PARC protein was detected by ELISA in the supernatants of eosinophils from seven of nine donors and in higher concentration in the supernatants of monocytes on day 1 of culture . Purified recombinant PARC activated human monocytes/macrophages kept in culture for 3-4 days but not freshly isolated monocytes . The threshold dose for Ca(2+) mobilization as determined fluorometrically in indo 1-AM-labeled monocytes was 5 nM; maximal response was reached with approximately 50 nM PARC . PARC was chemotactic for these cultured monocytes and caused actin polymerization determined by FITC-phalloidin binding and fluorescence-activated cell sorting analysis . In contrast, PARC activated neither neutrophils nor eosinophils . Eosinophil production of PARC, its chemotactic effect on monocytes and lymphocytes, and PARC's previously described localization to the lung suggest that this chemokine might play a role in pulmonary leukocyte trafficking.

Zhonghua Gan Zang Bing Za Zhi, 2003 Apr, 11(4), 209 - 11
{Prokaryocytic expression of the shortened hepatitis B surface antigen and antigenic analysis}; Ling N et al.; OBJECTIVE: To study the expression of the shortened hepatitis B surface antigen in prokaryocyte and detect the antigenic characters . METHODS: Firstly, the gene fragments coding the 152 and 124 amino acids of the carboxyl terminus of hepatitis B surface antigen (HBsAg) were amplified by polymerase chain reaction (PCR) . Secondly, they were cloned to plasmid pBKS+, and the accuracy of those constructions were confirmed by restriction enzyme digestion and DNA sequencing . Then, they were cloned to prokaryocytic expression vector-plasmid pET32a(+) . The recombinant plasmids were transfected into E.coli BL21 and induced to express with IPTG . RESULTS: The recombinant plasmids were successfully constructed . In E.coli BL21, the protein was expressed in a fusion fashion and could be recognized by monoclonal antibody against HBsAg with ELISA and Western blot . CONCLUSION: The shortened HBsAg can be expressed in prokaryocyte.

J Interferon Cytokine Res, 2003 Mar, 23(3), 163 - 70
The role of interferons in rotavirus infections and protection; Vancott JL et al.; Type I and type II interferons (IFNs) play a critical role in control of a number of viral infections . To study whether altered and reduced functional capacities of type I and type II IFNs would affect rotavirus-induced diarrhea and viral replication, we obtained signal transducers and activators of transcription 1 (Stat1) knock-out mice (Stat1(-/-)) that lack many IFN-induced responses . We found that suckling Stat1(-/-) and immunocompetent mice orally infected with rotavirus experienced diarrhea and shed rotavirus with similar intensity . However, adult Stat1(-/-) mice shed up to 100-fold more homologous murine rotavirus and heterologous rhesus rotavirus antigen in their stools than did immunocompetent mice 2-6 days after infection . Clearance of rotavirus in stools from adult Stat1(-/-) mice occurred at the same time as in wild-type (WT) control mice . Clearance in Stat1(-/-) mice correlated with a potent antibody response and a mixed Th1 and Th2 response, whereas in WT control mice, clearance correlated with a weaker antibody response and a polarized Th1 response . Stat1(-/-) mice were fully protected against subsequent challenge . Moreover, vaccination of adult Stat1(-/-) mice with a rotavirus VP6 protein and the mucosal adjuvant Escherichia coli heat-labile toxin LT (R192G) elicited 94% protection, as measured by the total reduction in viral shedding for the group in comparison to unimmunized controls . Thus, modulating IFN function through the loss of Stat1 caused a defective innate immune response in adult mice but had no effect on rotavirus-induced diarrhea and replication in suckling mice . Furthermore, adult Stat1(-/-), IFN-gamma, and IFN-alpha/beta receptor(-/-) (IFNAR-2(-/-)) mice infected with rotavirus or vaccinated with VP6 vaccine and adjuvant were fully protected against rotavirus shedding following a subsequent challenge with rotavirus.

IUBMB Life, 2003 Jan, 55(1), 37 - 41
Effectory site in Escherichia coli inorganic pyrophosphatase is revealed upon mutation at the intertrimeric interface; Sitnik TS et al.; Escherichia coli inorganic pyrophosphatase (E-PPase) is a homohexamer formed from two trimers related by a two-fold axis . The residue Asp26 participates in intertrimeric contacts . Kinetics of MgPPi hydrolysis by a mutant Asp26Ala E-PPase is found to not obey Michaelis-Menten equation but can be described within the scheme of activation of hydrolysis by a free PPi binding at an effectory subsite . Existence of such a subsite is confirmed by the finding that the free form of methylenediphosphonate activates MgPPi hydrolysis though its magnesium complex is a competitive inhibitor . The Asp26Ala variant is the first example of hexameric E-PPase demonstrated to have an activatory subsite.

Biol Chem, 2003 Mar, 384(3), 373 - 86
Determinants of enzymatic specificity in the Cys-Met-metabolism PLP-dependent enzymes family: crystal structure of cystathionine gamma-lyase from yeast and intrafamiliar structure comparison; Messerschmidt A et al.; The crystal structure of cystathionine gamma-lyase (CGL) from yeast has been solved by molecular replacement at a resolution of 2.6 A . The molecule consists of 393 amino acid residues and one PLP moiety and is arranged in the crystal as a tetramer with D2 symmetry as in other related enzymes of the Cys-Met-metabolism PLP-dependent family like cystathionine beta-lyase (CBL) . A structure comparison with other family members revealed surprising insights into the tuning of enzymatic specificity between the different family members . CGLs from yeast or human are virtually identical at their active sites to cystathionine gamma-synthase (CGS) from E . coli . Both CGLs and bacterial CGSs exhibit gamma-synthase and gamma-lyase activities depending on their position in the metabolic pathway and the available substrates . This group of enzymes has a glutamate (E333 in yeast CGL) which binds to the distal group of cystathionine (CTT) or the amino group of cysteine . Plant CGSs use homoserine phosphate instead of O-succinyl-homoserine as one substrate . This is reflected by a partially different active site structure in plant CGSs . In CGL and CBL the pseudosymmetric substrate must dock at the active site in different orientations, with S in gamma-position (CBL) or in delta-position (CGL) . The conserved glutamate steers the substrate as seen in other CGLs . In CBLs this position is occupied by either tyrosine or hydrophobic residues directing binding of CTT such that S is in the in gamma-position . In methionine gamma-lyase a hydrophic patch operates as recognition site for the methyl group of the methionine substrate.

Biol Chem, 2003 Mar, 384(3), 363 - 71
Hepatitis B virus core antigen: enhancement of its production in Escherichia coli, and interaction of the core particles with the viral surface antigen; Tan WS et al.; The core antigen (HBcAg) of hepatitis B Virus (HBV) can be expressed in Escherichia coil where it assembles into icosahedral particles containing 240 or 180 subunits . Analysis of the two kinds of particles by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a substantial proportion of their subunits were smaller than the full-length HBcAg monomer and of variable size, but all had the same N-terminal sequence showing that the smaller species were heterogeneous in their arginine-rich C-terminal regions . Around 50% of these arginine residues are encoded by the triplet AGA which is rare in E . coli . Supplementation of the level of AGA tRNA in the cell by transformation with plasmids expressing the T4 AGA tRNA gene significantly enhanced the yield of HBcAg . Fusion phage carrying a ligand specific for HBcAg showed no significant difference in the affinity for the two sizes of HBcAg particles, but in similar reactions in solution HBV surface antigen exhibited differential affinities for the same two HBcAg preparations.

Biol Chem, 2003 Mar, 384(3), 343 - 50
HIV-1 TAR as anchoring site for optimized catalytic RNAs; Puerta-Fernandez E et al.; Ribozymes have a great potential for developing specific gene silencing molecules . One of the main limitations to ensure the efficient application of ribozymes is to achieve effective binding to the target . Stem-loop domains support efficient formation of the kissing complex between natural antisense molecules and their target sequence . We have characterized catalytic antisense RNA hybrid molecules composed of a hammerhead ribozyme and a stem-loop antisense domain . A series of artificial RNA substrates containing the TAR-RNA stem-loop and a target for the hammerhead ribozyme were constructed and challenged with a catalytic antisense RNA carrying the TAR complementary stem-loop . The catalytic antisense RNA cleaves each of these substrates significantly more efficiently than the parental hammerhead ribozyme . Deletion of the TAR domain in the substrate abolishes the positive effect . These results suggest that the enhancement is due to the interaction of both complementary stem-loop motifs . A similar improvement was corroborated when targeting the LTR region of HIV-1 with either hammerhead- and hairpin-based catalytic antisense RNAs . Our results indicate that the TAR domain can be used as an anchoring site to facilitate the access of ribozymes to their specific target sequences within TAR-containing RNAs . Finally, we propose the addition of stable stem-loop motifs to the ribozyme domain as a rational way for constructing catalytic antisense RNAs.

