Eur J Biochem, 2002 Apr, 269(8), 2257 - 64 Effect of coenzymes and thyroid hormones on the dual activities of Xenopus cytosolic thyroid-hormone-binding protein (xCTBP) with aldehyde dehydrogenase activity; Yamauchi K et al.; A cytosolic thyroid-hormone-binding protein (xCTBP), predominantly responsible for the major binding activity of T3 in the cytosol of Xenopus liver, has been shown to be identical to aldehyde dehydrogenase class 1 (ALDH1) {Yamauchi, K., Nakajima, J., Hayashi, H., Horiuchi, R . & Tata, J.R . (1999) J . Biol . Chem . 274, 8460-8469} . Within this paper we surveyed which signaling, and other, compounds affect the thyroid hormone binding activity and aldehyde dehydrogenase activity of recombinant Xenopus ALDH1 (xCTBP/xALDH1) while examining the relationship between these two activities . NAD+ and NADH (each 200 microm), and two steroids (20 microm), inhibit significantly the T3-binding activity, while NADH and NADPH (each 200 microm), and iodothyronines (1 microm), inhibit the ALDH activity . Scatchard analysis and kinetic studies of xCTBP/xALDH1 indicate that NAD+ and T3 are noncompetitive inhibitors of thyroid-hormone-binding and ALDH activities, respectively . These results indicate the formation of a ternary complex consisting of the protein, NAD+ and thyroid hormone . Although the in vitro studies indicate that NAD+ and NADH markedly decrease T3-binding to xCTBP/xALDH1 at approximately 10-4 m, a concentration equal to the NAD content in various Xenopus tissues, photoaffinity-labeling of {125I}T3 using cultured Xenopus cells demonstrates xCTBP/xALDH1 bound T3 within living cells . These results raise the possibility that an unknown factor(s) besides NAD+ and NADH may modulate the thyroid-hormone-binding activity of xCTBP/xALDH1 . In comparison, thyroid hormone, at its physiological concentration, would poorly modulate the enzyme activity of xCTBP/xALDH1. Eur J Biochem, 2002 Apr, 269(8), 2186 - 93 Soluble guanylate cyclase is allosterically inhibited by direct interaction with 2-substituted adenine nucleotides; Ruiz-Stewart I et al.; Nitric oxide (NO), the principal endogenous ligand for soluble guanylate cyclase (sGC), stimulates that enzyme and accumulation of intracellular cGMP, which mediates many of the (patho) physiological effects of NO . Previous studies demonstrated that 2-substituted adenine nucleotides, including 2-methylthioATP (2MeSATP) and 2-chloroATP (2ClATP), allosterically inhibit guanylate cyclase C, the membrane-bound receptor for the Escherichia coli heat-stable enterotoxin in the intestine . The present study examined the effects of 2-substituted adenine nucleotides on crude and purified sGC . 2-Substituted nucleotides inhibited basal and NO-activated crude and purified sGC, when Mg2+ served as the substrate cation cofactor . Similarly, 2-substituted adenine nucleotides inhibited those enzymes when Mn2+, which activates sGC in a ligand-independent fashion, served as the substrate cation cofactor . Inhibition of sGC by 2-substituted nucleotides was associated with a decrease in Vmax, consistent with a noncompetitive mechanism . In contrast to guanylate cyclase C, 2-substituted nucleotides inhibited sGC by a guanine nucleotide-independent mechanism . These studies demonstrate that 2-substituted adenine nucleotides allosterically inhibit basal and ligand-stimulated sGC . They support the suggestion that allosteric inhibition by adenine nucleotides is a general characteristic of the family of guanylate cyclases . This allosteric inhibition is mediated by direct interaction of adenine nucleotides with sGC, likely at the catalytic domain in a region outside the substrate-binding site. Eur J Biochem, 2002 Apr, 269(8), 2178 - 85 Activation of a covalent outer membrane phospholipase A dimer; Kingma RL et al.; The activity of outer membrane phospholipase A (OMPLA) is regulated by reversible dimerization . However, native OMPLA reconstituted in phospholipid vesicles was found to be present as a dimer but nevertheless inactive . To investigate the importance of dimerization for control of OMPLA activity, a covalent OMPLA dimer was constructed and its properties were compared to native OMPLA both in a micellar detergent and after reconstitution in a phospholipid bilayer . Unlike native OMPLA, activity of the covalent OMPLA dimer was independent of type and concentration of detergent in micellar systems . In such systems, the covalent OMPLA dimer invariantly displayed high calcium affinity . In contrast, high calcium concentrations were required to activate a covalent OMPLA dimer when present in intact vesicles . Solubilization of the vesicles increased the affinity for calcium, suggesting that in an intact bilayer the dimer interface is not properly formed . This was supported by the observation that OMPLA variants having an impaired dimeric interface also lacked high affinity calcium binding . A covalent linkage was not able to restore high affinity calcium binding in these variants, demonstrating that a proper dimer interface is essential for optimal catalysis. Eur J Biochem, 2002 Apr, 269(8), 2075 - 82 Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system; Kitazume T et al.; Cytochrome P450foxy (P450foxy, CYP505) is a fused protein of cytochrome P450 (P450) and its reductase isolated from the fungus Fusarium oxysporum, which catalyzes the subterminal (omega-1 approximately omega-3) hydroxylation of fatty acids . Here, we produced, purified and characterized a fused recombinant protein (rP450foxy) using the Escherichia coli expression system . Purified rP450foxy was catalytically and spectrally indistinguishable from the native protein, but most of the rP450foxy was recovered in the soluble fraction of E . coli cells unlike the membrane-bound native protein . The results are consistent with our notion that the native protein is targeted to the membrane by a post-translational modification mechanism . We also discovered that P450foxy could use shorter saturated fatty acid chains (C9 and C10) as a substrate . The regiospecificity (omega-1 approximately omega-3) of hydroxylation due to the enzymatic reaction for the short substrates (decanoate, C10; undecanoate, C11) was the same as that for longer substrates . Steady state kinetic studies showed that the kcat values for all substrates tested (C9-C16) were of the same magnitude (1200-1800 min-1), whereas the catalytic efficiency (kcat/Km) was higher for longer fatty acids . Substrate inhibition was observed with fatty acid substrates longer than C13, and the degree of inhibition increased with increasing chain length . This substrate inhibition was not apparent with P450BM3, a bacterial counterpart of P450foxy, which was the first obvious difference in their catalytic properties to be identified . Kinetic data were consistent with the inhibition due to binding of the second substrate . We discuss the inhibition mechanism based on differences between P450foxy and P450BM3 in key amino acid residues for substrate binding. Biochem J, 2002 Aug 1, 365(Pt 3), 809 - 16 Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue; Saysell CG et al.; Copper amine oxidases are homodimeric enzymes containing one Cu(2+) ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer . Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383 . To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N . The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5-9.4 . For the wild-type enzyme, the plot of the binding constant for adduct formation (K(b)) against pH is bell-shaped, indicating two pK(a)s of 5.8 and approximately 8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP . For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant . Surprisingly, for the D383E variant, the K(b) versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP . The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics . The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event. Mol Reprod Dev, 2002 Jun, 62(2), 233 - 47 A novel asparaginase-like protein is a sperm autoantigen in rats; Bush LA et al.; A novel asparaginase-like protein (ALP) of spermatozoa was cloned from rat and human testis cDNA libraries on the basis of reactivity with antibodies produced after vasectomy . Although obstruction of the male reproductive tract is known to cause an immunologic response, few of the sperm antigens responsible for the generation of autoantibodies have been characterized . We are identifying proteins of interest by coring autoantigenic protein spots from two-dimensional (2-D) gels of rat sperm extracts and microsequencing them by mass spectrometry . The peptide sequences from ALP, a 28 kDa, pI 5.7 protein, matched to a single partial length rat EST . These peptide sequences were used to clone a cDNA encoding a novel 333 amino acid open reading frame . The new protein had a similarity to portions of L-asparaginases of plants (43%) and to glycosylasparaginases in animal cells (32%) . Human ALP cDNA was subsequently cloned . It showed 77% identity to the rat ALP sequence and the gene, ASRGL1 (asparaginase-like 1), mapped to chromosome locus 11q12.3 . Purified recombinant rat ALP (rALP), expressed in E . coli, was used to raise polyclonal antiserum in guinea pigs . Two observations verified that the correct protein had been cloned: 1) the anti-rALP antibody reacted with both rALP and rat sperm; and 2) post-vasectomy sera bound rALP . Anti-rALP antibody stained the midpiece of rat and human sperm coincident with staining by MitoTracker Green FM, suggesting that ALP is associated with the mitochondria . Northern analysis revealed that rat ALP message was abundantly expressed in the testis but was also present in heart, brain, liver, skeletal muscle, and kidney . Mol Med, 2002 Jan, 8(1), 16 - 23 Association of severe noncerebral Plasmodium falciparum malaria in Brazil with expressed PfEMP1 DBL1 alpha sequences lacking cysteine residues; Kirchgatter K et al.; BACKGROUND: Cytoadherence and rosetting contribute to the development of severe Plasmodium falciparum malaria . In Brazil,severe falciparum malaria is mostly associated with renal or pulmonary complications and very rarely with cerebral malaria . The most N-terminal DBL1 alpha domain of PfEMP1, a protein encoded by the var multigene family mediates rosetting . We analyzed parasites of Brazilian patients with severe malaria to determine whether there were particular DBL1 alpha var sequences predominantly expressed in such patients . MATERIALS AND METHODS: DBL1 alpha var sequences were obtained from parasites of Brazilian patients with severe and mild malaria and were analyzed by standard bioinformatic programs . Three hundred twenty var DBL1 alpha sequences obtained from 80 Brazilian patients with mild malaria were spotted in high-density filters and hybridized to probes representing predominantly expressed sequences in parasites from patients with severe malaria . A DBL1 alpha domain was expressed in bacteria and used to demonstrate its binding capacity to erythrocytes by immunofluorescence . RESULTS: Forty-three different and unreported DBL1 alpha amino acid sequences were obtained . Sequences predominantly expressed in patients with severe malaria could be subgrouped due to deletions of 1-2-cysteine residues . These sequences were commonly found in the var gene repertoire of parasites from patients with mild malaria, yet they were rarely expressed in these patients . A recombinant protein representing the most abundantly expressed sequence detected in one patient with severe malaria bound directly to uninfected erythrocytes . CONCLUSIONS: This is the first report showing an association of severe noncerebral malaria from Brazil with particular DBL1 alpha sequences. Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 5965 - 70 Modulation of tRNAAla identity by inorganic pyrophosphatase; Wolfson AD et al.; A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3'-(32)P-labeled tRNA . When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA(Ala) . The effect of tRNA(Ala) identity mutations on both aminoacylation efficiency (k(cat)/K(M)) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu . Significant levels of aminoacylation were achieved for tRNA mutants even when the k(cat)/K(M) value is reduced by as much as several thousandfold . These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA(Ala) identity. Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 5884 - 9 Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei; Roper JR et al.; The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease Nagana . The bloodstream form of the parasite uses a dense cell-surface coat of variant surface glycoprotein to escape the innate and adaptive immune responses of the mammalian host and a highly glycosylated transferrin receptor to take up host transferrin, an essential growth factor . These glycoproteins, as well as other flagellar pocket, endosomal, and lysosomal glycoproteins, are known to contain galactose . The parasite is unable to take up galactose, suggesting that it may depend on the action of UDP-glucose 4'-epimerase for the conversion of UDP-Glc to UDP-Gal and subsequent incorporation of galactose into glycoconjugates via UDP-Gal-dependent galactosyltransferases . In this paper, we describe the cloning of T . brucei galE, encoding T . brucei UDP-Glc-4'-epimerase, and functional characterization by complementation of a galE-deficient Escherichia coli mutant and enzymatic assay of recombinant protein . A tetracycline-inducible conditional galE null mutant of T . brucei was created using a transgenic parasite expressing the TETR tetracycline repressor protein gene . Withdrawal of tetracycline led to a cessation of cell division and substantial cell death, demonstrating that galactose metabolism in T . brucei proceeds via UDP-Glc-4'-epimerase and is essential for parasite growth . After several days without tetracycline, cultures spontaneously recovered . These cells were shown to have undergone a genetic rearrangement that deleted the TETR gene . The results show that enzymes and transporters involved in galactose metabolism may be considered as potential therapeutic targets against African trypanosomiasis. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6761 - 6 Epub 2002 Apr 30. Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE; Hu Z et al.; Selection of the division site in Escherichia coli is regulated by the min system and requires the rapid oscillation of MinD between the two halves of the cell under the control of MinE . In this study we have further investigated the molecular basis for this oscillation by examining the interaction of MinD with phospholipid vesicles . We found that MinD bound to phospholipid vesicles in the presence of ATP and, upon binding, assembled into a well-ordered helical array that deformed the vesicles into tubes . Stimulation of the MinD ATPase by addition of MinE led to disassembly of the tubes and the release of MinD from the vesicles . It is proposed that this MinE-regulated dynamic assembly of MinD underlies MinD oscillation. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 7060 - 5 Epub 2002 Apr 30. Collaborative signaling by mixed chemoreceptor teams in Escherichia coli; Ames P et al.; Chemoreceptors of the methyl-accepting chemotaxis protein family form clusters, typically at the cell pole(s), in both Bacteria and Archaea . To elucidate the architecture and signaling role of receptor clusters, we investigated interactions between the serine (Tsr) and aspartate (Tar) chemoreceptors in Escherichia coli by constructing Tsr mutations at the six hydrophobic and five polar residues implicated in "trimer of dimers" formation . Tsr mutants with proline replacements could not mediate serine chemotaxis, receptor clustering, or clockwise flagellar rotation . Alanine and tryptophan mutants, although also nonchemotactic, formed receptor clusters, and some produced clockwise flagellar rotation, indicating receptor-coupled activation of the signaling CheA kinase . The alanine and tryptophan mutants evidently assemble defective receptor complexes that cannot modulate CheA activity in response to serine stimuli . In cells containing wild-type Tar receptors, tryptophan replacements in Tsr interfered with Tar function, whereas four Tsr mutants with alanine replacements regained Tsr function . These epistatic and rescuable phenotypes imply interactions between Tsr and Tar dimers in higher-order signaling teams . The bulky side chain in tryptophan mutants may prevent stimulus-induced conformational changes in the team, whereas the small side chain in alanine mutants may permit signaling control when teamed with functional receptor molecules . Direct physical interactions between Tsr and Tar molecules were observed by in vivo chemical crosslinking . Wild-type Tsr crosslinked to Tar, whereas a clustering-defective proline replacement mutant did not . These findings indicate that bacterial chemoreceptor clusters are comprised of signaling teams, seemingly based on trimers of dimers, that can contain different receptor types acting collaboratively. J Biol Chem, 2002 Jul 12, 277(28), 25090 - 5 Epub 2002 Apr 30. Identification of the tRNA-dihydrouridine synthase family; Bishop AC et al.; 5,6-Dihydrouridine (D) is a modified base found abundantly in the D-loops of tRNA from Archaea, Bacteria, and Eukarya . D is thought to be formed post-transcriptionally by the reduction of uridines in tRNA transcripts . Despite its abundance, no enzymes that catalyze D-formation have been identified . Using comparative genomics and computational methods we have identified members of the cluster of orthologous genes, COG0042, as putative dihydrouridine synthase encoding genes . Escherichia coli contains three COG0042 family members (yjbN, yhdG, and yohI) . Strains were created where one, two, or all three of the COG0042 genes were deleted . Purified tRNA samples were investigated from the three single and the three double knockout strains, as well as from the triple deletion strain . The results showed that the COG0042 gene family is responsible for tRNA-dihydrouridine synthase activity in E . coli . They also suggest that the COG0042-encoded family members act site-specifically on the tRNA D-loop and contain non-redundant catalytic functions in vivo. J Biol Chem, 2002 Jul 5, 277(27), 24289 - 93 Epub 2002 Apr 30. A monomeric L-aspartase obtained by in vitro selection; Kong X et al.; By mimicking the partial spatial structure of the dimer of the l-aspartase subunit, the central ten-helix bundle, and an "active site" between the cleft of domain 1 (D1) and domain 3 (D3) from different subunits, we designed l-aspartase variants, in which D1D2 and D2D3 were ligated with a random hexapeptide loop . As expected, we obtained the variant with the highest activity (relative activity is 21.3% of the native enzyme, named as drAsp017) by in vitro selection . The molecular weight of this variant, obtained from size-exclusion column chromatography, is about 81 kDa, which indicates that it is indeed a monomer, whereas native l-aspartase is a tetramer . The activity-reversibility of drAsp017 (10(-7) m) was 80% after incubation for 30 min at 50 degrees C, while native enzyme only retained about 17% under the same conditions . Reactivation of drAsp017 denatured in 4 m guanidine HCl was independent of protein concentration at up to 20 x 10(-8) m at 25 degrees C, whereas the protein concentration of native enzyme strongly affected its reactivation under the above conditions . The sensitivity of drAsp017 (10(-7) m) to effective factors in the fumarate-amination reaction compared with native enzyme was also determined . Half-saturating concentrations of the activator l-aspartate and Mg2+ for drAsp017 (0.8 and 0.5 mm, respectively) are much higher than that of the native enzyme (0.10 and 0.15 mm, respectively) . The data show that a monomeric l-aspartase is obtained by in vitro selection . Thus, the conversion of oligomeric proteins into their functional monomers could have important applications. Biochim Biophys Acta, 2002 Apr 1, 1596(1), 95 - 107 Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor; Fong CC et al.; Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6) . Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate . In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine . Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer . The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram . The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined . Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4 . While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine . Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues . The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate . This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates . The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme. Biochim Biophys Acta, 2002 Apr 1, 1596(1), 1 - 5 Human phosphatidylcholine transfer protein: purification, crystallization and preliminary X-ray diffraction data; Chan WW et al.; We have expressed, purified and crystallized recombinant human phosphatidylcholine transfer protein (PC-TP) and selenomethionyl PC-TP bound to dilinoleoyl phosphatidylcholine . The biochemical properties of native and selenomethionyl PC-TP were indistinguishable, and the two proteins crystallized under similar conditions . Both native and selenomethionyl PC-TP crystallized in two distinct space groups and diffracted X-rays to 2.4 A resolution. J Immunol Methods, 2002 Apr 1, 262(1-2), 41 - 51 Quantitation of secretory group V phospholipase A(2) in human tissues by sandwich enzyme-linked immunosorbent assay; Munoz NM et al.; We have developed a sensitive sandwich ELISA (sELISA) for quantitative determination of group V phospholipase A(2) (gVPLA(2)) . This assay utilizes three monoclonal antibodies (mAbs) directed against human gVPLA(2) (MCL-1B7, MCL-2A5, and MCL-3G1), which recognize specifically different epitopes of gVPLA(2) . A mixture of MCL-1B7 and MCL-2A5 was used as the capture mAb, and MCL-3G1 as the detector mAb; purified human gVPLA(2) was used as the standard protein . The limit of detection of the sELISA is 2 ng/ml; the intra- and inter-coefficients of variation were 4.97+/-0.81% and 8.42+/-3.4% . The validity of the sELISA was assured by the recovery of exogenous recombinant gVPLA(2), which was 99.7% to 102%, and demonstration of noninterference of the gVPLA(2) assay by a high concentrations of other protein from murine lung and heart . To assess the usefulness of this sELISA for tissue measurements, the amount of gVPLA(2) in cultured human epithelial cells and isolated human eosinophils was determined . Total gVPLA(2) mass in epithelial cells was 2.83+/-0.33 ng/10(7) cells; gVPLA(2) was not detected in eosinophils . The presence of high concentration of gVPLA(2) in epithelial cells was confirmed by immunoprecipitation/Western blot analysis and by flow cytometry . This assay allows for convenient differentiation between the highly homologous 14-kDa secretory PLA(2)s, gVPLA(2), gIIaPLA(2), gIbPLA(2) and gXPLA(2), and accurate quantitation of gVPLA(2) in biological samples. Zhonghua Gan Zang Bing Za Zhi, 2002 Apr, 10(2), 113 - 5 {Expression and identification of specific autoantigens in autoimmune hepatitis}; Fan L et al.; OBJECTIVE: To express and identify soluble liver antigen (SLA) and cytochrome P-450 (CYP 2D6) . METHODS: SLA cDNA and CYP 2D6 cDNA were obtained from human liver tissue poly (A)+RNA by RT-PCR . The cDNAs were inserted into fusion expression vector PQE-30 site of BamH I and Hind III . SLA and CYP 2D6 were identified by the SDS-PAGE and Western blot . RESULTS: SDS-PAGE analysis showed that there was a very strong stained band at about 47 kd and 50 kd, respectively . The products could specifically band to anti-SLA or anti-CYP 2D6 autoantibodies . CONCLUSIONS: The clone and expression of SLA and CYP 2D6 provide useful substances for the diagnosis and research of pathogenesis on autoimmune hepatitis. Zhonghua Gan Zang Bing Za Zhi, 2002 Apr, 10(2), 93 - 5 {Expression of CD(14) protein in liver sinusoidal endothelial cells during endotoxemia}; Dai L et al.; OBJECTIVE: To observe the expression of CD(14) protein and CD(14) gene in liver sinusoidal endothelial cells (LSECs) of rats during endotoxemia and the role of CD(14) protein in the activation of lipopolysaccharide (LPS)-induced LSECs . METHODS: Wistar rat endotoxemia model was established by injection of a dose of LPS (5 mg/kg, Escherichia coli O111:B4) via the tail vein of the rats, then sacrificed immediately, at 3, 6, 12, and 24 h, respectively . LSECs were isolated from normal and LPS-injected rats by the in situ collagenase perfusion technique . The isolated LSECs were incubated with anti CD(14) polyclonal