Escherichia coli

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 gene is mediated by DNA methylation and the global regulator OxyR . Transcription of agn43 occurs (ON phase) when three Dam target sequences in the agn43 regulatory region are methylated, which prevents the repressor OxyR from binding . Conversely, transcription is repressed (OFF) when these Dam target sequences are unmethylated and OxyR binds . A change in expression phase requires a concomitant change in the DNA methylation state of these Dam target sequences . To gain insight into the process of inheritance of the expression phase and the DNA methylation state, protein-DNA interactions at agn43 were examined . Binding of OxyR at agn43 was sufficient to protect the three GATC sequences contained within its binding site from Dam-dependent methylation in vitro, suggesting that no other factors are required to maintain the unmethylated state and OFF phase . To maintain the methylated state of the ON phase, however, Dam must access the hemimethylated agn43 region after DNA replication, and OxyR binding must not occur . OxyR bound hemimethylated agn43 DNA, but the affinity was severalfold lower than for unmethylated DNA . This presumably contributes to the maintenance of the methylated state but, at the same time, may allow for infrequent OxyR binding and a switch to the OFF phase . Hemimethylated agn43 DNA was also a binding substrate for the sequestration protein SeqA . Thus, SeqA, OxyR and Dam may compete for the same hemimethylated agn43 DNA that is formed after DNA replication in an ON phase cell . In isolates with a mutant seqA allele, agn43 phase variation rates were altered and resulted in a bias to the OFF phase . In part, this can be attributed to the observed decrease in the level of DNA methylation in the seqA mutant.

Mol Microbiol, 2002 Apr, 44(2), 509 - 20
Competitive interaction of the OxyR DNA-binding protein and the Dam methylase at the antigen 43 gene regulatory region in Escherichia coli; Waldron DE et al.; The antigen 43 surface protein of Escherichia coli is expressed in a phase-variable manner by a mechanism involving alternative activation and repression of transcription of the agn43 gene . The repressor is the OxyR DNA-binding protein, and its binding site was found to be located downstream of the agn43 transcription start site in a region of DNA that encompasses three 5'-GATC-3' sequences that are subject to Dam-mediated DNA methylation . It has been suggested previously that the phase-variable expression of antigen 43 results from a competition between Dam methylase and the OxyR repressor for these sites . The 5'-GATC-3' sequences were inactivated for methylation by site-directed mutagenesis, and all possible combinations of inactive and active sites were assessed for effects on phase-variable expression of the agn43 gene . Inactivation of any 5'-GATC-3' site individually had no effect; at least two sites had to be inactivated to disrupt the normal pattern of expression . Studies of OxyR interaction with agn43 DNA showed that methylation of any two 5'-GATC-3' sites was necessary and sufficient to block binding of the repressor . It was also found that the adenines of the second and third 5'-GATC-3' sites are required for OxyR binding, demonstrating that the sites for Dam methylation and for repressor binding are intimately associated . This is consistent with a competition model in which Dam and OxyR share a preference for specific DNA sequences in the regulatory region of the agn43 gene.

Mol Microbiol, 2002 Apr, 44(2), 501 - 7
Polar localization of the Escherichia coli oriC region is independent of the site of replication initiation; Gordon GS et al.; The location of the origin-linked region of the Escherichia coli chromosome was analysed in strains lacking the core origin locus, oriC . In these strains, which initiate replication from F factors integrated at different locations around the chromosome, origin-linked DNA remains localized near the cell poles, as in wild-type cells . In contrast, minichromosomes containing 7 kb of chromosomal DNA including oriC are generally excluded from the ends of the cell . Thus, we propose that positioning of the wild-type origins at the poles is not a function of their order of replication but a sequence-specific phenomenon . It is proposed that there are centromere-like sequences, bordering the wild-type origin of replication, which are used by host mechanisms to direct the proper placement of the origin region of the chromosome . This function, combined with other host processes, may assure efficient segregation of the E . coli chromosome.

Mol Microbiol, 2002 Apr, 44(2), 461 - 78
The FtsH protease is involved in development, stress response and heat shock control in Caulobacter crescentus; Fischer B et al.; The ftsH gene of Caulobacter crescentus has been isolated and identified as a component of the general stress response of this organism . In C . crescentus, ftsH expression is transiently induced after temperature upshift and in stationary phase . Consistent with this, mutants deprived of the FtsH protease are viable at normal growth conditions, but are highly sensitive to elevated temperature, increased salt concentration or the presence of antibiotics . Overexpression of ftsH resulted in an increased salt but not thermotolerance, emphasizing the importance of the FtsH protease in stress response . Mutants lacking FtsH were unable to undergo morphological and physiological adaptation in stationary phase and, upon starvation, experienced a more pronounced loss of viability than cells containing FtsH . In addition, cells lacking FtsH had an increased cellular concentration of the heat shock sigma factor sigma32, indicating that, as in Escherichia coli, the FtsH protease is involved in the control of the C . crescentus heat shock response . In agreement with this, transcription of the heat-induced sigma32-dependent gene dnaK was derepressed at normal temperature when FtsH was absent . In contrast, the groEL gene, which is controlled in response to heat stress by both sigma32 and a HcrA/CIRCE mechanism, was not derepressed in an ftsH mutant . Finally, FtsH is involved in C . crescentus development and cell cycle control . ftsH mutants were unable to synthesize stalks efficiently and had a severe cell division phenotype . In the absence of FtsH, swarmer cells differentiated into stalked cells faster than when FtsH was present, even though the entire cell cycle was longer under these conditions . Thus, directly or indirectly, the FtsH protease is involved in the inherent biological clock mechanism, which controls the timing of cell differentiation in C . crescentus.