J Antibiot (Tokyo), 2003 Feb, 56(2), 143 - 53
Studies on a second and third ring cyclization in anthracycline biosynthesis; Hautala A et al.; This paper focuses on study of second and third ring cyclization in anthracycline biosynthesis by a heterologous gene expression . Firstly, anthracycline non-producing Streptomyces peucetius mutant, D2 was heterologously complemented to produce daunomycins with plasmids pSgs44 and pSYE66, which contain putative cyclase genes of S . galilaeus and S . nogalater, respectively . A point mutation in the cyclase gene dpsY of D2 has changed glycine to serine resulting inactivation of the enzyme . Secondly, the putative cyclase gene snoaM from S . nogalater, was expressed in a gene cassette in S . lividans TK24 and S . coelicolor CH999 to study the influence of the cyclase gene on auramycinone production and the impact of endogenous genes on production profiles . The results obtained confirms that a cyclase closing the second and third ring of a polyketide is essential in anthracycline biosynthesis.

Ugeskr Laeger . 2003 Apr 7;165(15):1569.
{Pyomyositis}; Jacobsen TW et al.; Pyomyositis is a bacterial infection of skeletal muscle . It is a rare condition which cannot easily be diagnosed . A fatal case of pyomyositis in a 78-year-old man is presented . The clinical signs and diagnostics of pyomyositis are discussed.

Invest Ophthalmol Vis Sci, 2003 May, 44(5), 2245 - 51
DNA is a target of the photodynamic effects elicited in A2E-laden RPE by blue-light illumination; Sparrow JR et al.; PURPOSE: When the pyridinium bisretinoid A2E, an age-related fluorophore in the retinal pigment epithelium (RPE), is irradiated with blue light, photochemical events are initiated that can ultimately provoke cell death . This study was designed to determine whether DNA is a target of the cellular damage . METHODS: ARPE-19 cells accumulated A2E before exposure to blue light . DNA damage was assayed in individual cells by alkaline gel electrophoresis (comet assay), with and without the addition of the repair enzymes formamidopyrimidine N-glycosylase (Fpg), endonuclease III (endo III) and T4-endonuclease V (T4-endo V) to characterize DNA lesions . Damage was quantified as comet tail moment . The base lesion 8-oxo-deoxyguanosine (8-oxo-dG) was detected by immunoperoxidase and histochemical methods . The singlet oxygen quencher, sodium azide, was tested for its ability to reduce DNA damage, and cell viability was quantified . RESULTS: DNA damage was induced in A2E-containing RPE exposed to 430-nm illumination . The extent of damage, measured as tail moment, was proportional to exposure duration and was reduced by preincubation with sodium azide . The detection of FPG- and endo III-sensitive DNA lesions revealed the presence of oxidized purine and pyrimidine bases, whereas labeling with specific antibody and binding of fluorescein-labeled avidin indicated that guanine bases were oxidatively modified to 8-oxo-dG . The ability of the cells to repair the DNA damage declined as the severity was increased, and kinetic studies disclosed rapid and slow stages of repair . CONCLUSIONS: DNA is one of the cellular constitutents that can be damaged by the interaction of A2E and blue light . At least some of the DNA lesions are oxidative base modifications.

Invest Ophthalmol Vis Sci, 2003 May, 44(5), 1993 - 7
Visualization of cell death in vivo during murine endotoxin-induced uveitis; Yang P et al.; PURPOSE: To develop a technology to image cell death in the eye of a live mouse and to apply that technology to characterize the role of apoptosis and necrosis in the evolution of endotoxin-induced uveitis (EIU), a standard model of intraocular inflammation . METHODS: To induce EIU, 250 ng Escherichia coli 055:B5 lipopolysaccharide was injected into the vitreous body of BALB/c mice . At 0, 6, 10, 16, 20, 24, 48, 72, and 96 hours and on day 7 after endotoxin injection, annexin V and propidium iodide were injected into the anterior chamber of these mice, and labeled cells were observed by using intravital epifluorescence video microscopy . Iris and corneal wholemounts isolated from the mice were also evaluated with standard and confocal fluorescence microscopy . TUNEL staining was performed on iris wholemounts taken from additional mice similarly injected with endotoxin, to confirm the in vivo results . RESULTS: Uveitis was induced in all the mice that received an endotoxin injection . The percentages of annexin V(+) propidium(-) cells, annexin V(+) propidium(+) cells, and annexin V(-) propidium(+) cells in the iris tissues visible by intravital microscopy were comparable to those observed by TUNEL staining in vitro . In addition, intravital microscopy allowed observation of labeled cells in the aqueous humor and on the surface of the lens . Both the number and the pattern of labeled cells changed dramatically over time . The cells stained with annexin V had a variety of morphologies, including small and round, round with a lobulated or a kidney-shaped nucleus, dendriform, and irregular . CONCLUSIONS: A technique was developed to image cell death in the anterior segment of the eye in vivo and used to demonstrate that the number and proportion of early apoptotic (annexin V(+) propidium(-)) and late apoptotic or necrotic (annexin V(+) propidium(+) and annexin V(-) propidium(+)) cells change over the course of EIU . A variety of inflammatory cells and resident cells undergo apoptosis, or possibly necrosis, which may contribute to the rapid resolution of EIU . This in vivo technique will be a valuable tool for future studies on the resolution of ocular inflammation.

J Biol Chem, 2003 Jul 4, 278(27), 24966 - 75 Epub 2003 Apr 24.
The plant biotin synthase reaction . Identification and characterization of essential mitochondrial accessory protein components; Picciocchi A et al.; In plants, the last step of the biotin biosynthetic pathway is localized in mitochondria . This chemically complex reaction is catalyzed by the biotin synthase protein, encoded by the bio2 gene in Arabidopsis thaliana . Unidentified mitochondrial proteins in addition to the bio2 gene product are obligatory for the reaction to occur . In order to identify these additional proteins, potato mitochondrial matrix was fractionated onto different successive chromatographic columns . Combination experiments using purified Bio2 protein and the resulting mitochondrial matrix subfractions together with a genomic based research allowed us to identify mitochondrial adrenodoxin, adrenodoxin reductase, and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction . Arabidopsis cDNAs encoding these proteins were cloned, and the corresponding proteins were expressed in Escherichia coli cells and purified . Purified recombinant adrenodoxin and adrenodoxin reductase proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex . Bio2 from Arabidopsis is the first identified protein partner for this specific plant mitochondrial redox chain.

Curr Opin Chem Biol, 2003 Apr, 7(2), 183 - 8
Di-iron-tyrosyl radical ribonucleotide reductases; Stubbe J; New insights have been gained into the formation of the di-iron tyrosyl radical cofactor, which is essential for nucleotide reduction in the class I ribonucleotide reductases . Recent advances include Insight into the function of the tyrosyl radical in initiation of nucleotide reduction.