antibody, then followed by staining with goat anti-rabbit IgG conjugated fluorescein isothiocyanate (FITC) . The percentage and mean fluorescence intensity (MFI) of CD(14)-positive cells were detected by the flow cytometric analysis (FCM) . LSECs were collected to measure the expression of CD(14) mRNA by the in situ hybridization analysis . The isolated LSECs from normal rats were divided into two groups . Group of LPS: LSECs were induced with different concentration of LPS (0, 0.01 microg/ml, 1 microg/ml, 10 microg/ml, and 100 microg/ml) . Group of anti-CD(14) blockade: LSECs were pre-incubated for 30 min with CD(14) antibody before different concentrations of LPS were added . The supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)- alpha and interleukin (IL)-6 . RESULTS: In rats with endotoxemia, LSECs displayed a strong MFI distinct from that of control rats . The number of FITC-CD(14) positive LSECs was 54.32%, 65.83%, 85.61%, and 45.65% at 3, 6, 12, and 24 h, respectively, which increased markedly when compared to control rats (4.45%, P<0.01) . The expression of CD(14) mRNA in LSECs was stronger than that in control rats . The levels of TNF-alpha were significantly increased in group of LPS (54.49 +/- 6.02 pg/ml, 84.65 +/- 10.16 pg/ml, 206.54 +/- 23.55 pg/ml, 349.87 +/- 39.47 pg/ml, and 365.76 +/- 40.31 pg/ml) than those in group of anti-CD(14) blockade (55.93 +/- 6.95 pg/ml, 63.32 +/- 7.81 pg/ml, 85.34 +/- 9.72 pg/ml, 112.75 +/- 13.54 pg/ml, and 198.66 +/- 21.54 pg/ml) (P<0.01) . The levels of IL-6 also increased significantly in group of LPS (103.34 +/- 12.52 pg/ml, 187.39 +/- 20.31 pg/ml, 243.87 +/- 27.83 pg/ml, 289.51 +/- 30.15 pg/ml, and 298.53 +/- 31.94 pg/ml) than those in group of anti-CD(14) blockade (104.37 +/- 11.49 pg/ml, 125.02 +/- 13.58 pg/ml, 164.59 +/- 19.47 pg/ml, 183.47 +/- 20.17 pg/ml, and 221.76 +/- 26.43pg/ml) (P<0.01) . CONCLUSIONS: LSEC can synthesize CD(14) protein and express CD(14) gene during endotoxemia . Anti CD(14) antibody can inhibit the production of TNF-alpha and IL-6 in LSECs induced by LPS . The expression of CD(14) protein may take an important part in the activation of LSECs induced by LPS. J Biochem (Tokyo), 2002 May, 131(5), 713 - 9 Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli; Tokumoto U et al.; The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster . Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery . We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E . coli extracts with a Ni-affinity column . Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system . In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA . In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx . We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS . Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3 . Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters. J Biochem (Tokyo), 2002 May, 131(5), 705 - 12 Heterologous expression and catalytic properties of the C-terminal domain of starfish cdc25 dual-specificity phosphatase, a cell cycle regulator; Deshimaru S et al.; The 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli . The C-terminal domain consisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate . The enzyme activity was strongly inhibited by SH inhibitors . Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not . These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases . The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2.cyclin B complex (preMPF) from extracts of immature starfish oocytes . Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity . Phosphotyrosine and phosphothreonine underwent dephosphorylation in this order . This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF . Monophosphopeptides having the same sequence served as much poorer substrates . As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase. J Biochem (Tokyo), 2002 May, 131(5), 679 - 85 Structure of external aldimine of Escherichia coli CsdB, an IscS/NifS homolog: implications for its specificity toward selenocysteine; Mihara H et al.; Escherichia coli CsdB is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes both cysteine desulfuration and selenocysteine deselenation . The enzyme has a high specific activity for L-selenocysteine relative to L-cysteine . On the other hand, its paralog, IscS, exhibits higher activity for L-cysteine, which acts as a sulfur donor during the biosynthesis of the iron-sulfur cluster and 4-thiouridine . The structure of CsdB complexed with L-propargylglycine was determined by X-ray crystallography at 2.8 A resolution . The overall polypeptide fold of the complex is similar to that of the uncomplexed enzyme, indicating that no significant structural change occurs upon formation of the complex . In the complex, propargylglycine forms a Schiff base with PLP, providing the features of the external aldimine formed in the active site . The Cys364 residue, which is essential for the activity of CsdB toward L-cysteine but not toward L-selenocysteine, is clearly visible on a loop of the extended lobe (Thr362-Arg375) in all enzyme forms studied, in contrast to the corresponding disordered loop (Ser321-Arg332) of the Thermotoga maritima NifS-like protein, which is closely related to IscS . The extended lobe of CsdB has an 11-residue deletion compared with that of the NifS-like protein . These facts suggest that the restricted flexibility of the Cys364-anchoring extended lobe in CsdB may be responsible for the ability of the enzyme to discriminate between selenium and sulfur. J Biochem (Tokyo), 2002 May, 131(5), 671 - 7 Catalysis-linked inactivation of fluoroacetate dehalogenase by ammonia: a novel approach to probe the active-site environment; Ichiyama S et al.; Fluoroacetate dehalogenase from Moraxella sp . B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates . Asp(105) of the enzyme acts as a nucleophile to attack the alpha-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His(272) {Liu, J.Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N . (1998) J . Biol . Chem . 273, 30897-30902} . In this study, we found that FAc-DEX is inactivated concomitantly with defluorination of fluoroacetate by incubation with ammonia . Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp(105), into an asparagine residue . The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity . Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity . In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme . Basic residues probably play a role in counterbalancing the electronegativity of the substrate . These results demonstrate that catalysis-linked inactivation is useful for characterizing the active-site environment as well as for identifying the catalytic residue. Clin Exp Immunol, 2002 Apr, 128(1), 83 - 7 Analysis of mitochondrial antigens reveals inner membrane succinate dehydrogenase flavoprotein subunit as autoantigen to antibodies in anti-M7 sera; Cicek G et al.; The role of mitochondrial proteins as antigens to antibodies of anti-M7 sera was analysed by flavin fluorescence, one- and two-dimensional Western blots and blue native gel electrophoresis . Flavin fluorescence of succinate dehydrogenase (SucDH, complex II of the respiratory chain) of rat liver inner mitochondrial membranes correlated with the immunoreactivity of a representative anti-M7 myocarditis serum . Antigens of isolated bovine heart mitochondria reacting with antibodies of myocarditis serum on two-dimensional Western blots were identified by MALDI-TOF and NanoESI mass spectrometry as myosin heavy chain beta and as dihydrolipoamide dehydrogenase of the mitochondrial 2-oxoacid dehydrogenase complexes . The SucDH-flavoprotein was not resolved as a discrete protein spot on two-dimensional polyacrylamide gels . However, separation of the rat liver inner mitochondrial membrane complexes by blue native gel electrophoresis followed by Western blotting, and Western blots of purified Escherichia coli SucDH complex revealed that anti-M7 sera contained antibodies directed against the SucDH-flavoprotein subunit. Plasmid, 2002 Mar, 47(2), 120 - 8 Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences; Coates L et al.; The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences . In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil . This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322 . {(3)H}Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug . Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil . We have also found that berenil induces DNA degradation in plasmids containing the homopolymer . Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids . Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect . Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected . Plasmid, 2002 Mar, 47(2), 88 - 93 pMH11, A tool for gene disruption and expression analysis in Azorhizobium caulinodans; D'Haeze W et al.; Tools for mutagenesis and expression analyses are needed to study the role of bacterial genes . Here, we report the construction of pMH11, a small, mobilizable plasmid that replicates in Escherichia coli, but not in Azorhizobium caulinodans, a nodulating microsymbiont of Sesbania rostrata, and that contains a unique BamHI restriction site upstream of a promoterless lacZ gene . pMH11 and two derivatives with the multiple cloning site of pBluescript (KS(II)) are useful for mutagenesis by gene disruption and for expression analyses after selection for cointegration by kanamycin resistance . Weakly constitutive promoter activity from the vector allowed transcription of genes downstream of the integration site, so that no polar effects were caused by gene disruption . Plasmid, 2002 Mar, 47(2), 69 - 78 A model for regulation of ColE1-like plasmid replication by uncharged tRNAs in amino acid-starved Escherichia coli cells; Wang Z et al.; It has been previously observed that various ColE1-like plasmids replicate differentially in Escherichia coli cells during the relaxed response to amino acid starvation . Here we develop a kinetic model to explain these observations based on the possibility of interaction of the 3' CCA-OH sequence with the UGG triplets in loops of RNA I and RNA II encoded by ColE1-like plasmids . According to our model, when the interaction of uncharged CCA with RNA I is possible, the replication of the ColE1-like plasmid is affected by differences in the concentration of various tRNAs in the starved cell, but it is not affected by the tRNA concentration if the hypothetical pairing occurs between the CCA-OH and RNA II . Using the previously determined parameters for the pBR322 plasmid, the concentration of uncharged tRNAs in the amino acid starved relaxed strains and the assumed efficiency of binding of tRNA and RNA I, we show that our model explains the differences in pBR322 copy number in the relaxed strain starved for several amino acids . J Basic Microbiol, 2002, 42(2), 91 - 103 The pab1 gene of Coprinus cinereus encodes a bifunctional protein for para-aminobenzoic acid (PABA) synthesis: implications for the evolution of fused PABA synthases; James TY et al.; The pab1 gene of the basidiomycete Coprinus cinereus encodes PABA synthase, necessary for para-aminobenzoic acid production . The C . cinereus protein is bifunctional with an N-terminal glutamine amidotransferase domain and a C-terminal chorismate amination domain . In most bacteria, these two functions are encoded in separate genes (e.g., pabA and pabB of E . coli) . Fused PABA synthases have so far been detected in actinomycetes, Plasmodium falciparum, fungi and Arabidopsis thaliana . Phylogenetic analysis shows that the fused PAB sequences form a tight group that also includes uncharacterized PabB homologues from several bacteria . Unfused bacterial PabA proteins group with the glutamine amidotransferase subunits of bacterial anthranilate synthases, independent of organismal systematics, indicating a complex and perhaps independent evolutionary origin . In contrast, unfused PabB group and fused PabA/B proteins form a monophyletic group on a branch separate from the chorismate amination subunits of anthranilate synthases, probably reflecting a need for recognition of different positions in the common substrate chorismate. Psychopharmacology (Berl), 2002 May, 161(2), 113 - 9 Epub 2002 Mar 22. Reversal of stress- and CRF-induced anorexia in rats by the synthetic nociceptin/orphanin FQ receptor agonist, Ro 64-6198; Ciccocioppo R et al.; RATIONALE: (1S,3aS)-8-(2,3,3,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triazaspiro{4.5}decan-4-one (Ro 64-6198), a non-peptidic agonist for the opioid receptor-like1 (ORL1) receptor, exhibits anxiolytic properties in stressful conditions . OBJECTIVE: The present study was aimed at evaluating whether activation of ORL1 receptors by Ro 64-6198 may reverse the anorectic effect of restraint stress or intracerebroventricular (ICV) CRF injection . METHODS: In body restraint experiments, 20-h food deprived rats were treated with intraperitoneal (IP) injection of Ro 64-6198 or vehicle . Ten minutes later, they were confined in cylindrical Plexiglas tubes for 60 min and then returned to their cage with food . In CRF experiments, 20-h food deprived rats were IP injected with Ro 64-6198 or vehicle . Ten minutes later, they received ICV CRF, 200 ng/rat or vehicle; food was offered after 20 min . RESULTS: Intraperitoneal (IP) pretreatment with Ro 64-6198 reversed the hypophagic effect induced by both restraint or CRF; the effect was statistically significant at the three doses tested (0.3, 1.0 or 2.5 mg/kg) . ICV administration of the selective ORL1 receptor antagonist {Nphe(1)}NC(1-13)NH(2)(two injections of 33 or 66 microg/rat) abolished the effect of Ro 64-6198 on CRF-induced anorexia . In freely feeding rats, Ro 64-6198 significantly increased feeding at 2.5, but not at 0.3 or 1.0 mg/kg; in food deprived rats, Ro 64-6198 (0.3 or 1.0 mg/kg) did not modify food intake . Thus, reversal of stress- and CRF-induced anorexia by Ro 64-6198 can be evoked at doses lower than those that are hyperphagic . Ro 64-6198 (1 or 2.5 mg/kg) did not modify the anorectic effect of E . coli lipopolysaccharide, suggesting that its effect is selective for stress- or CRF-induced anorexia . Lastly, the benzodiazepine diazepam was unable to reduce the anorectic effect of CRF at the anxiolytic dose of 0.3 mg/kg, and partially reduced it at the hyperphagic dose of 1 mg/kg . CONCLUSIONS: The results of this study show that the non-peptidic ORL1 receptor agonist Ro 64-6198 markedly and selectively inhibits the anorectic effect of stress and CRF, and provide evidence that this effect is mediated by ORL1 receptors . Thus, Ro 64-6198 may represent an interesting tool for treatment of stress-induced anorexia. J Endotoxin Res, 2002, 8(1), 17 - 26 Modulation of endotoxin-induced cardiopulmonary dysfunction by S-nitroso-albumin; Gluckman TL et al.; Nitric oxide (NO) is an endogenous vasodilator and modulator of inflammation . During endotoxemia, the beneficial effects of NO are overwhelmed by the inflammatory cascade, resulting in a functional depletion of NO . S-nitroso-albumin (S-NO-alb) exists as a novel and highly stable NO thiol complex that slowly releases NO into the vascular micro-environment . Using a porcine model, we examined the ability of intravenous S-NO-alb to modulate cardiopulmonary dysfunction characteristic of endotoxemia . Pigs were anesthetized, instrumented for standard cardiopulmonary function measurements, and randomly assigned to receive: (i) albumin + saline; (ii) albumin + LPS; or (iii) S-NO-alb + LPS . Cardiopulmonary parameters were evaluated every 30 min and ex vivo phorbol myristate acetate (PMA)-stimulated superoxide release was serially determined as a marker of in vivo neutrophil priming . Lung myeloperoxidase (MPO) activity was measured as a marker of neutrophil migration into the lung . LPS-induced cardiopulmonary dysfunction was characterized by a sustained elevation in mean pulmonary arterial pressure, pulmonary vascular resistance, and peak intratracheal pressure, as well as a reduction in cardiac index, stroke volume index and PaO(2) over 6 h . Pretreatment with S-NO-alb attenuated LPS-induced cardiopulmonary dysfunction without adversely affecting systemic hemodynamics . Moreover, S-NO-alb blunted the LPS-induced hypoxemic response and reduced neutrophil activation . S-NO-alb did not, however, attenuate LPS-induced increases in lung MPO . Our results suggest that S-NO-alb can selectively modulate endotoxin-induced pulmonary dysfunction, attenuate neutrophil priming and block the early mortality (40%) in this model. J Biol Chem, 2002 Jul 19, 277(29), 25992 - 6002 Epub 2002 Apr 29. Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator; Li S et al.; ArsD is a trans-acting repressor of the arsRDABC operon that confers resistance to arsenicals and antimonials in Escherichia coli . It possesses two-pairs of vicinal cysteine residues, Cys(12)-Cys(13) and Cys(112)-Cys(113), that potentially form separate binding sites for the metalloids that trigger dissociation of ArsD from the operon . However, as a homodimer it has four vicinal cysteine pairs . Titration of the steady-state fluorescence of ArsD with metalloids revealed positive cooperativity, with a Hill coefficient of 2, between these sites . Disruption of the Cys(112)-Cys(113) site by mutagenesis of arsD, but not the Cys(12)-Cys(13) site, largely abolished this cooperativity, indicative of interactions between adjacent Cys(112)-Cys(113) sites within the dimer . The kinetics of metalloid binding were determined by stopped flow spectroscopy; the rate increased in a sigmoidal manner, with a Hill coefficient of 4, indicating that the pre-steady-state measurements reported cooperativity between all four sites of the dimer rather than just the intermolecular interactions reported by the steady-state measurements . The kinetics of Sb(III) displacement by As(III) revealed that the metalloid-binding sites behave differentially, with the rapid exchange of As(III) for Sb(III) at one site retarding the release of Sb(III) from the other sites . We propose a model involving the sequential binding and release of metalloids by the four binding sites of dimeric ArsD, with only one site releasing free metalloids. J Biol Chem, 2002 Jun 28, 277(26), 23208 - 15 Epub 2002 Apr 29. The carboxyltransferase activity of the apicoplast acetyl-CoA carboxylase of Toxoplasma gondii is the target of aryloxyphenoxypropionate inhibitors; Jelenska J et al.; Inhibition of growth of the apicomplexan parasite Toxoplasma gondii by aryloxyphenoxypropionate herbicides has been correlated with the inhibition of its acetyl-CoA carboxylase (ACC) by these compounds . Here, full-length and C-terminal fragments of T . gondii apicoplast ACC as well as C-terminal fragments of the cytosolic ACC were expressed in Escherichia coli . The recombinant proteins that were soluble showed the expected enzymatic activities . Yeast gene-replacement strains depending for growth on the expressed T . gondii ACC were derived by complementation of a yeast ACC1 null mutation . In vitro and in vivo tests with aryloxyphenoxypropionates showed that the carboxyltransferase domain of the apicoplast T . gondii ACC is the target for this class of inhibitors . The cytosolic T . gondii ACC is resistant to aryloxyphenoxypropionates . Both T . gondii isozymes are resistant to cyclohexanediones, another class of inhibitors targeting the ACC of grass plastids. EMBO J, 2002 May 1, 21(9), 2272 - 81 Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic; Hirokawa G et al.; Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex . Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex . In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex . The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone . The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites . The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome . With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3. EMBO J, 2002 May 1, 21(9), 2253 - 62 Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA; Christian EL et al.; Interactions with divalent metal ions are essential for the folding and function of the catalytic RNA component of the tRNA processing enzyme ribonuclease P (RNase P RNA) . However, the number and location of specific metal ion interactions in this large, highly structured RNA are poorly understood . Using atomic mutagenesis and quantitative analysis of thiophilic metal ion rescue we provide evidence for metal ion interactions at the pro-R(P) and pro-S(P) non-bridging phosphate oxygens at nucleotide A67 in the universally conserved helix P4 . Moreover, second-site modifications within helix P4 and the adjacent single stranded region (J3/4) provide the first evidence for metal ion interactions with nucleotide base functional groups in RNase P RNA and reveal the presence of an additional metal ion important for catalytic function . Together, these data are consistent with a cluster of metal ion interactions in the P1-P4 multi-helix junction that defines the catalytic core of the RNase P ribozyme. EMBO J, 2002 May 1, 21(9), 2242 - 52 Structural basis of VDR-DNA interactions on direct repeat response elements; Shaffer PL et al.; The vitamin D receptor (VDR) forms homo- or heterodimers on response elements composed of two hexameric half-sites separated by 3 bp of spacer DNA . We describe here the crystal structures at 2.7-2.8 A resolution of the VDR DNA-binding region (DBD) in complex with response elements from three different promoters: osteopontin (SPP), canonical DR3 and osteocalcin (OC) . These structures reveal the chemical basis for the increased affinity of VDR for the SPP response element, and for the poor stability of the VDR-OC complex, relative to the canonical DR3 response element . The homodimeric protein-protein interface is stabilized by van der Waals interactions and is predominantly non-polar . An extensive alpha-helix at the C-terminal end of the VDR DBD resembles that found in the thyroid hormone receptor (TR), and suggests a mechanism by which VDR and TR discriminate among response elements . Selective structure-based mutations in the asymmetric homodimeric interface result in a VDR DBD protein that is defective in homodimerization but now forms heterodimers with the 9-cis retinoic acid receptor (RXR) DBD. EMBO J, 2002 May 1, 21(9), 2107 - 16 A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition; Bogdanov M et al.; To address the role of phospholipids in the topological organization of polytopic membrane proteins, the function and assembly of lactose permease (LacY) was studied in mutants of Escherichia coli lacking phosphatidylethanolamine (PE) . PE is required for the proper conformation and active transport function of LacY . The N-terminal half of LacY assembled in PE-lacking cells adopts an inverted topology in which normally non-translocated domains are translocated and vice versa . Post-assembly synthesis of PE triggers a conformational change, resulting in a lipid-dependent recovery of normal conformation and topology of at least one LacY subdomain accompanied by restoration of active transport . These results demonstrate that membrane protein topology once attained can be changed in a reversible manner in response to alterations in phospholipid composition, and may be subject to post-assembly proofreading to correct misfolded structures. Biochemistry, 2002 May 7, 41(18), 5938 - 44 Dissociation of the GroEL-GroES asymmetric complex is accelerated by increased cooperativity in ATP binding to the GroEL ring distal to GroES; Fridmann Y et al.; A kinetic analysis of the ATP-dependent dissociation of wild-type GroEL and mutants from immobilized GroES was carried out using surface plasmon resonance . Excellent fits of the data were obtained using a double-exponential equation with a linear drift . Both the fast and slow observed dissociation rate constants are found to have a sigmoidal dependence on the concentration of ATP . The values of the Hill coefficients corresponding to the fast and slow observed rate constants of dissociation of wild-type GroEL and the Arg197-->Ala mutant are in good agreement with the respective values of the Hill coefficients previously determined for these proteins from plots of initial rates of ATP hydrolysis as a function of ATP concentration, in the presence of GroES . Our results are consistent with a kinetic mechanism for dissociation of the GroEL-GroES complex according to which GroES release takes place after an ATP-induced conformational change in the trans ring that is preceded by ATP hydrolysis and a subsequent conformational change in the cis ring . It is shown that the rate of complex dissociation increases with increasing positive cooperativity in