Mol Microbiol, 2002 Apr, 44(2), 361 - 79
The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells; Giron JA et al.; Enteropathogenic Escherichia coli (EPEC) utilizes a type III protein secretion system to target effector molecules into the host cell leading to effacement of the intestinal mucosa . This secretion apparatus shares many structural features of the flagellar type III export system involved in flagella assembly and motility . We report here that fliC insertional mutants constructed in two wild-type EPEC strains were markedly impaired in adherence and microcolony formation on cultured cells . An E . coli K-12 strain harbouring the EPEC H6 fliC gene on a plasmid showed discrete adhering clusters on HeLa cells, albeit to less extent than the wild-type EPEC strain . Flagella purified from EPEC bound to cultured epithelial cells and antiflagella antibodies blocked adherence of several EPEC serotypes . We determined that eukaryotic cells in culture stimulate expression of flagella by motile and non-motile EPEC . Isogenic strains mutated in perA (a transcriptional activator), bfpA (a type IV pilin), luxS (a quorum-sensing autoinducer gene) and in the type III secretion genes were reduced for motility in Dulbecco's modified Eagle medium (DMEM) motility agar and produced none or few flagella when associated with epithelial cells . Growth of these mutants in preconditioned tissue culture medium restored motility and their ability to produce flagella, suggesting the influence of a signal provided by mammalian cells that triggers flagella production . This study shows for the first time that the flagella of EPEC are directly involved in the adherence of these bacteria and supports the existence of a molecular relationship between the two existing type III secretion pathways of EPEC, the EPEC adherence factor (EAF) plasmid-encoded regulator, quorum sensing and epithelial cells.

Mol Microbiol, 2002 Apr, 44(2), 309 - 24
Yeast Rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression; Angermayr M et al.; Rio1p was identified as a protein serine kinase founding a novel subfamily . It is highly conserved from Archaea to man and only distantly related to previously established protein kinase families . Nevertheless, analysis of multiple protein sequence alignments shows that those amino acid residues that are important for either structure or catalytic activity in conventional protein kinases are also conserved in members of the Rio1p family at the respective positions (corresponding to domains I-XI of protein kinases) . Recombinant Rio1p from Escherichia coli and tagged Rio1p from yeast has kinase activity in vitro, and mutation of amino acid residues that are conserved and indispensable for catalytic activity (i.e . ATP-binding motif, catalytic centre) abrogates activity . RIO1 is essential in yeast and plays a role in cell cycle progression . After sporulation of RIO1/rio1 diploids, RIO1-disrupted progeny cease growth after one to three cell divisions and arrest as either large unbudded or large-budded cells . Cells deprived of Rio1p are enlarged and arrest either in G1 or in mitosis mainly with the DNA at the bud neck and short spindles (a phenotype also seen in cells carrying a weak allele), suggesting that Rio1p activity is required for at least at two steps during the cell division cycle: for entrance into S phase and for exit from mitosis . The weak RIO1 allele leads to increased plasmid loss.

Immunology, 2002 May, 106(1), 60 - 70
Antigen binding to GM1 ganglioside results in delayed presentation: minimal effects of GM1 on presentation of antigens internalized via other pathways; Nashar TO et al.; Plasma membrane rafts are sphingolipid- and cholesterol-rich patches that function as membrane trafficking and surface signalling regions . Ganglioside GM1 is an integral component of these microdomains, and Escherichia coli enterotoxin B subunit (EtxB) is a pentamer that binds with high affinity to GM1 resulting in GM1 cross-linking . We previously demonstrated that antigen coupled directly to EtxB resulted in enhanced presentation relative to antigen taken up by fluid-phase endocytosis . Here we demonstrate a new role for GM1 in antigen presentation by examining the effects of cross-linking GM1 on the kinetics of presentation and processing of antigen by the B-cell receptor (BCR), fluid-phase endocytosis and GM1-targeted antigen . EtxB bound to B cells does not augment the subsequent kinetics or magnitude of presentation of either BCR-internalized antigen or soluble antigen . Moreover, presentation of GM1-bound antigen is significantly slower than antigen presentation following BCR-mediated uptake . In contrast to the rapid internalization of BCR-bound antigen (which has a half life of 60 min), the majority of EtxB-bound antigen forms a plasma membrane depot detectable for many hours after initial incubation (and with a half life of 12 hr) . We conclude that cross-linking of GM1 by EtxB minimally affects the processing and presentation of antigens internalized via other pathways . Nevertheless, binding of antigens to GM1 results in delayed presentation that has important implications for in vivo immunization using GM1-targeted adjuvants.

Nucleic Acids Res, 2002 May 1, 30(9), 2083 - 8
Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'; Thompson JR et al.; Although it has been recognized that PCR amplification of mixed templates may generate sequence artifacts, the mechanisms of their formation, frequency and potential elimination have not been fully elucidated . Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR . Nearly equal proportions of homoduplexes and heteroduplexes were observed after co-amplifying 16S rDNA from three bacterial genomes and analyzing products by constant denaturing capillary electrophoresis (CDCE) . Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased . A model exploring the fate of cloned heteroduplexes during MutHLS-mediated mismatch repair in the Escherichia coli host demonstrates that the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants . Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening . Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to 'reconditioning PCR', a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product . This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample.

Nucleic Acids Res, 2002 May 1, 30(9), 1886 - 94
Over-representation of repeats in stress response genes: a strategy to increase versatility under stressful conditions?
Rocha EP, Matic I, Taddei F.
The survival of individual organisms facing stress is enhanced by the induction of a set of changes . As the intensity, duration and nature of stress is highly variable, the optimal response to stress may be unpredictable . To face such an uncertain future, it may be advantageous for a clonal population to increase its phenotypic heterogeneity (bet-hedging), ensuring that at least a subset of cells would survive the current stress . With current techniques, assessing the extent of this variability experimentally remains a challenge . Here, we use a bioinformatic approach to compare stress response genes with the rest of the genome for the presence of various kinds of repeated sequences, elements known to increase variability during the transfer of genetic information (i.e . during replication, but also during gene expression) . We investigated the potential for illegitimate and homologous recombination of 296 Escherichia coli genes related to repair, recombination and physiological adaptations to different stresses . Although long repeats capable of engaging in homologous recombination are almost absent in stress response genes, we observed a significant high number of short close repeats capable of inducing phenotypic variability by slipped-mispair during DNA, RNA or protein synthesis.

Nucleic Acids Res, 2002 May 1, 30(9), 1879 - 85
Charge neutralization and DNA bending by the Escherichia coli catabolite activator protein; Hardwidge PR et al.; We are interested in the role of asymmetric phosphate neutralization in DNA bending induced by proteins . We describe an experimental estimate of the actual electrostatic contribution of asymmetric phosphate neutralization to the bending of DNA by the Escherichia coli catabolite activator protein (CAP), a prototypical DNA-bending protein . Following assignment of putative electrostatic interactions between CAP and DNA phosphates based on X-ray crystal structures, appropriate phosphates in the CAP half-site DNA were chemically neutralized by methylphosphonate substitution . DNA shape was then evaluated using a semi-synthetic DNA electrophoretic phasing assay . Our results confirm that the unmodified CAP DNA half-site sequence is intrinsically curved by 26 degrees in the direction enhanced in the complex with protein . In the absence of protein, neutralization of five appropriate phosphates increases DNA curvature to 32 degrees (approximately 23% increase), in the predicted direction . Shifting the placement of the neutralized phosphates changes the DNA shape, suggesting that sequence-directed DNA curvature can be modified by the asymmetry of phosphate neutralization . We suggest that asymmetric phosphate neutralization contributes favorably to DNA bending by CAP, but cannot account for the full DNA deformation.

Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 5999 - 6004 Epub 2002 Apr 23.
Conformational pathways in the gating of Escherichia coli mechanosensitive channel; Kong Y et al.; The pathway of the gating conformational transition of Escherichia coli mechanosensitive channel was simulated, using the recently modeled open and closed structures, by targeted molecular dynamics method . The transition can be roughly viewed as a four-stage process . The initial motion under a lower tension load is predominantly elastic deformation . The opening of the inner hydrophobic pore on a higher tension load takes place after the major expansion of the outer channel dimension . The hypothetical N-terminal S1 helical bundle has been confirmed to form the hydrophobic gate, together with the M1 helices . The sequential breaking of the tandem hydrophobic constrictions on the M1 and S1 helices makes the two parts of the gate strictly coupled, acting as a single gate . The simulation also revealed that there is no significant energetic coupling between the inner S1 bundle and the outer M2 transmembrane helices . The molten-globular-like structural features of the S1 bundle in its intermediate open states may account for the observed multiple subconductance states . Moreover, the intermediate open states of mechanosensitive channels are not symmetric, i.e., the opening does not follow iris-like motion, which sharply contrasts to the potassium channel KcsA.

Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 6085 - 90 Epub 2002 Apr 23.
Evolutionary recruitment of a flavin-dependent monooxygenase for the detoxification of host plant-acquired pyrrolizidine alkaloids in the alkaloid-defended arctiid moth Tyria jacobaeae; Naumann C et al.; Larvae of Tyria jacobaeae feed solely upon the pyrrolizidine alkaloid-containing plant Senecio jacobaea . Ingested pyrrolizidine alkaloids (PAs), which are toxic to unspecialized insects and vertebrates, are efficiently N-oxidized in the hemolymph of T . jacobaeae by senecionine N-oxygenase (SNO), a flavin-dependent monooxygenase (FMO) with a high substrate specificity for PAs . Peptide microsequences obtained from purified T . jacobaeae SNO were used to clone the corresponding cDNA, which was expressed in active form in Escherichia coli . T . jacobaeae SNO possesses a signal peptide characteristic of extracellular proteins, and it belongs to a large family of mainly FMO-like sequences of mostly unknown function, including two predicted Drosophila melanogaster gene products . The data indicate that the gene for T . jacobaeae SNO, highly specific for toxic pyrrolizidine alkaloids, was recruited from a preexisting insect-specific FMO gene family of hitherto unknown function . The enzyme allows the larvae to feed on PA-containing plants and to accumulate predation-deterrent PAs in the hemolymph.

Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 5948 - 52 Epub 2002 Apr 23.
Cys-328 of IscS and Cys-63 of IscU are the sites of disulfide bridge formation in a covalently bound IscS/IscU complex: implications for the mechanism of iron-sulfur cluster assembly; Kato S et al.; IscS and IscU from Escherichia coli cooperate with each other in the biosynthesis of iron-sulfur clusters . IscS catalyzes the desulfurization of L-cysteine to produce L-alanine and sulfur . Cys-328 of IscS attacks the sulfur atom of L-cysteine, and the sulfane sulfur derived from L-cysteine binds to the Sgamma atom of Cys-328 . In the course of the cluster assembly, IscS and IscU form a covalent complex, and a sulfur atom derived from L-cysteine is transferred from IscS to IscU . The covalent complex is thought to be essential for the cluster biogenesis, but neither the nature of the bond connecting IscS and IscU nor the residues involved in the complex formation have been determined, which have thus far precluded the mechanistic analyses of the cluster assembly . We here report that a covalent bond is formed between Cys-328 of IscS and Cys-63 of IscU . The bond is a disulfide bond, not a polysulfide bond containing sulfane sulfur between the two cysteine residues . We also found that Cys-63 of IscU is essential for the IscU-mediated activation of IscS: IscU induced a six-fold increase in the cysteine desulfurase activity of IscS, whereas the IscU mutant with a serine substitution for Cys-63 had no effect on the activity . Based on these findings, we propose a mechanism for an early stage of iron-sulfur cluster assembly: the sulfur transfer from IscS to IscU is initiated by the attack of Cys-63 of IscU on the Sgamma atom of Cys-328 of IscS that is bound to sulfane sulfur derived from L-cysteine.