Gynecol Oncol, 2003 May, 89(2), 243 - 50
Transduction of adenovirus-mediated wild-type p53 after radiotherapy in human cervical cancer cells; Huh JJ et al.; OBJECTIVE: Radiotherapy is the mainstay of treatment for advanced cervical cancer and most cervical cancers have evidence of HPV (human papilloma virus) infection, which inactivates the p53 gene . The goal of this study was to determine the effect of combining radiotherapy and Adp53 infection on the growth of cervical carcinoma cells . METHODS: The silta cervical carcinoma cell line contains wild-type p53 and HPV 16 infection . The C33A cell line has a p53 mutation . The Adp53 recombinant adenovirus contains the cytomegalovirus promoter, wild-type p53 cDNA, and a polyadenylation signal in a minigene cassette inserted into the El-deleted region of a modified adenovirus 5 . Transduction efficiency was assessed by using an adenovirus containing the Escherichia coli beta-galactosidase gene and expression of wild-type p53 in infected cells was evaluated by Western blot analysis . One group of cells was irradiated prior to infection, the other group received no irradiation, but were either infected with virus or mock-infected . Cells were analyzed for viability 1 to 7 days after infection by using the sulforhodamine B assay . The percentage of cells undergoing apoptosis was determined by using a TUNEL assay . RESULTS: Fifty percent of C33A cells were transduced with 5 muthplicities of infection of virus whereas SiHa cells required 25 multiplicities of infection . Adp53 expression was found 48 h after infection . In the cells treated with both radiation and Adp53 infection growth inhibition was increased compared with inhibition resulting with either treatment alone . The combination treatment also increased the percentage of cells undergoing apoptosis . CONCLUSION: Combining radiotherapy with Adp53 infection increases the inhibition of growth of cervical cancer cells in vitro . This combination treatment has the potential of increasing efficacy and of therapy.

J Struct Biol, 2003 May, 142(2), 301 - 10
An approach to examining model dependence in EM reconstructions using cross-validation; Shaikh TR et al.; Reference bias refers to a common problem in fitting experimental data to an initial model . Given enough free parameters, a good fit of any experimental data to the model can be obtained, even if the experimental data contain only noise . Reference-based alignment methods used in electron microscopy (EM) are subject to this type of bias, in that images containing pure noise can regenerate the reference . Cross-validation is based on the idea that the experimental data used to assess the validity of the fitting should not be the same data as were used to do the fitting . Here we present the application of cross-validation to one form of reference-based alignment: 3D-projection matching in single-particle reconstructions . Our results show that reference bias is indeed present in reconstructions, but that the effect is small for real data compared to that for random noise, and that this difference in behavior is magnified, rather than diminished, during iterative refinement.

J Struct Biol, 2003 May, 142(2), 256 - 65
Underlying regularity in the shapes of nucleoids of Escherichia coli: implications for nucleoid organization and partition; Zimmerman SB; The genomic DNA of Escherichia coli is localized in one or a few compact nucleoids . Nucleoids in rapidly grown cells appear in complex shapes; the relationship of these shapes to underlying arrangements of the DNA is of structural interest and of potential importance in gene localization and nucleoid partition studies . To help assess this variation in shape, limited three-dimensional information on individual nucleoids was obtained by DNA fluorescence microscopy of cells as they reoriented in solution or by optical sectioning . These techniques were also applied to enlarged nucleoids within swollen cells or spheroplasts . The resulting images indicated that much of the apparent variation was due to imaging from different directions and at different focal planes of more regular underlying nucleoid shapes . Nucleoid images could be transformed into compact doublet shapes by exposure of cells to chloramphenicol or puromycin, consistent with a preexisting bipartite nucleoid structure . Isolated nucleoids and nucleoids in stationary-phase cells also assumed a doublet shape, supporting such a structure . The underlying structure is suggested to be two subunits joined by a linker . Both the subunits and the linker appear to deform to accommodate the space available within cells or spheroplasts ("flexible doublet" model).

Biosens Bioelectron, 2003 Jul, 18(7), 907 - 16
Rapid identification of viable Escherichia coli subspecies with an electrochemical screen-printed biosensor array; Ertl P et al.; Rapid identification of Escherichia coli strains is an important diagnostic goal in applied medicine as well as the environmental and food sciences . This paper reports an electrochemical, screen-printed biosensor array, where selective recognition is accomplished using lectins that recognize and bind to cell-surface lipopolysaccharides and coulometric transduction exploits non-native external oxidants to monitor respiratory cycle activity in lectin-bound cells . Ten different lectins were separately immobilized onto porous membranes that feature activated surfaces (ImmunodyneABC) . Modified membranes were exposed to untreated E . coli cultures for 30 min, rinsed, and layered over the individual screen-printed carbon electrodes of the sensor array . The membranes were were incubated 5 min in a reagent solution that contained the oxidants menadione and ferricyanide as well as the respiratory substrates succinate and formate . Electrochemical oxidation of ferrocyanide for 2 min provided chronocoulometric data related to the quantities of bound cells . These screen-printed sensor arrays were used in conjunction with factor analysis for the rapid identification of four E . coli subspecies (E . coli B, E . coli Neotype, E . coli JM105 and E . coli HB101) . Systematic examination of lectin-binding patterns showed that these four E . coli subspecies are readily distinguished using only five essential lectins.

DNA Repair (Amst), 2003 May 13, 2(5), 623 - 7
A novel cytidine deaminase AIDs in the delivery of error-prone polymerases to immunoglobulin genes; Diaz M et al.; Immunoglobulin genes undergo somatic hypermutation in activated B lymphocytes . The mutations are targeted to the variable region of the gene, apparently by co-transcriptional deposition of a mutation complex . The novel activation induced deaminase (AID) gene is required for this process and recent work indicates that it is the cytidine deaminase activity of the AID protein rather than an associated RNA editing function that is required {Curr . Biol . 12 (2002) 1748; Nature 419 (2002) 1; Nature 418 (2002) 99} . The authors review the implications of this work and argue that deamination of deoxy-cytidine to deoxy-uracil seems to initiate several pathways in which error-prone polymerases are postulated to create mutations.

DNA Repair (Amst), 2003 May 13, 2(5), 609 - 22
Regulated over-expression of DNA polymerase beta mediates early onset cataract in mice; Sobol RW et al.; Base excision repair (BER) is a tightly coordinated mechanism for repair of DNA base damage (via alkylation and oxidation) and base loss . From E . coli to yeast to human cells, subtle alterations in expression of BER proteins lead to mutagenic or genome instability phenotypes . DNA polymerase beta (beta-pol), the major BER polymerase, has been found to be over-expressed in human tumor tissues and more recently it has been shown that over-expression of beta-pol results in a mutator and genome instability phenotype . These previous reports imply that beta-pol over-expression is deleterious and suggests that such an imbalance may cause an overall functional deficiency in the BER pathway . In the present study, we have developed a bicistronic tetracycline-responsive transgenic system to over-express beta-pol in mice . We find that over-expression of beta-pol in the lens epithelium results in the early onset of severe cortical cataract, with cataractogenesis beginning within 4 days after birth . In utero and post-natal suppression of transgenic Flag-beta-pol expression by doxycycline administration completely prevents cataract formation through adulthood, yet cataract is subsequently observed following removal of doxycycline and re-expression of the transgene . Cataract development accompanies increased expression of cyclooxygenase-2 in the lenticular fibers of the lens, implicating oxidative stress in the development of this cataractous phenotype . Although the mechanism for the transgene mediated cataractogenesis is not clear at this time, it is nevertheless intriguing that increased expression of beta-pol leads to such a phenotype . These results suggest that either a beta-pol expression imbalance negatively affects overall fidelity and/or BER capacity or that beta-pol has a role in lens epithelial cell differentiation.

DNA Repair (Amst), 2003 May 13, 2(5), 581 - 91
The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death; Rosenquist TA et al.; DNA damage mediated by reactive oxygen species generates miscoding and blocking lesions that may lead to mutations or cell death . Base excision repair (BER) constitutes a universal mechanism for removing oxidatively damaged bases and restoring the integrity of genomic DNA . In Escherichia coli, the DNA glycosylases Nei, Fpg, and Nth initiate BER of oxidative lesions; OGG1 and NTH1 proteins fulfill a similar function in mammalian cells . Three human genes, designated NEIL1, NEIL2 and NEIL3, encode proteins that contain sequence homologies to Nei and Fpg . We have cloned the corresponding mouse genes and have overexpressed and purified mNeil1, a DNA glycosylase that efficiently removes a wide spectrum of mutagenic and cytotoxic DNA lesions . These lesions include the two cis-thymineglycol(Tg) stereoisomers, guanine- and adenine-derived formamidopyrimidines, and 5,6-dihydrouracil . Two of these lesions, fapyA and 5S,6R thymine glycol, are not excised by mOgg1 or mNth1 . We have also used RNA interference technology to establish embryonic stem cell lines deficient in Neil1 protein and showed them to be sensitive to low levels of gamma-irradiation . The results of these studies suggest that Neil1 is an essential component of base excision repair in mammalian cells; its presence may contribute to the redundant repair capacity observed in Ogg1 -/- and Nth1 -/- mice.