ATP binding by the GroEL ring distal to GroES in the GroEL-GroES complex. Biochemistry, 2002 May 7, 41(18), 5730 - 42 Structure of the beta subunit of translation initiation factor 2 from the archaeon Methanococcus jannaschii: a representative of the eIF2beta/eIF5 family of proteins; Cho S et al.; The beta subunit of archaeal translation initiation factor 2 (aIF2beta) is a representative of a family of proteins whose members include the beta subunit of eukaryotic translation initiation factor 2 (eIF2beta) and the N-terminal domain within translation initiation factor 5 (eIF5); no members of this family of proteins have been structurally characterized up to this time . In the work presented here, aIF2beta from Methanococcus jannaschii was expressed in Escherichia coli, purified, and analyzed using multidimensional NMR methods . The aIF2beta was found to contain two independent structural domains . The N-terminal domain contains a four-stranded antiparallel beta sheet and two alpha helices, and is structurally similar to the DNA-binding domain of a yeast heat shock transcription factor and a domain within ribosomal protein S4 . This structural similarity was an unanticipated result, since no significant homology was detected at the level of primary sequence . The C-terminal domain of aIF2beta contains a zinc-binding motif of three antiparallel beta strands, with four conserved cysteines arranged as two CXXC units separated by 17 residues . Conserved residues on the surface of each domain that are likely candidates for direct interaction with other components of the translational apparatus were identified . The significant primary sequence homology between archaeal aIF2beta and the eukaryotic eIF2beta and eIF5, when combined with the structural results in the work presented here, permitted structural features to be predicted for these latter two eukaryotic proteins. Biopolymers, 2002, 67(3), 186 - 96 Binding modes of cyclic AMP and environments of tryptophan residues in 1:1 and 1:2 complexes of cyclic AMP receptor protein and cyclic AMP; Fujimoto N et al.; Cyclic AMP (cAMP) receptor protein (CRP) forms 1:1 and 1:2 complexes with cAMP, and the former complex is considered to be the most active form of CRP in binding to specific DNA sequences and in modulating gene transcription by RNA polymerases . We examine the cAMP binding modes and structural changes of CRP upon cAMP binding by UV resonance Raman spectroscopy . The Raman spectra of CRP-(cAMP)(1) and CRP-(cAMP)(2) extracted from those of CRP-cAMP mixtures at varied mixing ratios clearly show that the hydrogen bonding state and the conformation of cAMP in both complexes in solution are very similar to those found in the X-ray crystal structure of CRP-(cAMP)(2), which is evidence that the cAMP binding mode does not differ between the two complexes . The environmental hydrophobicity of Trp85 monitored by UV resonance Raman intensity shows a significant decrease upon binding of the first cAMP molecule, whereas no further change occurs in the second cAMP binding step . The environmental change of Trp85 suggests an opening of the cleft between the N-terminal cAMP and C-terminal DNA binding domains in the process of CRP activation by binding of a single cAMP molecule . Biopolymers, 2002 Jul 5, 64(2), 106 - 14 A metal binding in the polypeptide chain improves the folding efficiency of a denatured and reduced protein; Ohkuri T et al.; In order to examine the effect of a metal binding to the polypeptide chain on the aggregation of a protein in the refolding process, we prepared a mutant hen lysozyme possessing the same Ca(2+) binding site as in human alpha-lactalbumin by Escherichia coli expression system (Ser(-1) CaB lysozyme) . In the presence of 2 mM CaCl(2), the refolding yield of Ser(-1) CaB lysozyme at a low protein concentration (25 microg/mL) was similar to that of the wild-type lysozyme (80%), but that at high protein concentration (200 microg/mL) decreased (15%) due to aggregation comparing to that of the wild-type lysozyme (45%) . However, the refolding yield of Ser(-1) CaB lysozyme in the presence of 100 mM CaCl(2) even at a protein concentration of 200 microg/mL was 80% and was higher than that of the wild-type lysozyme . From analysis of chemical shift changes of the cross peaks in the backbone region of total correlated spectroscopy (TOCSY) spectra of a decapeptide possessing the same calcium binding site as in Ser(-1) CaB lysozyme in the presence of various concentrations of Ca(2+), it was suggested that the dissociation constant of Ca(2+)-peptide complex was estimated to be 20-36 mM . Moreover, the solubility of the denatured Ser(-1) CaB lysozyme in the presence of 100 mM CaCl(2) was higher than that in the presence of 2 mM CaCl(2) whereas the solubility of the denatured Ser(-1) lysozyme in the presence of 100 mM CaCl(2) was not higher than that in the presence of 2 mM CaCl(2) . Therefore, it was concluded that the reduced lysozyme possessing the Ca(2+) binding site was efficiently folded in the presence of high concentration of Ca(2+) (100 mM) even at high protein concentration due to depression of aggregation by the binding of Ca(2+) to the polypeptide chain in Ser(-1) CaB lysozyme . Int Arch Allergy Immunol, 2002 Mar, 127(3), 181 - 90 cDNA cloning and immunological characterization of a newly identified enolase allergen from Penicillium citrinum and Aspergillus fumigatus; Lai HY et al.; BACKGROUND: Penicillium citrinum and Aspergillus fumigatus are prevalent indoor airborne fungal species that have been implicated in human respiratory allergic disorders . It is important to understand the allergenic profile of these fungal species . The purpose of the present study is to characterize a newly identified enolase allergen from P . citrinum and A . fumigatus . METHODS: Fungal proteins were separated by two-dimensional (2D) gel electrophoresis and blotted onto polyvinylidene difluoride membranes . Protein spots that reacted with IgE antibodies in serum samples from asthmatic patients were identified and the N-terminal amino acid sequences were determined by Edman degradation . The peptide sequences obtained were utilized in cloning the cDNA of the allergen genes by reverse transcriptase-polymerase chain reaction and the 5'- and 3'-rapid amplification cDNA end reactions . RESULTS: Our results from 2D immunoblotting identified a 47-kD IgE-reactive component in the extracts of P . citrinum and A . fumigatus . The N-terminal amino acid sequences of the 47-kD proteins are homologous to those of fungal enolases . The corresponding enolase cDNA from P . citrinum contains 1,552 bp and encodes a protein of 438 residues . In A . fumigatus, the isolated enolase cDNA has 1,649 bp and contains a 438-amino acid open reading frame . The deduced amino acid sequences of these two enolases have 94% identity . These enolases from P . citrinum and A . fumigatus were expressed in Escherichia coli as a His-tagged protein and designated as rPen c 22 and rAsp f 22, respectively . Sera from 7 (30%) of the 23 Penicillium-sensitized asthmatic patients showed IgE binding to the 47-kD P . citrinum component (Pen c 22) and rPen c 22 . In addition, six of seven Pen c 22-positive serum samples have IgE immunoblot reactivity to the 47-kD A . fumigatus component (Asp f 22) and rAsp f 22 . A polyclonal rabbit antiserum generated against the N-terminal peptide of Pen c 22 can react with Pen c 22, rPen c 22, Asp f 22 and rAsp f 22 . In addition, the presence of IgE cross-reactivity between rPen c 22 and rAsp f 22 and between enolases from A . fumigatus and Alternaria alternata was also detected by immunoblot inhibition . CONCLUSIONS: These results demonstrated that a novel enolase allergen from P . citrinum (Pen c 22) and A . fumigatus (Asp f 22) was identified . In addition, IgE cross-reactivity between enolase allergens from A . fumigatus and P . citrinum and between enolases from A . fumigatus and A . alternata was also detected . Results obtained provide more information on fungal enolase allergens . Plant Cell Physiol, 2002 Apr, 43(4), 429 - 39 Comparison of the structure of the extrinsic 33 kDa protein from different organisms; Tohri A et al.; The psbO gene encoding the extrinsic 33 kDa protein of oxygen-evolving photosystem II (PSII) complex was cloned and sequenced from a red alga, Cyanidium caldarium . The gene encodes a polypeptide of 333 residues, of which the first 76 residues served as transit peptides for transfer across the chloroplast envelope and thylakoid membrane . The mature protein consists of 257 amino acids with a calculated molecular mass of 28,290 Da . The sequence homology of the mature 33 kDa protein was 42.9-50.8% between the red alga and cyanobacteria, and 44.7-48.6% between the red alga and higher plants . The cloned gene was expressed in Escherichia coli, and the recombinant protein was purified, subjected to protease-treatments . The cleavage sites of the 33 kDa protein by chymotrypsin or V8 protease were determined and compared among a cyanobacterium (Synechococcus elongatus), a euglena (Euglena gracilis), a green alga (Chlamydomonas reinhardtii) and two higher plants (Spinacia oleracea and Oryza sativa) . The cleavage sites by chymotrypsin were at 156F and 190F for the cyanobacterium, 159M, 160F and 192L for red alga, 11Y and 151F for euglena, 10Yand 150F for green alga, and 16Y for spinach, respectively . The cleavage sites by V8 protease were at 181E (cyanobacterium), 182E and 195E (red alga), 13E, 67E, 69E, 153D and 181E (euglena), 176E and 180E (green alga), and 18E or 19E (higher plants) . Since most of the residues at these cleavage sites were conserved among the six organisms, the results indicate that the structure of the 33 kDa protein, at least the structure based on the accessibility by proteases, is different among these organisms . In terms of the cleavage sites, the structure of the 33 kDa protein can be divided into three major groups: cyanobacterial and red algal-type has cleavage sites at residues around 156-195, higher plant-type at residues 16-19, and euglena and green algal-type at residues of both cyanobacterial and higher plant-types. J Biol Chem, 2002 Jul 19, 277(29), 26003 - 11 Epub 2002 Apr 26. Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1; Warton K et al.; CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms . The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration . Soluble E . coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells . The process of membrane integration of CLIC1 is pH-dependent . Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer . These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics . This has four times the conductance of the SCSK and fast kinetics that characterize the native channel . This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins. Free Radic Biol Med, 2002 May 1, 32(9), 813 - 21 Involvement of mammalian OGG1(MMH) in excision of the 8-hydroxyguanine residue in DNA; Nishimura S; 8-Hydroxyguanine (7,8-dihydro-8-oxoguanine, abbreviated as 8-OH-G or 8-oxoG) is the site of a frequent mutagenic DNA lesion produced by oxidative damage . MutM of E . coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase and apurinic (AP) site lyase activity . cDNA clones of four isoforms (types 1a, 1b, 1c, and 2) of human OGG1 homologs (hMMH) were isolated . In order to examine whether expression of hMMH (hOGG1) protein actually occurs in human cells, we prepared type 1a specific antibody, and by using this antibody, we showed that type 1a protein isolated from HeLaS3 has 8-OH-G glycosylase/lyase activity . Furthermore, we showed that type 1a protein is a major enzyme for repair of the 8-OH-G lesion in human cells . In our second study, we generated a mouse line carrying an inactivated mutant Mmh allele by targeted gene disruption . Liver extracts of Mmh homozygous mutant mice were found to have loss of the nicking activity for the 8-OH-G site . In addition, the amount of endogenous 8-OH-G in liver DNA of the homozygous mice increased linearly with age, reaching 7-fold increase in 14 week old mice, over that of wild-type or heterozygous mice . Furthermore, when homozygous mice were fed the oxygen radical-forming agent KBrO3, to provide oxidative stress, the level of 8-OH-G in kidney DNA was tremendously increased: more than 200-fold as that of control mice without oxidative stress after 12 weeks of age . These results indicate that Ogg1/Mmh plays an essential role in the repair of the 8-OH-G residue in DNA produced by oxidative stress. Biochem J, 2002 Aug 15, 366(Pt 1), 63 - 71 Truncation of Arabidopsis thaliana and Selaginella lepidophylla trehalose-6-phosphate synthase unlocks high catalytic activity and supports high trehalose levels on expression in yeast; Van Dijck P et al.; Plants, such as Arabidopsis thaliana and Selaginella lepidophylla, contain genes homologous with the trehalose-6-phosphate synthase (TPS) genes of bacteria and fungi . Most plants do not accumulate trehalose with the desert resurrection plant S . lepidophylla, being a notable exception . Overexpression of the plant genes in a Saccharomyces cerevisiae tps1 mutant results in very low TPS-catalytic activity and trehalose accumulation . We show that truncation of the plant-specific N-terminal extension in the A . thaliana AtTPS1 and S . lepidophylla SlTPS1 homologues results in 10-40-fold higher TPS activity and 20-40-fold higher trehalose accumulation on expression in yeast . These results show that the plant TPS enzymes possess a high-potential catalytic activity . The growth defect of the tps1 strain on glucose was restored, however, the proper homoeostasis of glycolytic flux was not restored, indicating that the plant enzymes were unable to substitute for the yeast enzyme in the regulation of hexokinase activity . Further analysis of the N-terminus led to the identification of two conserved residues, which after mutagenesis result in strongly enhanced trehalose accumulation upon expression in yeast . The plant-specific N-terminal region may act as an inhibitory domain allowing modulation of TPS activity. Biochem J, 2002 Aug 15, 366(Pt 1), 121 - 7 Biochemical adaptations of two sugar kinases from the hyperthermophilic archaeon Pyrococcus furiosus; Verhees CH et al.; The hyperthermophilic archaeon Pyrococcus furiosus possesses a modified Embden-Meyerhof pathway, including an unusual ADP-dependent glucokinase (ADP-GLK) and an ADP-dependent phosphofructokinase . In the present study, we report the characterization of a P . furiosus galactokinase (GALK) and its comparison with the P . furiosus ADP-GLK . The pyrococcal genes encoding the ADP-GLK and GALK were functionally expressed in Escherichia coli, and the proteins were subsequently purified to homogeneity . Both enzymes are specific kinases with an optimal activity at approx . 90 degrees C . Biochemical characterization of these enzymes confirmed that the ADP-GLK is unable to use ATP as the phosphoryl group donor, but revealed that GALK is ATP-dependent and has an extremely high affinity for ATP . There is a discussion about whether the unusual features of these two classes of kinases might reflect adaptations to a relatively low intracellular ATP concentration in the hyperthermophilic archaeon P . furiosus. Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 63 - 8 {Cloning and expression of a thermostable beta-glycosidase gene from Thermus nonproteolyticus HG102}; He XY et al.; The gene coding for beta-glycosidase (EC3.2.1.21) from Thermus nonproteolyticus HG102 has been cloned and expressed in E . coli . The gene open reading frame was 1311 bp and it codes for 436 amino acids . The deduced amino acid sequence of the enzyme showed identity with members of glycosyl hydrolase family I . The enzyme had high content of hydrophobic amino acid (Ala 12.8%, Leu 10.9%), Arg(9.6%), Glu(9.4%) and Pro(8.0%), but low content Cys(0.45%) and Met (0.9%) . From the alignment of enzyme amino acid sequence with other glycosyl hydrolase family I members, Glu164 and Glu338 were predicated as the proton donor and nucleophile group . The DNASTAR program was used to predict the secondary structure . According to the Chou-Fasman model, the enzyme has 41.4% of alpha-helics, 16.2%, beta-strands, 14.4%, beta-turns . 14 of the 35 Pro were located at the second sites of beta-turns . Hydrophobic interaction, ion bond, alpha-helics and Pro had important contribution to Tn-gly thermostability. Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 35 - 9 {Cloning and expression of extracellular domain of prostate specific membrane antigen in Escherichia coli and preparation of polyclonal antibody}; Ye CZ et al.; Human Prostate Specific Membrane Antigen(PSMA) cDNA was amplified using total RNA extracted from prostate carcinoma tissue by RT-PCR . The cDNA fragment of extracellular domain of PSMA(edPSMA) gene was amplified by PCR and cloned into expression vector pMAL-c2x . Sequence analysis of both PSMA and edPSMA revealed identity to the GenBank reported . The edPSMA was expressed in E . coli as part of a fusion protein with MBP as the induction of IPTG . Western blot analysis showed the recombinant protein could react with PSMA monocloned antibodies 4G5 . MBP-edPSMA fusion protein were purified by amylose resin affinity chromatography and showed to be homogeneity in SDS-PAGE(120 kD) . BALB/C mice were immunized with the purified protein for the preparation of polyclonal antibody . The polyclonal antibody, which had a title of 1:12,800, were indicated the specificity to prostate tissue. Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 106 - 8 {Cloning and expression of Buthus martensii Karsch scorpion toxin gene (BmK IT3) in Escherichia coli}; Yu JB et al.; According to the reported sequence of Buthus martensii Karsch scorpion toxin gene (BmK IT3), we synthesized two primers, which were complementary in a region . By the means of PCR, we got the gene . The gene was fused in expression vector pET-28a, which gave rise to a recombinant plasmid pET(IT3R) . Then it was transformed into E . coli BL21 (DE3) . With IPTG induction, the gene was efficiently expressed . And the fusion product was soluble. Life Sci Space Res, 1976, 14, 355 - 8 Effect of space factors on Escherichia coli B/r cells; Bucker H et al.; Stationary phase cells of Escherichia coli B/r were inactivated when they were exposed to high vacuum (10(-6) torr) . About 5% of the cells were still able to form a colony after 45 min exposure . Vacuum dried cells (0.5 torr, 120 min) show colony forming ability of 30% or more . They were inactivated to about 5% by further vacuum treatment . Vacuum treated cells showed higher permeability for various cell components . UV irradiated E . coli B/r cells in vacuum showed increased UV sensitivity . DNA-protein cross-links were preferentially formed in a vacuum . To obtain 63% of DNA cross linked with protein required 852 erg mm-2 (D37) of UV irradiation in suspension and only 72 erg mm-2 of UV irradiation in vacuum . The protein components of DNA-protein cross-links were hydrolysed with pronase E and the amino acids directly bonding to DNA were determined . The most important amino acids concerned in DNA-protein cross-links seem to be glycine and alanine, followed by aspartic acid (asparagine), glutamic acid (glutamine) and histidine . The sensitivity to X-rays of stationary phase E . coli B/r cells seems to depend on the remaining gas atmospheric in the vacuum since it varies with different pumping systems. Mol Genet Genomics, 2002 Apr, 267(2), 241 - 53 Epub 2002 Mar 21. Molecular and biochemical characterization of the Neurospora crassa glycogen synthase encoded by the gsn cDNA; de Paula R et al.; Glycogen synthases catalyze the transfer of a glucosyl moiety from a nucleotide phosphosugar to a nascent glycogen chain via an alpha1-->4 linkage . Although many genes coding for glycogen synthases have been described, the enzymes from rabbit and yeast are the best characterized . The fungus Neurospora crassa accumulates glycogen during exponential growth, and mobilizes it at the onset of stationary phase, or when placed at high temperature or starved for carbon . Through a PCR methodology, the gsn cDNA coding for the N . crassa glycogen synthase was isolated, and the amino acid sequence of the protein was deduced . The product of the cDNA seems to be the only glycogen synthase present in N . crassa . Characterization of the gsn cDNA revealed that it codes for a 706-amino acids protein, which is very similar to mammalian and yeast glycogen synthases . Gene expression increased during exponential growth, reaching its maximal level at the end of the exponential growth phase, which is consistent with the pattern of glycogen synthase activity and glycogen level . Expression of the gsn is highly regulated at the transcriptional level . Under culture conditions that induce heat shock, conidiation, and carbon starvation, expression of the gsn gene was decreased, and glycogen synthase activity and glycogen content behaved similarly. Mol Genet Genomics, 2002 Apr, 267(2), 142 - 53 Epub 2002 Mar 23. Development of a high-throughput yeast two-hybrid screening system to study protein-protein interactions in plants; Fang Y et al.; We have developed a high-throughput yeast two-hybrid screening system (HTP-YTH) that incorporates yeast gap-repair cloning, multiple positive ( ADE2, HIS3, lacZ) and negative ( URA3-based) selection schemes to reduce the incidence of negative and false positive clones, and automation of laboratory procedures to increase throughput . This HTP-YTH system has been applied to the study of protein-protein interactions that are involved in rice defense signal transduction pathways . More than 100 genes involved in plant defense responses were selected from DuPont's rice expressed sequence tag (EST) databases as baits for HTP-YTH screening . Results from YTH screening of eight of these rice genes are presented in this paper . Not only have we identified known protein-protein interactions, but we have also discovered novel interactions, which may ultimately reveal the regulatory network of host defense signal transduction pathways . We have demonstrated that our HTP-YTH method can be used to map protein-protein interaction networks and signal transduction pathways in any system . In combination with other approaches, such efficient YTH screens can help us systemically to study the functions of known and unknown genes in the genomics era. Acta Crystallogr D Biol Crystallogr, 2002 May, 58(Pt 5), 870 - 1 Epub 2002 Apr 26. Crystallization and preliminary X-ray analysis of recombinant histone HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii OT3; Li T et al.; Recombinant archaeal histone from the hyperthermophile Pyrococcus horikoshii OT3 (HPhA) was crystallized by the hanging-drop vapour-diffusion method . Crystals grew at 291 K in 200 mM (NH(4))(2)SO(4), 100 mM sodium acetate buffer pH 4.6, 19% PEG 4000 . Diffraction data were obtained to a resolution of 2.3 A from a single frozen crystal, which belonged to space group P2(1) with unit-cell parameters a = 34.99, b = 46.89, c = 35.02 A, alpha = gamma = 90, beta = 104 degrees . The asymmetric unit contained two molecules and had a solvent content of approximately 35%. Acta Crystallogr D Biol Crystallogr, 2002 May, 58(Pt 5), 853 - 5 Epub 2002 Apr 26. Crystallization and preliminary X-ray analysis of the hydroperoxidase I C-terminal domain from Escherichia coli; Carpena X et al.; Hydroperoxidases (HP) are normally large haem-containing bifunctional enzymes capable of acting as both catalases and peroxidases . The C-terminal domain of HPI from Escherichia coli (KatG), extending from residue Tyr422 to Leu726, was found to be resistant to trypsin proteolysis . The segment of katG encoding this domain was cloned and overexpressed to produce a haemless protein that is soluble even at concentrations above 30 mg ml(-1) . This protein shows a 25% sequence identity with cytochrome c peroxidase (CCP) from Saccharomyces cerevisae, despite lacking the characteristic catalytic and iron-binding residues . Crystals from this protein were grown in 0.6 M sodium citrate buffered to pH 7.5 with HEPES by the hanging-drop vapour-diffusion method at 293 K . These crystals diffracted beyond 2.0 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.2, b = 98.7, c = 302.8 A . Three pseudo-origin peaks in the Patterson maps indicate an unusual packing compatible with the presence of three molecules in the crystal asymmetric unit and a solvent content of about 80% by volume. Acta Crystallogr D Biol Crystallogr, 2002 May, 58(Pt 5), 849 - 52 Epub 2002 Apr 26. Crystallization and preliminary X-ray analysis of human carbonic anhydrase III; Duda DM et al.; Carbonic anhydrases catalyze the interconversion of carbon dioxide