Mol Cell Biol, 2002 May, 22(10), 3474 - 87
Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2; Ancelin K et al.; We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2 . To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced . We demonstrate that their targeting leads to telomere shortening . This indicates that these proteins act in cis to repress telomere elongation . Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation . In contrast, TRF2 targeting and telomerase inhibition have additive effects . The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase . In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.

J Exp Bot, 2002 May, 53(371), 1005 - 15
Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation; Waterworth WM et al.; DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways . Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA . Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein . This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge . CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants . White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues . This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light . Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures . These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.

J Biol Chem, 2002 Jun 28, 277(26), 23764 - 72 Epub 2002 Apr 23.
Central region of the human splicing factor Hprp3p interacts with Hprp4p; Gonzalez-Santos JM et al.; Human splicing factors Hprp3p and Hprp4p are associated with the U4/U6 small nuclear ribonucleoprotein particle, which is essential for the assembly of an active spliceosome . Currently, little is known about the specific roles of these factors in splicing . In this study, we characterized the molecular interaction between Hprp3p and Hprp4p . Constructs were created for expression of Hprp3p or its mutants in bacterial or mammalian cells . We showed that antibodies against either Hprp3p or Hprp4p were able to pull-down the Hprp3p-Hprp4p complex formed in Escherichia coli lysates . By co-immunoprecipitation and isothermal titration calorimetry, we demonstrated that purified Hprp3p and its mutants containing the central region, but lacking either the N-terminal 194 amino acids or the C-terminal 240 amino acids, were able to interact with Hprp4p . Conversely, Hprp3p mutants containing only the N- or C-terminal region did not interact with Hprp4p . In addition, by co-immunoprecipitation, we showed that intact Hprp3p and its mutants containing the central region interacted with Hprp4p in HeLa cell nuclear extracts . Primer extension analysis illustrated that the central region of Hprp3p is required to maintain the association of Hprp3p-Hprp4p with U4/U6 small nuclear RNAs, suggesting that this Hprp3p/Hprp4p interaction allows the recruitment of Hprp4p, and perhaps other protein(s), to the U4/U6 small nuclear ribonucleoprotein particle.

J Am Chem Soc, 2002 May 1, 124(17), 4602 - 9
Spectroscopic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: molecular mechanism and CO(2) activation in the biosynthesis of ethylene; Zhou J et al.; 1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyzes the last step in the biosynthesis of the gaseous plant hormone ethylene, which is involved in development, including germination, fruit ripening, and senescence . ACCO is a mononuclear non-heme ferrous enzyme that couples the oxidation of the cosubstrate ascorbate to the oxidation of substrate ACC by dioxygen . In addition to substrate and cosubstrate, ACCO requires the activator CO(2) for continuous turnover . NIR circular dichroism and magnetic circular dichroism spectroscopies have been used to probe the geometric and electronic structure of the ferrous active site in ACCO to obtain molecular-level insight into its catalytic mechanism . Resting ACCO/Fe(II) is coordinatively saturated (six-coordinate) . In the presence of CO(2), one ferrous ligand is displaced to yield a five-coordinate site only when both the substrate ACC and cosubstrate ascorbate are bound to the enzyme . The open coordination position allows rapid O(2) activation for the oxidation of both substrates . In the absence of CO(2), ACC binding alone converts the site to five-coordinate, which would react with O(2) in the absence of ascorbate and quickly deactivate the enzyme . These studies show that ACCO employs a general strategy similar to other non-heme iron enzymes in terms of opening iron coordination sites at the appropriate time in the reaction cycle and define the role of CO(2) as stabilizing the six-coordinate ACCO/Fe(II)/ACC complex, thus preventing the uncoupled reaction that inactivates the enzyme.

Plant Cell, 2002 Apr, 14(4), 877 - 88
Functional significance of the alternative transcript processing of the Arabidopsis floral promoter FCA; Macknight R et al.; The Arabidopsis gene FCA encodes an RNA binding protein that functions to promote the floral transition . The FCA transcript is alternatively processed to yield four transcripts, the most abundant of which is polyadenylated within intron 3 . We have analyzed the role of the alternative processing on the floral transition . The introduction of FCA intronless transgenes resulted in increased FCA protein levels and accelerated flowering, but no role in flowering was found for products of the shorter transcripts . The consequences of the alternative processing on the FCA expression pattern were determined using a series of translational FCA-beta-glucuronidase fusions . The inclusion of FCA genomic sequence containing the alternatively processed intron 3 restricted the expression of the transgene predominantly to shoot and root apices and young flower buds . Expression of this fusion also was delayed developmentally . Therefore, the alternative processing of the FCA transcript limits, both spatially and temporally, the amount of functional FCA protein . Expression in roots prompted an analysis of root development, which indicated that FCA functions more generally than in the control of the floral transition.