DNA Repair (Amst), 2003 May 13, 2(5), 455 - 70
Endonuclease IV enhances base excision repair of endonuclease III from Methanobacterium thermoautotrophicum; Back JH et al.; Damaged DNA strands are repaired by base excision (BER) in organisms, a process initiated by repair enzymes, which include DNA glycosylases and endonucleases . We expressed and characterized two putative endonuclease genes from Methanobacterium thermoautotrophicum, Mt0764 and Mt1010, encoding homologues of endonuclease III (endo III) and endonuclease IV (endo IV) of Escherichia coli . The Mt0764 and Mt1010 proteins showed endo III activity by removing thymine glycol from DNA strand and AP endonuclease activity, respectively . The Mt0764 protein not only cleaved the oligonucleotide duplex, containing a thymine glycol/adenine pair efficiently, but also showed activity on the 8-oxoguanine-containing oligonucleotide duplex . In this study, we report upon the stimulation of endo III activity by endo IV using two recombinant proteins (Mt1010 and Mt0764) from M . thermoautotrophicum . Mt1010 stimulated the DNA glycosylase activity of Mt0764 for DNA substrates containing 8-oxoguanine residues and increasing the formation of the Mt0764 protein-DNA complex . The interaction between Mt1010 and Mt0764 was observed by using an in vitro binding assay . These results suggest that association between endo III and endo IV may occur in vivo, and this contributes to efficient base excision repair for the oxidative damage of DNA.

J Org Chem, 2003 May 2, 68(9), 3486 - 93
Biosynthesis of Vitamin B(6) in yeast . Incorporation pattern of trioses; Zeidler J et al.; The biosynthetic origin of the C(3) unit, C-6,5,5', of pyridoxamine was investigated in two yeasts, Candida utilis ATCC 9256 and Saccharomyces cerevisiae ATCC 7752 . The incorporation patterns within pyridoxamine bishydrochloride derived from variously multiply (13)C- and (2)H-labeled samples of glycerol and glyceraldehyde, established by NMR spectroscopy, indicate that the three-carbon unit C-6,5,5' of pyridoxamine is derived intact from a triose.

Avian Dis, 2003 Jan-Mar, 47(1), 79 - 86
Monoclonal antibodies to avian Escherichia coli Iss; Foley SL et al.; Escherichia coli infections are a major problem for the poultry industry in the United States . Yet, the virulence mechanisms operative in avian E . coli are poorly understood . In the present studies, monoclonal antibodies (MAbs) have been generated that may facilitate study of the pathogenesis of avian colibacillosis . These MAbs are directed against the Iss protein because results from our laboratory have shown that the possession of iss DNA sequences is strongly correlated with the E . coli implicated in avian colibacillosis . As part of an overall effort to explore the role of iss/Iss in colibacillosis pathogenesis, Iss protein has been purified, MAbs to Iss have been generated, and the MAbs are being evaluated . B cells from mice immunized with an Iss fusion to glutathione-S-transferase produced antibodies specifically against Iss, and these cells were used to generate the MAbs . These anti-Iss MAbs, when used in western blotting assays, can be used to distinguish iss-positive and -negative E . coli isolates, suggesting that they may be useful as reagents in the detection and study of virulent avian E . coli.

Avian Dis, 2003 Jan-Mar, 47(1), 21 - 31
Evaluation of the adhesive capacity of Escherichia coli isolates associated with avian cellulitis; Leclerc B et al.; It has been shown that Escherichia coli isolates from lesions of cellulitis belong to a limited number of clonal groups distinct from those of isolates found in the environment of these birds . In this study, different in vitro methods were used to evaluate adherence properties of E . coli isolates from cellulitis lesions and environments of high- and low-cellulitis prevalence broiler flocks . One hundred isolates were tested by hemagglutination . Adherence to frozen sections of chicken skin and binding to soluble fibronectin were examined for 40 of these 100 isolates by immunofluorescence and by immunocytofluorometry, respectively . Localization of bacterial adherence to skin tissues was confirmed by immunohistochemistry . It was demonstrated that O78:K80 isolates from cellulitis lesions adhered to skin sections to a much greater extent in deeper than in superficial tissue layers . A greater bacterial adherence following growth in TSB at 37 C was demonstrated for isolates from flocks with high prevalence of cellulitis than for isolates from flocks with low prevalence of cellulitis . MANOVA analysis results showed a significant difference between superficial and deep tissue layers only for one set of isolates from flocks with high prevalence of cellulitis . Hemagglutinating activity was variable among the O78:K80 isolates obtained from flocks with high prevalence of cellulitis . The results obtained for some O78:K80 isolates following growth in TSB suggest a role for type 1 fimbriae or F1 in adherence to skin sections . This was reinforced by the finding that adherence was inhibited by D-mannose . Poultry E . coli isolates that express F1 had no affinity for soluble fibronectin, although localization of the adherence in skin sections suggested a role for extracellular matrix components such as collagen and insoluble fibronectin.

Arch Environ Contam Toxicol, 2003 Apr, 44(3), 336 - 42
Compost-mediated removal of polycyclic aromatic hydrocarbons from contaminated soil; Sasek V et al.; Compost-assisted remediation of a manufactured-gas plant soil contaminated with polycyclic aromatic hydrocarbons (PAHs) was performed in thermally insulated composting chamber using mushroom compost consisting wheat straw, chicken manure, and gypsum . The degradation of individual PAHs was in range of 20-60% at the end of 54 days of composting followed by further increase of PAH removal (37-80%) after another 100 days of maturation . Both chemical analysis of the contaminated soil for PAHs and ecotoxicity tests on bioluminescent bacteria, earthworms, and plant seeds were performed before and after the composting . After the composting, inhibition of bioluminescence decreased, whereas no significant change in toxicity was observed for earthworm survival and seed germination . Using bacterial culture of Escherichia coli K12 genotoxicity tests were performed on samples taken from different parts of the composting pile; after the composting the decrease in genotoxicity was observed only in the sample taken from upper part of the composted pile.

Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5694 - 9 Epub 2003 Apr 23.
Mimicking natural evolution in vitro: an N-acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity; Joerger AC et al.; N-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS) belong to the NAL subfamily of (betaalpha)(8)-barrels . They share a common catalytic step but catalyze reactions in different biological pathways . By rational design, we have introduced various mutations into the NAL scaffold from Escherichia coli to switch the activity toward DHDPS . These mutants were tested with respect to their catalytic properties in vivo and in vitro as well as their stability . One point mutation (L142R) was sufficient to create an enzyme that could complement a bacterial auxotroph lacking the gene for DHDPS as efficiently as DHDPS itself . In vitro, this mutant had an increased DHDPS activity of up to 19-fold as defined by the specificity constant k(cat)K(M) for the new substrate l-aspartate-beta-semialdehyde when compared with the residual activity of NAL wild-type, mainly because of an increased turnover rate . At the same time, mutant L142R maintained much of its original NAL activity . We have solved the crystal structure of mutant L142R at 1.8 A resolution in complex with the inhibitor beta-hydroxypyruvate . This structure reveals that the conformations of neighboring active site residues are left virtually unchanged by the mutation . The high flexibility of R142 may favor its role in assisting in catalysis . Perhaps, nature has exploited the catalytic promiscuity of many enzymes to evolve novel enzymes or biological pathways during the course of evolution.

Nucleic Acids Res, 2003 May 1, 31(9), 2451 - 9
Stimulation of human DNA polymerase epsilon by MDM2; Asahara H et al.; The human DNA polymerase epsilon catalytic subunit consists of a 140-kDa N-terminal domain that contains the catalytic activity and a 120-kDa C-terminal domain that binds to the other subunits and to exogenous peptides, including PCNA and MDM2 . We report here that recombinant human MDM2 purified from insect cells or Escherichia coli stimulated the activity of DNA polymerase epsilon up to 10- and 40-fold, respectively, but not those of DNA polymerase beta or Klenow fragment of E.coli DNA polymerase I . Kinetic studies indicated that MDM2 increased the maximum velocity of the reaction, but did not change substrate affinities . The stimulation depended upon the interaction of the N-terminal 166 amino acid residues of MDM2 with the C-terminal domain of the full-length catalytic subunit, since the deletion of 166 amino acids from N-terminal of MDM2 or the removal of the C-terminal domain of DNA polymerase epsilon by trypsin digestion or competition for binding to it by the addition of excess C-terminal fragment eliminated the stimulation . Since DNA polymerase epsilon appears to be involved in DNA replication, recombination and repair synthesis, we suggest that MDM2 binding to DNA polymerase epsilon might be part of a reconfiguration process that allows DNA polymerase epsilon to associate with repair/recombination proteins in response to DNA damage.