to bicarbonate . Human carbonic anhydrase isozyme III with a C-terminal hexahistidine tag was overexpressed in Eschericha coli, purified and crystallized . Diffraction data (93.4% completeness) were collected to 2.2 A resolution on an in-house R-AXIS IV++ image-plate system with Osmic mirrors and a Rigaku HU-H3R CU rotating-anode generator operating at 50 kV and 100 mA . A 60 degrees sweep of data were collected from a single crystal with a crystal-to-detector distance of 150 mm and a 0.5 degrees oscillation angle per frame using an exposure of 60 s per frame at 293 K . The crystals were shown to conform to the Laue hexagonal crystal system P6, with unit-cell parameters a = 44.7, c = 222.5 A and a scaling R(sym) of 0.087 for 11 962 unique reflections . Using the known crystal structure of the rat form of carbonic anhydrase isozyme III, a molecular-replacement model was built . This model was used for rotation and translation searches and uniquely defined the space group as P6(5) . Rigid-body refinement of the model was used to generate an initial phased electron-density map with an R(work) of 31.17%. Acta Crystallogr D Biol Crystallogr, 2002 May, 58(Pt 5), 846 - 8 Epub 2002 Apr 26. Cloning, high-level expression, purification and crystallization of peptide deformylase from Leptospira interrogans; Li Y et al.; A new peptide deformylase (PDF; EC 3.5.1.27) gene from Leptospira interrogans was identified and cloned into expression plasmid pET22b(+) and was highly expressed in Escherichia coli BL21(DE3) . With DEAE-Sepharose anion-exchange chromatography followed by Superdex G-75 size-exclusion chromatography, 60 mg of PDF from L . interrogans was purified from 1 l of cell culture . Crystallization screening of the purified enzyme resulted in two crystal forms, from one of which a 3 A resolution X-ray diffraction data set has been collected. Acta Crystallogr D Biol Crystallogr, 2002 May, 58(Pt 5), 824 - 32 Epub 2002 Apr 26. The structure of L-rhamnulose-1-phosphate aldolase (class II) solved by low-resolution SIR phasing and 20-fold NCS averaging; Kroemer M et al.; The enzyme L-rhamnulose-1-phosphate aldolase catalyzes the reversible cleavage of L-rhamnulose-1-phosphate to dihydroxyacetone phosphate and L-lactaldehyde . It is a homotetramer with an M(r) of 30 000 per subunit and crystallized in space group P3(2)21 . The enzyme shows a low sequence identity of 18% with the structurally known L-fuculose-1-phosphate aldolase that splits a stereoisomer in a similar reaction . Structure analysis was initiated with a single heavy-atom derivative measured to 6 A resolution . The resulting poor electron density, a self-rotation function and the working hypothesis that both enzymes are C(4) symmetric with envelopes that resemble one another allowed the location of the 20 protomers of the asymmetric unit . The crystal-packing unit was a D(4)-symmetric propeller consisting of five D(4)-symmetric octamers around an internal crystallographic twofold axis . Presumably, the propellers associate laterally in layers, which in turn pile up along the 3(2) axis to form the crystal . The non-crystallographic symmetry was used to extend the phases to the 2.7 A resolution limit and to establish a refined atomic model of the enzyme . The structure showed that the two enzymes are indeed homologous and that they possess chemically similar active centres. J Biol Chem, 2002 Jul 5, 277(27), 24212 - 9 Epub 2002 Apr 25. The ATPase activity and the functional domain of PotA, a component of the sermidine-preferential uptake system in Escherichia coli; Kashiwagi K et al.; The ATPase activity of PotA, a component of the spermidine-preferential uptake system consisting of PotA, -B, -C, and -D, was studied using purified PotA and a PotABC complex on inside-out membrane vesicles . It was found that PotA can form a dimer by disulfide cross-linking but that each PotA molecule functions independently . When PotA was associated with the membrane proteins PotB and PotC, the K(m) value for ATP increased and PotA became much more sensitive to inhibition by spermidine . It was also shown that spermidine uptake in cells was gradually inhibited in parallel with spermidine accumulation in cells . The results suggest that spermidine functions as a feedback inhibitor of spermidine transport . The function of PotA was analyzed using PotA mutants obtained by random mutagenesis . There are two domains in PotA . The NH2-terminal domain (residues 1-250) contains the ATP binding pocket formed in part by residues Cys26, Phe27, Phe45, Cys54, Leu60, and Leu76, the active center of ATPase that includes Val135 and Asp172, and amino acid residues necessary for the interaction with a second PotA subunit (Cys26) and with PotB (Cys54) . The COOH-terminal domain (residues 251-378) of PotA contains a site that regulates ATPase activity and a site involved in the spermidine inhibition of ATPase activity. J Bacteriol, 2002 May, 184(10), 2850 - 3 The RcsCB His-Asp phosphorelay system is essential to overcome chlorpromazine-induced stress in Escherichia coli; Conter A et al.; The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown . We show here that treatment of an exponentially growing culture of E . coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system . This induction is abolished in rcsB or rcsC mutant strains . In addition, treatment with CPZ inhibits growth . The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation . In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ . These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress . This is the first report of the essential role of the RcsCB system in a stress situation . These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor. J Bacteriol, 2002 May, 184(10), 2837 - 40 The secE gene of Helicobacter pylori; Medigue C et al.; Despite extensive annotation by two independent teams, the Helicobacter pylori genome appeared to lack a complete secretion machinery . The use of clinical isolates to substantiate in silico annotation is used here to identify the missing secE component of the major secretion machinery of Helicobacter pylori. J Bacteriol, 2002 May, 184(10), 2767 - 79 TraG-like proteins of DNA transfer systems and of the Helicobacter pylori type IV secretion system: inner membrane gate for exported substrates? Schroder G, Krause S, Zechner EL, Traxler B, Yeo HJ, Lurz R, Waksman G, Lanka E. TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation . They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems . TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori . Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H . pylori) . These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity . Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity . Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4 . Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus . We predict that multimeric inner membrane protein TraG forms a pore . A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented. J Bacteriol, 2002 May, 184(10), 2692 - 8 Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase; Tan J et al.; The Escherichia coli RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits . An in-frame deletion of the rrmJ (ftsJ) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes . To investigate whether overexpression of other E . coli genes can restore the severe growth defect observed in rrmJ null mutants, we constructed an overexpression library from the rrmJ deletion strain and cloned and identified the E . coli genes that were capable of rescuing the rrmJ mutant phenotype . Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed . To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the rrmJ deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes . 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification . The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage . Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an rrmJ strain . These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA. J Bacteriol, 2002 May, 184(10), 2682 - 91 Mutational scanning and affinity cleavage analysis of UhpA-binding sites in the Escherichia coli uhpT promoter; Olekhnovich IN et al.; UhpA, a member of the NarL family of response regulators, activates transcription of the Escherichia coli uhpT gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate . UhpA binds with different affinities to adjacent regions in the uhpT promoter, termed the strong-binding (S) region from -80 to -50 and the weak-binding (W) region from -50 to -32 . Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits . Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding . Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a uhpT-lacZ reporter, and to support UhpA-dependent in vitro transcription . These W region variant promoters were strongly stimulated by CAP . Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro . Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region . None of these substitutions eliminated promoter function . The orientation of UhpA-binding sites was assessed by the affinity cleavage method . The iron chelate FeBABE {iron (S)-1-(p-bromoacetamidobenzyl) EDTA} was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA . Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions . These results are consistent with the binding of two dimers of UhpA . Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp . It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets. J Bacteriol, 2002 May, 184(10), 2674 - 81 Escherichia coli DNA polymerase III can replicate efficiently past a T-T cis-syn cyclobutane dimer if DNA polymerase V and the 3' to 5' exonuclease proofreading function encoded by dnaQ are inactivated; Borden A et al.; Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coli in the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (DeltaumuDC) strains if the 3' to 5' exonuclease proofreading activity of the Pol III epsilon subunit was also disabled by mutD5 . This observation might be explained in at least two ways . In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases . Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function . We have investigated this issue by examining dimer bypass frequencies in DeltaumuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations . Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V . Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself . This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally. J Bacteriol, 2002 May, 184(10), 2642 - 53 Functional analysis of the signal recognition particle in Escherichia coli by characterization of a temperature-sensitive ffh mutant; Park SK et al.; The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP) . Efforts to understand the function of Ffh in bacteria have depended largely on the use of E . coli strains that allow depletion of the wild-type gene product . As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated . The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42 degrees C as well as show reduced growth at the permissive temperature of 30 degrees C . While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired . The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42 degrees C . It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein . Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function. J Bacteriol, 2002 May, 184(10), 2634 - 41 Using disulfide bond engineering to study conformational changes in the beta'260-309 coiled-coil region of Escherichia coli RNA polymerase during sigma(70) binding; Anthony LC et al.; RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell . Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences . We have previously defined a region of the beta' subunit (beta'260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with sigma(70) both in vitro and in vivo . However, it was not known if the coiled-coil conformation was maintained upon binding to sigma(70) . In this work, we engineered a disulfide bond within beta'240-309 that locks the beta' coiled-coil region in the coiled-coil conformation, and we show that this "locked" peptide is able to bind to sigma(70) . We also show that the locked coiled-coil is capable of inducing a conformational change within sigma(70) that allows recognition of the -10 nontemplate strand of DNA . This suggests that the coiled-coil does not adopt a new conformation upon binding sigma(70) or upon recognition of the -10 nontemplate strand of DNA. J Bacteriol, 2002 May, 184(10), 2603 - 13 Functional characterization and regulation of gadX, a gene encoding an AraC/XylS-like transcriptional activator of the Escherichia coli glutamic acid decarboxylase system; Tramonti A et al.; The Escherichia coli chromosome contains two distantly located genes, gadA and gadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad) . The Gad reaction contributes to pH homeostasis by consuming intracellular H(+) and producing gamma-aminobutyric acid . This compound is exported via the protein product of the gadC gene, which is cotranscribed with gadB . Here we demonstrate that transcription of both gadA and gadBC is positively controlled by gadX, a gene downstream of gadA, encoding a transcriptional regulator belonging to the AraC/XylS family . The gadX promoter encompasses the 67-bp region preceding the gadX transcription start site and contains both RpoD and RpoS putative recognition sites . Transcription of gadX occurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of the gad structural genes . However, P(T5)lacO-controlled gadX expression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when the gad system is normally repressed . Autoregulation of the whole gad system is inferred by the positive effect of GadX on the gadA promoter and gadAX cotranscription . Transcription of gadX is derepressed in an hns mutant and strongly reduced in both rpoS and hns rpoS mutants, consistent with the expression profile of gad structural genes in these genetic backgrounds . Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX binds gadA and gadBC promoters at different sites and with different binding affinities. J Bacteriol, 2002 May, 184(10), 2595 - 602 Proteolytic activity of YibP protein in Escherichia coli; Ichimura T et al.; Escherichia coli YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family . A yibP disrupted mutant grows in rich medium at 37 degrees C but not at 42 degrees C . In the yibP null mutant, cell division and FtsZ ring formation are inhibited at 42 degrees C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis . Five percent betaine suppresses the temperature sensitivity of the yibP disrupted mutation . The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain . YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction . Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space . Purified YibP has a protease activity that split the substrate beta-casein. Appl Environ Microbiol, 2002 May, 68(5), 2619 - 23 Cloning of Escherichia coli lacZ and lacY genes and their expression in Gluconobacter oxydans and Acetobacter liquefaciens; Mostafa HE et al.; An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium . Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G . oxydans and A . liquefaciens . Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose . However, this ability strictly depended on the presence of a plasmid expressing both lac genes . Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose. Appl Environ Microbiol, 2002 May, 68(5), 2453 - 60 Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library; Lopez-Garcia B et al.; The purpose of the present study was to improve the antifungal activity against selected phytopathogenic fungi of the previously identified hexapeptide PAF19 . We describe some properties of a set of novel synthetic hexapeptides whose D-amino acid sequences were obtained through screening of a synthetic peptide combinatorial library in a positional scanning format . As a result of the screening, 12 putative bioactive peptides were identified, synthesized, and assayed . The peptides PAF26 (Ac-rkkwfw-NH(2)), PAF32 (Ac-rkwhfw-NH(2)), and PAF34 (Ac-rkwlfw-NH(2)) showed stronger activity than PAF19 against isolates of Penicillium digitatum, Penicillium italicum, and Botrytis cinerea . PAF26 and PAF32, but not PAF34, were also active against Fusarium oxysporum . Penicillium expansum was less susceptible to all four PAF peptides, and only PAF34 showed weak activity against it . Assays were also conducted on nontarget organisms, and PAF26 and PAF32 showed much-reduced toxicity to Escherichia coli and Saccharomyces cerevisiae, demonstrating selectivity towards certain filamentous fungi . Thus, the data showed distinct activity profiles for peptides differentiated by just one or two residue substitutions . Our conclusion from this observation is that a specificity factor is involved in the activity of these short peptides . Furthermore, PAF26 and PAF32 displayed activities against P . digitatum, P . italicum, and B . cinerea similar to that of the hemolytic 26-amino acid melittin, but they did not show the high toxicity of melittin towards bacteria and yeasts . The four peptides acted additively, with no synergistic interactions among them, and PAF26 was shown to have improved activity over PAF19 in in vivo orange fruit decay experiments. Biophys Chem, 2002 Apr 10, 96(1), 33 - 51 Thermodynamics of reactions catalyzed by PABA synthase; Tewari YB et al.; Microcalorimetry and high-performance liquid chromatography (HPLC) have been used to conduct a thermodynamic investigation of reactions catalyzed by PABA synthase, the enzyme located at the first step in the shikimic acid metabolic pathway leading from chorismate to 4-aminobenzoate (PABA) . The overall biochemical reaction catalyzed by the PabB and PabC components of PABA synthase is: chorismate(aq)+ammonia(aq)=4-aminobenzoate(aq)+pyruvate(aq)+H(2)O(l) . This reaction can be divided into two partial reactions involving the intermediate 4-amino-4-deoxychorismate (ADC): chorismate(aq)+ammonia(aq)=ADC(aq)+H(2)O(l) and ADC(aq)=4-aminobenzoate(aq)+pyruvate(aq) . Microcalorimetric measurements were performed on all three of these reactions at a temperature of 298.15 K and pH values in the range 8.72-8.77 . Equilibrium measurements were performed on the first partial (ADC synthase) reaction at T=298.15 K and at pH=8.78 . The saturation molality of 4-aminobenzoate(cr) in water is (0.00382+/-0.0004) mol kg(-1) at T=298.15 K . The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products . These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms . For the reaction: chorismate(2-)(aq)+NH(4)(+)(aq)=ADC(-)(aq)+H(2)O(l), K=(10.8+/-4.2) and Delta(r)H(m)(o)=-(35+/-15) kJ mol(-1) . For the reaction: ADC(-)(aq)=4-aminobenzoate(-)(aq)+pyruvate(-)(aq)+H(+)(aq), Delta(r)H(m)(o)=-(139+/-23) kJ mol(-1) . For the reaction: chorismate(2-)(aq)+NH(4)(+)(aq)=4-aminobenzoate(-)(aq)+pyruvate(-)(aq)+H(2)O(l)+H(+)(aq), Delta(r)H(m)(o)=-(174+/-6) kJ mol(-1) . Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that utilize L-glutamine rather than ammonia and that are pertinent to this branch point of the shikimic acid pathway . The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength . Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes Delta(r)G'(m)(o) under approximately physiological conditions are given. J Nat Prod, 2002 Apr, 65(4), 630 - 1 A new biologically active malyngamide from a New Zealand collection of the sea hare Bursatella leachii; Appleton DR et al.; A new malyngamide, S (1), was isolated from a New Zealand collection of the sea hare Bursatella leachii and structurally characterized by spectroscopic methods and chemical degradation . Malyngamide S exhibited cytotoxicity and antiinflammatory properties. Gene Ther, 2002 May, 9(9), 584 - 91 Double suicide gene therapy using a replication defective herpes simplex virus vector reveals reciprocal interference in a malignant glioma model; Moriuchi S et al.; Herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli cytosine deaminase (CD) are non-mammalian enzymes capable of converting innocuous prodrugs into cytotoxic metabolites . Both enzymes have been utilized independently, as well as together in 'suicide' gene therapy protocols to eliminate tumor cells in vitro and in vivo . We have used a set of replication defective HSV vectors expressing either or both enzymes to compare the efficacies of single and double suicide gene therapies in the 9L gliosarcoma model in vitro and in vivo . In cell culture experiments at high and low multiplicities of infection, combined expression of the two genes by vector TOCD/TK along with exposure to the matching prodrugs (ganciclovir and 5-fluorocytosine) showed increased cytotoxicity compared with exposure to either prodrug alone . However, the two gene combination was inferior to single gene treatments, suggesting that HSVtk and CD are mutually counteractive in the prodrug-dependent killing of glioma cells . In animal experiments, survival was not significantly prolonged by administration of both prodrugs to TOCD/TK-treated animals, while each single gene/prodrug pair resulted in increased survival . These results indicate that single suicide gene systems employing HSVtk or CD may be preferable over combinations of the two. J Biol Chem, 2002 Jul 19, 277(29), 25983 - 91 Epub 2002 Apr 24. A selenoprotein in the plant kingdom . Mass spectrometry confirms that an opal codon (UGA) encodes selenocysteine in Chlamydomonas reinhardtii gluththione peroxidase; Fu LH et al.; Selenoproteins that contain the rare amino acid selenocysteine in their primary structure have been identified in diverse organisms such as viruses, bacteria, archea, and mammals, but so far not in yeast or plants . Among the most thoroughly investigated families of selenoenzymes are the animal glutathione peroxidases (GPXs) . In the last few years, genes encoding GPX-like homologues from Chlamydomonas and higher plants have been isolated, but, unlike the animal ones, all of them have cysteine (rather than selenocysteine) residues in their catalytic site . In all organisms investigated that contain selenoproteins, selenocysteine is encoded by a UGA opal codon, which is usually a stop codon . We report here that, in Chlamydomonas reinhardtii, the cDNA-cloned sequence of a GPX homologue contains an internal TGA codon in frame to the ATG . Specific mRNA expression, protein production, and enzyme activity are selenium-dependent . Sequence analysis of the peptides produced by proteolytic digestion, performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), confirmed the presence of a selenocysteine residue at the predicted site and suggest its location in the mitochondria . Thus, our data present the first direct proof that a UGA opal codon is decoded in the plant kingdom to incorporate selenocysteine. BMC Biotechnol . 2002 Apr 24;2(1):7. Selective permeation and organic extraction of recombinant green fluorescent protein (gfpuv) from Escherichia coli; Vessoni Penna TC et al.; BACKGROUND: Transformed cells of Escherichia coli DH5-alpha with pGFPuv, induced by IPTG (isopropyl-beta-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases . E . coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E . coli on releasing gfpuv from the over-expressing cells . MATERIAL AND METHODS: Cultures (37 degrees C/100 rpm/ 24 h; mu = 0.99 h(-1)-1.10 h(-1)) of transformed (pGFP) Escherichia coli DH5-alpha, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells . For selective permeation, the transformed previously frozen (-75 degrees C) cells were subjected to three freeze/thaw (-20 degrees C/ 0.83 degrees C/min) cycles interlaid by sonication (3 pulses/6 seconds/25 vibrations) . The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM beta-mercaptoethanol beta-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate . Sonication efficiency was verified on the application to the cells previously treated by the TPP method . The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0) . RESULTS: The sonication maximum released amount obtained from the cells was 327.67 microg gfpuv/mL (20.73 microg gfpuv/mg total proteins-BSA), after 9 min of treatment . Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 microg gfpuv/mL (29.74 microg gfpuv/mg BSA) was obtained . The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 microg/mg and 135.10 microg/mg . CONCLUSIONS: The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column. Mol Microbiol, 2002 Apr, 44(2), 521 - 32 Dam-dependent phase variation of Ag43 in Escherichia coli is altered in a seqA mutant; Correnti J et al.; In Escherichia coli, phase variation of the outer membrane protein Ag43 encoded by the agn43 ge