Plant Cell, 2002 Apr, 14(4), 805 - 15
A gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis; He Y et al.; SAG101, a leaf senescence-associated gene, was cloned from an Arabidopsis leaf senescence enhancer trap line and functionally characterized . Reporter gene and RNA gel blot analyses revealed that SAG101 was not expressed until the onset of senescence in leaves . A recombinant SAG101 fusion protein overexpressed in Escherichia coli displayed acyl hydrolase activity . Antisense RNA interference in transgenic plants delayed the onset of leaf senescence for approximately 4 days . Chemically induced overexpression of SAG101 caused precocious senescence in both attached and detached leaves of transgenic Arabidopsis plants . These data suggest that SAG101 plays a significant role in leaf senescence.

Plant Cell, 2002 Apr, 14(4), 795 - 803
Rice TATA binding protein interacts functionally with transcription factor IIB and the RF2a bZIP transcriptional activator in an enhanced plant in vitro transcription system; Zhu Q et al.; TATA binding protein (TBP) and transcription factor IIB (TFIIB) are key factors for the assembly of eukaryotic transcription initiation complexes . We used a rice whole-cell extract in vitro transcription system to characterize the functional interactions of recombinant plant TBP and TFIIB . Bacterially expressed rice TBP (OsTBP2) bound to the TATA box of the rice pal gene encoding phenylalanine ammonia-lyase, caused DNA bending, and enhanced basal transcription from the pal promoter in a TATA box-dependent manner . Recombinant rice TFIIB (OsTFIIB) stimulated the DNA binding and bending activities of OsTBP2 and synergistically enhanced OsTBP2-mediated transcription from the pal promoter and the promoter of Rice tungro bacilliform virus but not from the barley pr1 promoter . We also demonstrate a physical interaction between OsTBP2 and RF2a, a rice bZIP transcription factor that bound to the box II cis element of the promoter of Rice tungro bacilliform virus, resulting in enhanced transcription from the viral promoter . Enhancement of rice whole-cell extracts with recombinant transcription factors thus provides a powerful tool for the in vitro determination of plant gene regulation mechanisms . We conclude that OsTBP2 undergoes promoter-specific functional interactions with both the basal transcription factor OsTFIIB and the accessory transcription factor RF2a.

J Immunol, 2002 May 1, 168(9), 4576 - 84
Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy; Swoboda I et al.; IgE-mediated reactions to fish allergens represent one of the most frequent causes of food allergy . We have constructed an expression cDNA library from carp (Cyprinus carpio) muscle in phage lambda gt11 and used serum IgE from a fish allergic patient to isolate 33 cDNA clones that coded for two parvalbumin isoforms (Cyp c 1.01 and Cyp c 1.02) with comparable IgE binding capacities . Both isoforms represented calcium-binding proteins that belonged to the beta-lineage of parvalbumins . The Cyp c 1.01 cDNA was overexpressed in Escherichia coli, and rCyp c 1.01 was purified to homogeneity . Circular dichroism analysis and mass spectroscopy showed that rCyp c 1.01 represented a folded protein with mainly alpha-helical secondary structure and a molecular mass of 11,416 Da, respectively . rCyp c 1.01 reacted with IgE from all fish-allergic patients tested (n = 60), induced specific and dose-dependent basophil histamine release, and contained most of the IgE epitopes (70%) present in natural allergen extracts from cod, tuna, and salmon . Therefore, it may be used to identify patients suffering from IgE-mediated fish allergy . The therapeutic potential of rCyp c 1.01 is indicated by our findings that rabbit Abs raised against rCyp c 1.01 inhibited the binding of IgE (n = 25) in fish-allergic patients to rCyp c 1.01 between 35 and 97% (84% mean inhibition) and that depletion of calcium strongly reduced IgE recognition of rCyp c 1.01 . The latter results suggest that it will be possible to develop strategies for immunotherapy for fish allergy that are based on calcium-free hypoallergenic rCyp c 1.01 derivatives.

J Biol Chem, 2002 Jun 28, 277(26), 23821 - 7 Epub 2002 Apr 22.
Characterization of a direct oxygen sensor heme protein from Escherichia coli . Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure; Sasakura Y et al.; A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme . In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E . coli, and examined its structure-function relationships for the first time . Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex . Its alpha-helix content was calculated as 53% by CD spectroscopy . The redox potential of the heme was found to be +67 mV versus SHE . Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands . Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min(-1) with cAMP, which was optimal at pH 8.5 in the presence of Mg(2+) and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor . Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme . Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.

J Biol Chem, 2002 Jun 28, 277(26), 23374 - 81 Epub 2002 Apr 22.
Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase; Angove HC et al.; The cytochrome c nitrite reductases perform a key step in the biological nitrogen cycle by catalyzing the six-electron reduction of nitrite to ammonium . Graphite electrodes painted with Escherichia coli cytochrome c nitrite reductase and placed in solutions containing nitrite (pH 7) exhibit large catalytic reduction currents during cyclic voltammetry at potentials below 0 V . These catalytic currents were not observed in the absence of cytochrome c nitrite reductase and were shown to originate from an enzyme film engaged in direct electron exchange with the electrode . The catalytic current-potential profiles observed on progression from substrate-limited to enzyme-limited nitrite reduction revealed a fingerprint of catalytic behavior distinct from that observed during hydroxylamine reduction, the latter being an alternative substrate for the enzyme that is reduced to ammonium in a two electron process . Cytochrome c nitrite reductase clearly interacts differently with these two substrates . However, similar features underlie the development of the voltammetric response with increasing nitrite or hydroxylamine concentration . These features are consistent with coordinated two-electron reduction of the active site and suggest that the mechanisms for reduction of both substrates are underpinned by common rate-defining processes.