Nucleic Acids Res, 2003 May 1, 31(9), 2272 - 8
A candidate prostate cancer susceptibility gene encodes tRNA 3' processing endoribonuclease; Takaku H et al.; tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from precursor tRNA (pre-tRNA) . We purified approximately 85 kDa 3' tRNase from pig liver and determined its partial sequences . BLAST search of them suggested that the enzyme was the product of a candidate human prostate cancer susceptibility gene, ELAC2, the biological function of which was totally unknown . We cloned a human ELAC2 cDNA and expressed the ELAC2 protein in Escherichia coli . The recombinant ELAC2 was able to cleave human pre-tRNA(Arg) efficiently . The 3' tRNase activity of the yeast ortholog YKR079C was also observed . The C-terminal half of human ELAC2 was able to remove a 3' trailer from pre-tRNA(Arg), while the N-terminal half failed to do so . In the human genome exists a gene, ELAC1, which seems to correspond to the C-terminal half of 3' tRNase from ELAC2 . We showed that human ELAC1 also has 3'-tRNase activity . Furthermore, we examined eight ELAC2 variants that seem to be associated with the occurrence of prostate cancer for 3'-tRNase activity . Seven ELAC2 variants which contain one to three amino acid substitutions showed efficient 3'-tRNase activities, while one truncated variant, which lacked a C-terminal half region, had no activity.

J Biol Chem, 2003 Jun 27, 278(26), 23996 - 4002 Epub 2003 Apr 23.
Involvement of single residue tryptophan 548 in the quaternary structural stability of pigeon cytosolic malic enzyme; Chang HC et al.; Pigeon cytosolic malic enzyme has a double dimer quaternary structure with three tryptophanyl residues in each monomer distributed in different structural domains . The enzyme showed a three-state unfolding phenomenon upon increasing the urea concentration (Chang, H . C., Chou, W . Y., and Chang, G . G . (2002) J . Biol . Chem . 277, 4663-4671) . At urea concentration of 4-4.5 m, where the intermediate form was detected, the enzyme existed as partially unfolded dimers, which were easily polymerized . Mn2+ provided full protection against the polymerization . To further characterize this phenomenon, three mutants of the enzyme (W129, W321, and W548), each with only one tryptophanyl residue left, were constructed . All these mutants were successfully overexpressed in Escherichia coli cells and purified to homogeneity . Changes in the circular dichroism spectra of all mutants revealed a three-state urea-unfolding process in the absence of Mn2+ . In the presence of 4 mm Mn2+, W548 and wild type (WT) enzymes shifted to monophasic, while W129 and W321 were still biphasic . Similar results were obtained from the fluorescence spectral changes, except for W321, which showed monophasic denaturation curve with or without Mn2+ . Analytical ultracentrifugation analysis indicated that the mutant enzymes were polymerized at 4.5 m urea, and Mn2+ provided protective effect on W548 and WT enzymes only . Other mutants with mutated Trp-548 polymerized at 4.5 m urea in the absence or presence of 4 mm Mn2+ . The above results indicate that a single residue, Trp-548, in the subunit interface region, is responsible for the integrity of the quaternary structure of the pigeon cytosolic malic enzyme.

Gene, 2003 Apr 10, 308, 115 - 28
NusA modulates intragenic termination by different pathways; Carlomagno MS et al.; In bacteria, conditions that uncouple translation from transcription activate intragenic terminators located within cistrons . We analyzed the function of NusA in intragenic termination, making use of two tandem terminators located within the hisG cistron, GTTE1 and GTTE2 . GTTE2 is a canonical Rho site, capable to terminate with Rho alone in vitro . By contrast, GTTE1 is a suboptimal terminator, featuring a boxA element and requiring a functional NusB to terminate efficiently in vivo . We found that a functional NusA is necessary for efficient termination events to occur at both GTTE1 and 2 . To enhance termination at GTTE1 in conditions in which the transcript is free of ribosomes, NusA acts at the same step as NusB and NusE/S10 . In the presence of concomitant translation, termination at GTTE1 is dependent on the relative position of the translation stop codon and boxA . If translation stops upstream of boxA, NusA acts at the same step as NusB enhance termination . Ribosomes terminating translation at boxA influence termination at GTTE1 . Interactions of NusA and/or NusB with ribosomal components, including NusE/S10, might facilitate termination . Differently from what observed at GTTE1, the NusA-stimulated pausing seems to be sufficient for the occurrence of complete termination events at GTTE2 . A functional NusA is also necessary to prevent premature termination of normally translated transcripts . Our data support the hypothesis that NusA may program a fraction of the RNA polymerase to terminate transcription upon interactions with specific sites on the nascent mRNA and either other Nuses or ribosomes.

Biochem Biophys Res Commun, 2003 May 2, 304(2), 333 - 8
Rabies virus nucleoprotein is phosphorylated by cellular casein kinase II; Wu X et al.; It has been reported that phosphorylation of rabies virus N plays an important role in the process of viral transcription and replication . Rabies virus N is phosphorylated when expressed alone, indicating that cellular kinase phosphorylates rabies virus N . To identify what cellular kinase phosphorylates rabies virus N, the N was expressed in Escherichia coli and purified by metal affinity chromatography . The recombinant N was phosphorylated by BHK cellular extracts and by purified CK-II . In addition, the phosphorylation of the recombinant N in vitro can be blocked by a CK-II inhibitor, heparin . Furthermore, N phosphorylation in the virus-infected cells can be inhibited by a CK-II specific inhibitor, 5,6-dichloro-beta-D-ribofuranosyl benzimidazole . However, PKC did not phosphorylate the recombinant N in vitro; nor did staurosporine, a PKC and other kinase inhibitor, prevent rabies virus N from phosphorylation . Thus, our data demonstrate that cellular CK-II phosphorylates rabies virus N.

Biochem Biophys Res Commun, 2003 May 2, 304(2), 279 - 84
Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli; Barrett CM et al.; The Tat system mediates the transport of folded proteins across the bacterial cytoplasmic membrane . To study the properties of the Escherichia coli Tat-system, we used green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA) . In the presence of arabinose, low levels of this protein rapidly saturate the translocase and cause the accumulation of inactive, membrane-bound TorA-GFP; fluorescence microscopy also showed active TorA-GFP to be distributed throughout the cytoplasm . However, the efficiency of export can be massively increased by alteration of the growth conditions, and further increased by overexpression of the tatABC genes . Under these conditions, the levels of GFP in the periplasm are raised over 20-fold and the export efficiency nears 100% . These results show that the Tat-system is relatively inactive under some growth conditions and the data suggest that the system may be applicable for the larger-scale export of heterologous proteins.

J Virol Methods, 2003 May, 109(2), 143 - 52
Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients; Gehringer H et al.; A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors . The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells . The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol . The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined . The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation . The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population . Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed . There was a good correlation between clinical parameters and the results obtained by this phenotypic assay . For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay . In a minority of patients our phenotypic results diverged from the genotypic ones . This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

J Virol Methods, 2003 May, 109(2), 139 - 42
Recombinant M protein-based ELISA test for detection of antibodies to canine coronavirus; Elia G et al.; The membrane (M) protein of canine coronavirus (CCoV) was cloned and expressed in E . coli . The purified recombinant protein was then evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for detection of CCoV antibodies in dog sera . Fifty serum samples, screened previously by whole virus ELISA and Western blotting, were tested . When the performance of the new test was compared with those of whole virus ELISA and Western blotting, an excellent correlation was found with the latter two assays . The ELISA based on recombinant M protein represents an alternative and valid test for detection of antibodies to CCoV in dog sera.

Biochem J, 2003 Jul 15, 373(Pt 2), 539 - 45
A novel amphibian Pi-class glutathione transferase isoenzyme from Xenopus laevis: importance of phenylalanine 111 in the H-site; De Luca A et al.; Screening of a liver tumour cDNA library from Xenopus laevis resulted in the isolation of a full-length cDNA clone encoding a novel Pi-class amphibian glutathione transferase (GST) isoenzyme (designated as XlGSTP1-1) . The gene encodes a protein of 212 amino acids with a calculated molecular mass of 24428 Da . The product of the gene has been overexpressed in Escherichia coli and characterized . XlGSTP1-1 has one of the highest specific activities towards 1-chloro-2,4-dinitrobenzene (1310 micromol/min per mg of protein) obtained with any GST . A notable feature of XlGSTP1-1 is the presence in the H-site of Phe(111) and Pro(208) in place of tyrosine and glycine residues respectively, present in other mammalian Pi-class GSTs . Site-directed mutagenesis indicate that Phe(111) is involved in substrate specificity of XlGSTP1-1 . We provide evidence showing that XlGSTP1-1 is present only in the embryo and its expression might be associated with cellular proliferation.

Z Naturforsch {C}, 2003 Mar-Apr, 58(3-4), 288 - 94
Fluorescence studies on denaturation and stability of recombinant human interferon-gamma; Christova P et al.; Unfolding/folding transitions of recombinant human interferon-gamma (hIFNgamma) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy . At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 M and deltaG0 = 13.4 kJ/mol) than urea (C* = 2.8 M and deltaG0 = 11.7 kJ/mol) . The close deltaG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNgamma is insignificant . Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNgamma remains native in the pH range of 4.8-9.5 . Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified . Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNgamma, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNgamma.

Z Naturforsch {C}, 2003 Mar-Apr, 58(3-4), 244 - 8
Mitomycin C-activity effected by vitamins B1, C, E and beta-carotene under irradiation with gamma-rays; Heinrich E et al.; Vitamin B1 (thiamine) can essentially effect the activity of mitomycin C (MMC), added individually or in combination with antioxidant vitamins (C, E-acetate, beta-carotene) as found in experiments in vitro (Escherichia coli bacteria, AB 1157) under irradiation with gamma-rays . The environment plays a crucial role . In airfree media vitamin B1 leads to a 2-fold increase of the MMC-efficiency, but adding vitamin C it decreases . In the presence of all vitamins (B1, C, E-ac., and beta-carotene) the MMC-action increases about 1.8-fold . In aerated media vitamin B1 causes an about 4-times increase of the MMC-efficiency, but by adding vitamin B1 and C the MMC-activity decreases by a factor of two, whereas in the presence of B1, C, E-ac., and beta-carotene it rises again to 2.6-fold . In environment saturated with N2O (conversion of e(-)aq into OH radicals) a different picture is observed . The presence of vitamin B1 or vitamin B1 + C causes a strong decrease of the MMC-efficiency, but the addition of all vitamins (B1, C, E-ac., and beta-car.) leads to a small increase of the cytostatic action . The results demonstrate the influence of vitamin B1 used individually or in combination with other antioxidants on the MMC-efficiency and the strong effect of the environment . The results are of interest for the application of MMC in radiotherapy.

Acta Paediatr, 2003, 92(2), 254 - 7
Cerebral venous thrombosis and Escherichia coli infection in neonates; Farstad H et al.; AIM: To present a possible association between cerebral venous thrombosis (CVT) and infection with Escherichia coli . METHODS: Four neonates with deep CVT occurring during an E . coli infection are presented . RESULTS: In these patients the thrombotic disease was found by Doppler ultrasonography . The thrombosis involved at least the sagittal sinus and the transverse sinus according to subsequent MRI scans . The E . coli strains did not produce verotoxin or haemolysin . Disseminated intravascular coagulation was not demonstrated . Three patients presented with seizures . At discharge, all of the patients had signs of neurological damage, but two of them have improved significantly since then . None of the patients has had recurrent (venous) thrombosis . CONCLUSION: E . coli infections in neonates may predispose to CVT, a finding that has clinical implications.

J Neurovirol, 2003, 9 Suppl 1, 21 - 4
Analysis of DNA-binding activity of the JC virus minor capsid protein VP2; Huang YL et al.; To investigate the DNA binding activity of the JC virus minor capsid protein, VP2, both wild-type and mutant VP2 were cloned and expressed in Escherichia coli . Southwestern blotting was employed for the DNA-binding assay . The results showed that VP2 was able to bind to DNA, except when either the last 13 or the last 29 amino acids were truncated . The results indicate that the DNA-binding domain of VP2 is located within the last 13 amino acids . Furthermore, we also demonstrated that Lys(332) and Lys(336) within the DNA-binding domain are crucial for DNA binding . The findings may provide further information for understanding the mechanism of virion assembly of the JC virus.

FASEB J, 2003 Jun, 17(9), 1135 - 7 Epub 2003 Apr 22.
Silencing of ubiquinone biosynthesis genes extends life span in Caenorhabditis elegans; Asencio C et al.; Ubiquinone (coenzyme Q; Q) is a key factor in the mitochondria electron transport chain, but it also functions as an antioxidant and as a cofactor of mitochondrial uncoupling proteins . Furthermore, Q isoforms balance in Caenorhabiditis elegans is determined by both dietary intake and endogenous biosynthesis . In the absence of synthesis, withdrawal of dietary Q8 in adulthood extends life span . Thus, Q plays an important role in the aging process and understanding its synthesis acquires a new impetus . We have identified by RNA interference (RNAi) eight genes, including clk-1, involved in ubiquinone biosynthesis in C . elegans feeding animals with dsRNA-containing Escherichia coli HT115 strains . Silenced C . elegans showed lower levels of both endogenous Q9 and Q8 provided by diet, produced less superoxide without a significant modification of mitochondrial electron chain, and extended life span compared with non-interfered animals . E . coli strains harboring dsRNA also interfered with their own Q8 biosynthesis . These findings suggest that more efficient electron transport between a lower amount of Q and electron transport capacity of the mitochondrial complexes leads to less production of reactive oxygen species that contributes to extension of life span in the nematode C . elegans.

Eur J Biochem, 2003 May, 270(9), 2101 - 7
Expression, purification and characterization of the second Kunitz-type protease inhibitor domain of the human WFIKKN protein; Nagy A et al.; Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of protease inhibitory modules, including two tandem Kunitz-type protease inhibitor-domains . On the basis of its homologies we have suggested that the WFIKKN protein is a multivalent protease inhibitor that may control the action of different proteases . In the present work we have expressed the second Kunitz-type protease inhibitor domain of the human protein WFIKKN in Escherichia coli, purified it by affinity chromatography on trypsin-Sepharose and its structure was characterized by CD spectroscopy . The recombinant protein was found to inhibit trypsin (Ki = 9.6 nm), but chymotrypsin, elastase, plasmin, pancreatic kallikrein, lung tryptase, plasma kallikrein, thrombin, urokinase or tissue plasminogen activator were not inhibited by the recombinant protein even at 1 microm concentration . In view of the marked trypsin-specificity of the inhibitor it is suggested that its physiological target may be trypsin.

Eur J Biochem, 2003 May, 270(9), 1958 - 68
The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa alphabeta heterodimer in an undissociated form; Fernandez FJ et al.; The isopenicillin N acyltransferases (IATs) of Aspergillus nidulans and Penicillium chrysogenum differed in their ability to maintain the 40-kDa proacyltransferase alphabeta heterodimer in an undissociated form . The native A . nidulans IAT exhibited a molecular mass of 40 kDa by gel filtration . The P . chrysogenum IAT showed a molecular mass of 29 kDa by gel filtration (corresponding to the beta subunit of the enzyme) but the undissociated 40-kDa heterodimer was never observed even in crude extracts . Heterologous expression experiments showed that the chromatographic behaviour of IAT was determined by the source of the penDE gene used in the expression experiments and not by the host itself . When the penDE gene of A . nidulans was expressed in P . chrysogenum npe6 and npe8 or in Acremonium chrysogenum, the IAT formed had a molecular mass of 40 kDa . On the other hand, when the penDE gene originating from P . chrysogenum was expressed in A . chrysogenum, the active IAT had a molecular mass of 29 kDa . The intronless form of the penDE gene cloned from an A . nidulans cDNA library and overexpressed in Escherichia coli formed the enzymatically active 40-kDa proIAT, which was not self-processed as shown by immunoblotting with antibodies to IAT . This 40-kDa protein remained unprocessed even when treated with A . nidulans crude extract . In contrast, the P . chrysogenum penDE intronless gene cloned from a cDNA library was expressed in E . coli, and the IAT was self-processed efficiently into its alpha (29 kDa) and beta (11 kDa) subunits . It is concluded that P . chrysogenum and A . nidulans differ in their ability to self-process their respective proIAT protein and to maintain the alpha and beta subunits as an undissociated heterodimer, probably because of the amino-acid sequence differences in the proIAT which affect the autocatalytic activity.