Biochemistry, 2002 Apr 30, 41(17), 5685 - 94
Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces; O'Connor SE et al.; The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value . Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli . EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis . The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction . The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB . The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain . The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible . These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.

Biochemistry, 2002 Apr 30, 41(17), 5637 - 43
Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase; Huang YC et al.; Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure {Hurley et al . (1991) Biochemistry 30, 8671-8688}, and porcine His(315) and His(319) are close to that site . The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E . coli, and purified . The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type . Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis . In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type . These results implicate His(315) in the region of the NADP site . Replacement of His(309) by Q or F yields enzyme with no detectable activity . The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme . The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes . However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally . Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes . In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure . We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.

Biochemistry, 2002 Apr 30, 41(17), 5605 - 12
Transbilayer movement of fluorescent phospholipid analogues in the cytoplasmic membrane of Escherichia coli; Kubelt J et al.; We investigated the transmembrane movement of fluorescent labeled phospholipids in inverted inner membrane vesicles (IIMV) of Escherichia coli (E . coli) wild-type strain (MG1655), as well as in proteoliposomes reconstituted from detergent extracts of the IIMV . The transbilayer movement of 1-myristoyl-2-{6-{(7-nitro-2,1,3-benzoxadiazol-4-yl)amino}caproyl}-sn-glycero-3-phosphoethanolamine (M-C6-NBD-PE) and -phosphocholine (M-C6-NBD-PC) was measured by a fluorescence stopped-flow back-exchange assay . Both analogues were rapidly translocated across the IIMV membrane, with half-times of <1 min (outward movement) and approximately 3 min (inward movement) . No flip-flop was detected in protein-free liposomes, but in IIMV-derived proteoliposomes flip-flop of M-C6-NBD-PE occurred similarly to IIMV and could be largely eliminated by proteinase K treatment.

Biochemistry, 2002 Apr 30, 41(17), 5573 - 80
SecA specificity for different signal peptides; Kebir MO et al.; SecA performs a critical function in the recognition, targeting, and transport of secretory proteins across the cytoplasmic membrane of Escherichia coli . In this study we investigate the substrate specificity of SecA, including the influence of the early mature region of the preprotein on SecA interactions, and the extent to which SecA recognizes targeting signals from different transport pathways . A series of fusion proteins were generated which involved the tandem expression of GST, signal peptide, and the first 30 residues from alkaline phosphatase . These were purified and evaluated for their ability to promote SecA ATPase activity . No significant difference in the stimulation of SecA-lipid ATPase activity between the synthetic wild-type alkaline phosphatase signal peptide and a fusion that also contains the first 30 residues of alkaline phosphatase was observed . The incorporation of sequence motifs in the mature region, which confer SecB dependence in vivo, had no impact on SecA activation in vitro . These results suggest that the early mature region of alkaline phosphatase does not affect the interactions between SecA and the signal peptide . Sec, Tat, and YidC signal peptide fusions were also assayed for their ability to stimulate SecA ATPase activity in vitro and further analyzed in vivo for the Sec dependence of the transport of the corresponding signal peptide mutants of alkaline phosphatase . Our results demonstrate that E . coli Sec signals give the highest level of SecA activation; however, SecA-signal peptide interactions in vitro are not the only arbiter of whether the preprotein utilizes the Sec pathway in vivo.

Biochemistry, 2002 Apr 30, 41(17), 5556 - 65
Binding of enzyme IIAGlc, a component of the phosphoenolpyruvate:sugar phosphotransferase system, to the Escherichia coli lactose permease; Sondej M et al.; Enzyme IIA(Glc), encoded by the crr gene of the phosphoenolpyruvate:sugar phosphotransferase system, plays an important role in regulating intermediary metabolism in Escherichia coli ("catabolite repression") . One function involves inhibition of inducible transport systems ("inducer exclusion"), and with lactose permease, a galactoside is required for unphosphorylated IIA(Glc) binding to cytoplasmic loops IV/V and VI/VII {Sondej, M., Sun, J . et al . (1999) Proc . Natl . Acad . Sci . U.S.A . 96, 3525-3530} . With inside-out membrane vesicles containing the permease, {(125)I}IIA(Glc) binding promoted by melibiose exhibits an affinity (K(D)(IIA)) of approximately 1 microM and a stoichiometry of one mole of IIA(Glc) per six moles of lactose permease . Both the quantity of {(125)I}IIA(Glc) bound and the sugar concentration required for half-maximal IIA(Glc) binding (K(0.5)(IIA)(sug)) was measured for eight permease substrates . Differences in maximal IIA(Glc) binding are observed, and the K(0.5)(IIA)(sug) does not correlate with the affinity of LacY for sugar . Furthermore, K(0.5)(IIA)(sug) does not correlate with sugar affinities for various permease mutants . IIA(Glc) does not bind to a mutant (Cys154 --> Gly), which is locked in an outwardly facing conformation, binds with increased stoichiometry to mutant Lys131 --> Cys, and binds only weakly to two other mutants which appear to be predominantly in either an outwardly or an inwardly facing conformation . When the latter two mutations are combined, sugar-dependent IIA(Glc) binding returns to near wild-type levels . The findings suggest that binding of various substrates to lactose permease results in a collection of unique conformations, each of which presents a specific surface toward the inner face of the membrane that can interact to varying degrees with IIA(Glc).