Allergy, 2003 Apr, 58(4), 352 - 6
Cloning of a group 3 allergen from Blomia tropicalis mites; Cheong N et al.; BACKGROUND: Blomia tropicalis is an important mite species in the tropics and subtropical regions of the world . It is well established that the allergen from this species of mite is one of the triggering factors for allergic asthma . The isolation and characterization of allergens in this mite species is desired to provide sensitive and specific reagents for diagnostic as well as therapeutic purposes . METHODS: The SMART (Clontech Laboratories, Palo Alto, CA, USA) rapid amplification of complementary DNA ends (RACE cDNA amplification) method was used to isolate the putative Blo t 3 gene . Polymerase chain reactions (PCR) were performed in the presence of specific gene primers to obtain the full-length gene, and were confirmed by DNA sequencing . The putative gene was cloned into E . coli expression vector GST-4T-1 and expressed as a fusion protein with glutathione-S-transferase (GST) . The allergenicity of the GST-Blo t 3 recombinant protein was evaluated by human IgE enzyme-linked immunoassay (ELISA) and skin pricks tests . RESULTS: The full length Blo t 3 gene had 1138 base pairs, including a 105-bp long 5' nontranslated region, an ATG start codon at positions 106-108, and a stop codon TAA at positions 904-906, with an open reading frame coding for a polypeptide of 266 amino acids . Protein analysis revealed that it was a serine protease that had a prepro-mature structure that shared high sequence homology with group 3 dust mite allergens . The predicted molecular weight of the matured protein was approximately 23.8 kD with a theoretical pI of 8.87 . The frequency of IgE reactivity of the recombinant protein showed up to 50% of IgE reactivity with mite allergic subjects but IgE titer was generally low . CONCLUSION: We had isolated and fully characterized the cDNA encoding an important B . tropicalis allergen that was highly homologous to Group 3 dust mite allergens and we proposed that it should be designated as Blo t 3 . Its clinical importance was implicated by the high frequency of IgE reactivity with allergic sera.

J Mol Model (Online), 2003 Apr, 9(2), 88 - 98 Epub 2003 Mar 01.
Computational docking of L-arginine and its structural analogues to C-terminal domain of Escherichia coli arginine repressor protein (ArgRc); Kueh R et al.; The arginine repressor (ArgR) of Escherichia coli binds to six L-arginine molecules that act as its co-repressor in order to bind to DNA . The binding of L-arginine molecules as well as its structural analogues is compared by means of computational docking . A grid-based energy evaluation method combined with a Monte Carlo simulated annealing process was used in the automated docking . For all ligands, the docking procedure proposed more than one binding site in the C-terminal domain of ArgR (ArgRc) . Interaction patterns of ArgRc with L-arginine were also observed for L-canavanine and L-citrulline . L-lysine and L-homoarginine, on the other hand, were shown to bind poorly at the binding site . Figure A general overview of the sites found from docking the various ligands into ArgRc ( grey ribbons) . Red coloured sticks: residues in binding site H that was selected for docking

J Ind Microbiol Biotechnol, 2003 Aug, 30(8), 448 - 55 Epub 2003 Apr 18.
Polyketide-nonribosomal peptide epothilone antitumor agents: the EpoA, B, C subunits; Walsh CT et al.; The epothilones are a family of macrolactone natural products from the myxobacterial species Sorangium cellulosum . Similar to taxol, they are of current clinical interest as anticancer agents . Sequence analysis of the epothilone gene cluster allowed the identification of polyketide synthase and nonribosomal peptide synthetase modules involved in catalyzing epothilone biosynthesis . Given this information, it has been possible to test the predicted functions of several modules to date . EpoA ACP, EpoB, and EpoC have been overproduced in Escherichia coli, allowing in vitro reconstitution of the EpoA/B/C interface and production of the expected epothilone precursor . Further experiments probed the tolerance of EpoB and EpoC for unnatural substrates . These studies of the first three modules of the epothilone biosynthetic cluster suggest that combinatorial biosynthesis may lead to the production of a variety of epothilone analogs that incorporate diversity into the heterocycle starter unit . Additional efforts with the remaining modules, coupled with increased understanding of the macrocyclizing thioesterase domain, may lead to the production of epothilone variants with improved clinical properties.

Chromosoma, 2003 Apr, 111(7), 461 - 9 Epub 2003 Feb 26.
Deletion and site-specific mutagenesis of nucleolin's carboxy GAR domain; Pellar GJ et al.; Vertebrate nucleolin is an abundant RNA-binding protein in the dense fibrillar component of active nucleoli . Nucleolin is modular in composition . Its amino-terminal third contains alternating acidic and basic domains, its middle section contains four consensus RNA-binding domains (cRBDs), and its carboxy-terminus contains a distinctive glycine/arginine-rich (GAR) domain with several RGG motifs . The arginines within these motifs are asymmetrically dimethylated . Several laboratories have shown that the GAR domain is necessary but not sufficient for the efficient localization of nucleolin to nucleoli . We examined the distribution of endogenous fibrillarin, Nopp140, and B23 when full-length and DeltaGAR nucleolin were expressed exogenously as enhanced green fluorescent protein (EGFP)-tagged fusions . Only B23 redistributed when DeltaGAR-EGFP was expressed at moderate to high levels, suggesting an in vivo interaction between nucleolin and B23 . Next we substituted all ten arginines within the GAR domain of Chinese hamster ovary (CHO) nucleolin with lysines to test the hypothesis that methylation of the carboxy GAR domain is necessary for the nucleolar association of nucleolin . The lysine-substituted mutant was not an in vitro substrate for the yeast protein methyltransferase, Hmt1p/Rmt1 . It was, however, able to associate properly with interphase nucleoli and with interphase pre-nucleolar bodies upon recovery from hypotonic shock . We conclude, therefore, that although the GAR domain is necessary for the efficient localization of nucleolin to nucleoli, methylation of this domain is not required for proper nucleolar localization.

J Biol Chem, 2003 Jul 11, 278(28), 25919 - 25 Epub 2003 Apr 21.
Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei: photoreduction of the redox disulfide using synchrotron radiation and evidence for a conformational switch implicated in function; Alphey MS et al.; Tryparedoxin (TryX) is a member of the thioredoxin (TrX) fold family involved in the regulation of oxidative stress in parasitic trypanosomatids . Like TrX, TryX carries a characteristic Trp-Cys-Xaa-Xaa-Cys motif, which positions a redox-active disulfide underneath a tryptophan lid . We report the structure of a Crithidia fasciculata tryparedoxin isoform (CfTryX2) in two crystal forms and compare them with structures determined previously . Efforts to chemically generate crystals of reduced TryX1 were unsuccessful, and we carried out a novel experiment to break the redox-active disulfide, formed between Cys-40 and Cys-43, utilizing the intense x-radiation from a third generation synchrotron undulator beamline . A time course study of the S-S bond cleavage is reported with the structure of a TryX1 C43A mutant as the control . When freed from the constraints of a disulfide link to Cys-43, Cys-40 pivots to become slightly more solvent-accessible . In addition, we have determined the structure of Trypanosoma brucei TryX, which, influenced by the molecular packing in the crystal lattice, displays a significantly different orientation of the active site tryptophan lid . This structural change may be of functional significance when TryX interacts with tryparedoxin peroxidase, the final protein in the trypanothione-dependent peroxidase pathway . Comparisons with chloroplast TrX and its substrate fructose 1,6-bisphosphate phosphatase suggest that this movement may represent a general feature of redox regulation in the trypanothione and thioredoxin peroxidase pathways.