Biochemistry, 2002 Apr 30, 41(17), 5537 - 47
Structure of Ala24/Asp61 --> Asp24/Asn61 substituted subunit c of Escherichia coli ATP synthase: implications for the mechanism of proton transport and rotary movement in the F0 complex; Dmitriev OY et al.; The structure of the A24D/D61N substituted subunit c of Escherichia coli ATP synthase, in which the essential carboxylate has been switched from residue 61 of the second transmembrane helix (TMH) to residue 24 of the first TMH, has been determined by heteronuclear multidimensional NMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture . As in the case of the wild-type protein, A24D/D61N substituted subunit c forms a hairpin of two extended alpha-helices (residues 5-39 and 46-78), with residues 40-45 forming a connecting loop at the center of the protein . The structure was determined at pH 5, where Asp24 is fully protonated . The relative orientation of the two extended helices in the A24D/D61N structure is different from that in the protonated form of the wild-type protein, also determined at pH 5 . The C-terminal helix is rotated by 150 degrees relative to the wild-type structure, and the N-terminal helix is rotated such that the essential Asp24 carboxyl group packs on the same side of the molecule as Asp61 in the wild-type protein . The changes in helix-helix orientation lead to a structure that is quite similar to that of the deprotonated form of wild-type subunit c, determined at pH 8 . When a decameric ring of c subunits was modeled from the new structure, the Asp24 carboxyl group was found to pack in a cavity at the interface between two subunits that is similar to the cavity in which Asp61 of the wild-type protein is predicted to pack . The interacting faces of the packed subunits in this model are also similar to those in the wild-type model . The results provide further evidence that subunit c is likely to fold in at least two conformational states differing most notably in the orientation of the C-terminal helix . Based upon the structure, a mechanistic model is discussed that indicates how the wild-type and A24D/D61N subunits could utilize similar helical movements during H(+) transport-coupled rotation of the decameric c ring.

Biochemistry, 2002 Apr 30, 41(17), 5505 - 14
The C terminus of apocytochrome b562 undergoes fast motions and slow exchange among ordered conformations resembling the folded state; D'Amelio N et al.; The present work describes the dynamics of the apo form of cytochrome b(562), a small soluble protein consisting of 106 amino acid residues {Itagaki, E., and Hager, L . P . (1966) J . Biol . Chem . 241, 3687-3695} . The presence of exchange in the millisecond time scale is demonstrated for the last part of helix IV (residues 95-105 in the holo form) . The chemical shift index analysis {Wishart, D . S., and Sykes, B . D . (1994) J . Biomol . NMR 4, 171-180} based on H(alpha), C(alpha), C(beta), and C' chemical shifts suggests a larger helical content than shown in the NMR structure based on NOEs . These results indicate the presence of helical-like conformations participating in the exchange process . This hypothesis is consistent with amide deuterium exchange rates and the presence of some hydrogen bonds identified from amide chemical shift temperature coefficients {Baxter, N . J., and Williamson, M . P . (1997) J . Biomol . NMR 9, 359-369} . (15)N relaxation indicates limited mobility for the amide protons of this part of the helix in the picosecond time scale . A 30 ns stochastic dynamics simulation shows small fluctuations around the helical conformation on this time scale . These fluctuations, however, do not result in a significant decrease of the calculated order parameters which are consistent with the experimental (15)N relaxation data . These results resolve an apparent discrepancy in the NMR structures between the disorder observed in helix IV due to a lack of NOEs and the secondary structure predictions based on H(alpha) chemical shifts {Feng, Y., Wand, A . J., and Sligar, S . G . (1994) Struct . Biol . 1, 30-35}.

Biochemistry, 2002 Apr 30, 41(17), 5333 - 9
A combinatorial approach toward analyzing functional elements of the Escherichia coli hemolysin signal sequence; Hui D et al.; Secretion of hemolysin is directed by a signal sequence located within its C-terminal 60 amino acids . Deletion analyses have indicated that the extreme end of this C-terminus is critical for transport; however, it is not known if this region contains structural features necessary for function . In this study, we have used a combinatorial approach to generate two contiguous 8-residue random libraries (Cterm1 and Cterm2) in the signal sequence to investigate the functional specificity of the last 16 residues . The large number of variants generated had provided us with a rich data set to determine if a restricted subset of sequences was actually required for function in the extreme C-terminus . We observed that over 90% of the random sequences in the Cterm1 region were secreted at close to wild-type level, while the Cterm2 region was more restricted with only 50% of the random sequences supporting wild-type-like transport . It appeared that, in the Cterm2 region, the relative lack of positive charge is favored for function . These findings, along with previous results, allow us to propose a model for recognition and transport of hemolysin that emphasizes secondary structure and general biophysical properties over primary sequence . This model may have implications for understanding the broad substrate specificity common among ATP-binding cassette transporters.

Biofizika, 2002 Mar-Apr, 47(2), 315 - 7
{Membrane potential before and after deep freezing of Escherichia coli in the presence of dimethyl sulfoxide and diethyl sulfoxide}; Markarian ShA et al.; It was shown by the method of penetrating tetraphenylphosphonium cations that low-temperature freezing (-196 degrees C) of Escherichia coli leads to a sharp decrease (from 198 to 85 mV) in membrane potential . Incubation of bacteria in a medium containing dimethyl sulfoxide and diethyl sulfoxide as cryoprotectors results in a reduction of the potential by 16 and 27 mV, respectively . It was also shown that diethyl sulfoxide is more effective in maintaining the membrane potential after freezing--thawing than dimethyl sulfoxide.