J Biol Chem, 2003 Jun 27, 278(26), 23497 - 501 Epub 2003 Apr 21.
Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution; Boulanger RR Jr et al.; Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure . Each monomer contains an active site located 32 A away from the active site in the second subunit . Indirect evidence has previously suggested that the monomeric form of AP is inactive . Molecular modeling studies indicated that destabilization of the dimeric interface should occur if Thr-59, located near the 2-fold axis of symmetry, were replaced by a sterically large and charged residue such as arginine . The T59R enzyme was constructed and characterized by sucrose-density gradient sedimentation, size-exclusion chromatography, and circular dichroism (CD) and compared with the previously constructed T59A enzyme . The T59A enzyme was found to exist as a dimer, whereas the T59R enzyme was found to exist as a monomer . The T59A, T59R, and wild-type APs exhibited almost identical secondary structures as judged by CD . The T59R monomeric AP has a melting temperature (Tm) of 43 degrees C, whereas the wild-type AP dimer has a Tm of 97 degrees C . The catalytic activity of the T59R enzyme was reduced by 104-fold, whereas the T59A enzyme exhibited an activity similar to that of the wild-type enzyme . The T59A and wild-type enzymes contained similar levels of zinc and magnesium, whereas the T59R enzyme has almost undetectable amounts of tightly bound metals . These results suggest that a significant conformational change occurs upon dimerization, which enhances thermal stability, metal binding, and catalysis.

J Biol Chem, 2003 Jun 27, 278(26), 24174 - 80 Epub 2003 Apr 21.
Characterization of novel hexadecameric thioredoxin peroxidase from Aeropyrum pernix K1; Jeon SJ et al.; A gene (APE2278) encoding the peroxiredoxin (Prx) homologous protein of yeast and human was identified in the genome data base of the aerobic hyperthermophilic archaeon Aeropyrum pernix . We cloned the gene and produced the encoded protein in Escherichia coli cells . The isolated recombinant protein showed peroxidase activity in vitro and used the thioredoxin system of A . pernix as an electron donor . These results indicate that the recombinant protein is in fact thioredoxin peroxidase (ApTPx) of A . pernix . Immunoblot analysis revealed that the expression of ApTPx was induced as a cellular adaptation in response to the addition of exogenous H2O2 and may exert an antioxidant activity in vivo . An analysis of the ApTPx oligomers by high pressure liquid chromatography and electron microscopic studies showed that ApTPx exhibited the hexadecameric protein forming 2-fold toroid-shaped structure with outer and inner diameters of 14 and 6 nm, respectively . These results indicated that ApTPx is a novel hexadecameric protein composed of two identical octamers . Although oligomerization of individual subunits does not take place through an intersubunit-disulfide linkage involving Cys50 and Cys213, Cys50 is essential for the formation of the hexadecamer . Mutagenesis studies suggest that the sulfhydryl group of Cys50 is the site of oxidation by peroxide and that oxidized Cys50 reacts with the sulfhydryl group of Cys213 of another subunit to form an intermolecular disulfide bond . The resulting disulfide can then be reduced by thioredoxin . In support of this hypothesis, ApTPx mutants lacking either Cys50 or Cys213 showed no TPx activity, whereas the mutant lacking Cys207 had a TPx activity . This is the first report on the biochemical and structural features of a novel hexadecameric thioredoxin peroxidase from the archaea.

J Biol Chem, 2003 Jun 27, 278(26), 23295 - 300 Epub 2003 Apr 21.
Conditional lethal mutations separate the M13 procoat and Pf3 coat functions of YidC: different YIDC structural requirements for membrane protein insertion; Chen M et al.; Conditional lethal YidC mutants have been isolated to decipher the role of YidC in the assembly of Sec-dependent and Sec-independent membrane proteins . We now show that the membrane insertion of the Sec-independent M13 procoat-lep protein is inhibited in a short time in a temperature-sensitive mutant when shifted to the nonpermissive temperature . This provides an additional line of evidence that YidC plays a direct role in the insertion of the Sec-independent M13 procoat protein . However, in the temperature-sensitive mutant, the insertion of the Sec-independent Pf3 phage coat protein and the Sec-dependent leader peptidase were not strongly inhibited at the restricted temperatures . Conversely, using a cold-sensitive YidC strain, we find that the membrane insertion of the Sec-independent Pf3 coat protein is blocked, and the Sec-dependent leader peptidase is inhibited at the nonpermissive temperature, whereas the insertion of the M13 procoat protein is nearly normal . These data show that the YidC function for procoat and its function for Pf3 coat and possibly leader peptidase are genetically separable and suggest that the YidC structural requirements are different for the Sec-independent M13 procoat and Pf3 coat phage proteins that insert by different mechanisms.

Trends Microbiol, 2003 Apr, 11(4), 152 - 5
A new turn in Rho GTPase activation by Escherichia coli cytotoxic necrotizing factors; Aktories K et al.; Various bacterial protein toxins and effectors target Rho GTPases, which are eukaryotic regulators of signal transduction pathways . Many toxins inactivate these GTPases but some, such as the cytotoxic necrotizing factors (CNFs) from Escherichia coli, activate them by deamidation . Recent studies have provided exciting new clues to the functional consequences of the activation of Rho GTPases by CNFs and its effect on the host-pathogen interaction.

FEBS Lett, 2003 Apr 24, 541(1-3), 115 - 20
Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a {4Fe-4S} protein; Wolff M et al.; The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate . This enzyme possesses a dioxygen-sensitive {4Fe-4S} cluster . This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein . Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.

FEBS Lett, 2003 Apr 24, 541(1-3), 97 - 101
DNA target sequence and FNR-dependent gene expression; Scott C et al.; FNR proteins are global transcription regulators that respond to fluctuations in environmental oxygen . They recognise a DNA target consisting of an inverted repeat, TTGATN(1)N(2)N(3)N(4)ATCAA (where N(1-4) represents a non-conserved tetrad, NCT) . Analysis of 68 known and predicted FNR sites from the Escherichia coli K12 genome revealed a bias toward A or T at positions N(2) and N(3) of the NCT . The effect of the NCT sequence on FNR-dependent transcription in vivo was assessed using a series of class II and class I model promoters with different NCT sequences . Changing the NCT sequence did not affect basal activity but altered anaerobic induction by as much as an order of magnitude . Thus, the NCT sequence is a fundamental component in setting the dynamic range of the FNR switch.

FEBS Lett, 2003 Apr 24, 541(1-3), 16 - 20
Cytotoxicity of ribosome-inactivating protein saporin is not mediated through alpha2-macroglobulin receptor; Bagga S et al.; Saporin is a single chain ribosome-inactivating protein produced by the plant Saponaria officinalis . Several isoforms of saporin have been isolated from various parts of the plant . In the present study recombinant saporin isoforms 5 and 6 were produced in Escherichia coli . Saporin-6 was found to be more active than saporin-5 in its N-glycosidase, cytotoxic, and genomic DNA fragmentation activities . Earlier, saporin has been shown to bind low-density lipoprotein receptor-related protein (LRP), however, in this study the sensitivities of LRP-negative and LRP-positive cell lines were found to be similar towards saporin-6 toxicity suggesting the internalization of saporin not to be solely dependent on the expression of LRP on eukaryotic cells.

What Is Antibiotic?, What Is Nitrification?, What Is Yeast?, What Is Listeria Monocytogenes?, What Is Growth Medium?, s, Microbiology, a, Bacterium, o, Microorganism, s, Bacteria, i, Bacteriology, a, Salmonella typhimurium, n, Antimicrobial, o, Antibiotic prophylaxis, n, Escherichia coli, s, Microorganism, n, Antimicrobial, e, Bacteria, o, Bioremediation, i, Antibiotics, i, Minimum inhibiting concentration, r, Rhizobacter, r, Escherichia coli, a, Culture medium, c, Halophilic bacterium, i, Salmonella, o, Microbial, c, Beta lactamase, o, Antibiotic prophylaxis, i, Escherichia coli, a, Escherichia coli, r, Yeasts

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005