Biofizika, 2002 Mar-Apr, 47(2), 295 - 9
{The role of reactive oxygen species in copper-induced permeability of plasma membranes in Escherichia coli}; Lebedev VS et al.; The role of active oxygen species in the induction of nonselective cationic permeability of the plasma membrane of bacteria Escherichia coli B by the action of Cu2+ ions was studied . It was found that the increase in the amount of active oxygen species in the suspension after treating cells with copper occurred synchronously with the leakage of K+ cations from them . Evidence is presented that active oxygen species formed during the interaction of copper ions with bacteria under aerobic conditions are not involved in the induction of channel conductivity in the membrane . Moreover, the ability of oxygen to protect the membrane from the toxic action of copper was shown, and the activation of membrane damage by external reductants was confirmed . These data suggest that the barrier properties of the membrane are disturbed during the interaction of Cu+ ions with critical targets on the surface, the concentration of Cu+ being determined by all redox processes in the near-membrane space.

Surg Endosc . 2002 Feb;16(2):360 . Epub 2001 Nov 16.
Abdominal wall sinus due to impacting gallstone during laparoscopic cholecystectomy: an unusual complication; Pavlidis TE et al.; During laparoscopic cholecystectomy, perforation of the gallbladder can occurs in < or = 20% of cases, while gallstone spillage occurs in < or = 6% of cases . In most cases, there are no consequences . Gallstones can be lost in the abdominal wall as well as the abdomen during extraction of the gallbladder . The fate of such lost gallstones, which can lead to the formation of an abscess, an abdominal wall mass, or a persistent sinus, has not been studied adequately . Herein we report the case of a persistent sinus of the abdominal wall after an emergent laparoscopic cholecystectomy in an 82-year-old woman with gangrenous cholecystitis and perforation of the friable wall in association with an empyema of the gallbladder . The culture of the obtained pus was positive for Escherichia coli . After a small leak of dirty fluid from the wound of the epigastric port site of 4 months' duration, surgical exploration under local anesthesia revealed that the sinus was caused by spilled gallstones impacting into the abdominal wall between the posterior sheath and left rectus abdominalis muscle . The removal of the stones resulted in complete healing . Long-term complications after laparoscopic cholecystectomy involving the abdominal wall are rare but important possible consequences that could be avoided.

Nat Genet, 2002 May, 31(1), 64 - 8 Epub 2002 Apr 22.
Network motifs in the transcriptional regulation network of Escherichia coli; Shen-Orr SS et al.; Little is known about the design principles of transcriptional regulation networks that control gene expression in cells . Recent advances in data collection and analysis, however, are generating unprecedented amounts of information about gene regulation networks . To understand these complex wiring diagrams, we sought to break down such networks into basic building blocks . We generalize the notion of motifs, widely used for sequence analysis, to the level of networks . We define 'network motifs' as patterns of interconnections that recur in many different parts of a network at frequencies much higher than those found in randomized networks . We applied new algorithms for systematically detecting network motifs to one of the best-characterized regulation networks, that of direct transcriptional interactions in Escherichia coli . We find that much of the network is composed of repeated appearances of three highly significant motifs . Each network motif has a specific function in determining gene expression, such as generating temporal expression programs and governing the responses to fluctuating external signals . The motif structure also allows an easily interpretable view of the entire known transcriptional network of the organism . This approach may help define the basic computational elements of other biological networks.

Am J Obstet Gynecol, 2002 Apr, 186(4), 696 - 700
Defective regulation of the proinflammatory immune response in women with vulvar vestibulitis syndrome; Gerber S et al.; OBJECTIVE: The cause of vulvar vestibulitis syndrome is unknown . To determine a possible role for defective immune regulation in this chronic condition, proinflammatory and anti-inflammatory immune responses to the 70-kd heat shock protein and to lipopolysaccharide were compared in women with and without vulvar vestibulitis syndrome . STUDY DESIGN: Whole blood cultures from 62 women with vulvar vestibulitis syndrome and 48 control subjects were incubated in the presence or absence of 5 microg/mL human recombinant 70-kd heat shock protein or 0.1 ng/mL lipopolysaccharide for 18 hours . The culture supernatants were then assayed for interleukin-1 beta and interleukin-1 receptor antagonist by enzyme-linked immunosorbent assay . RESULTS: Median levels of interleukin-1 beta were higher in response to heat shock protein in cultures from patients with vulvar vestibulitis syndrome (median, 1.07 ng/mL) as opposed to control subjects (median, 0.40 ng/mL; P =.006) . Conversely, levels of interleukin-1 receptor antagonist were higher in response to heat shock protein in control subjects (median, 39.21 ng/mL) than in patients (median, 29.25 ng/mL; P =.009).In response to lipopolysaccharide, median levels of interleukin-1 beta were similar in patients (1.00 ng/mL) and control subjects (1.15 ng/mL); median interleukin-1 receptor antagonist concentrations were higher in control subjects (70.0 ng/mL) than in patients (44.3 ng/mL; P <.0001) . The ratio of interleukin-1 receptor antagonist to interleukin-1 beta was higher in control subjects than in women with vulvar vestibulitis syndrome in response to both heat shock protein (P =.0002) and lipopolysaccharide (P =.01) . In uninduced cultures, interleukin-1 receptor antagonist levels were also higher in control subjects (median, 1.60 ng/mL) than in patients with vulvar vestibulitis syndrome (median, 0.62 ng/mL; P <.0001) . CONCLUSION: A relative inability to down-regulate proinflammatory interleukin-1 beta activity by interleukin-1 receptor antagonist may contribute to the pathophysiologic features of vulvar vestibulitis syndrome.

Protein Sci, 2002 May, 11(5), 1192 - 8
Stability and interactions of the amino-terminal domain of ClpB from Escherichia coli; Tek V et al.; ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates aggregated proteins . The sequence of ClpB contains two ATP-binding regions that are enclosed between the N- and C-terminal extensions . Whereas it has been found that the N-terminal region of ClpB is essential for the chaperone activity, the structure of this region is not known, and its biochemical properties have not been studied . We expressed and purified the N-terminal fragment of ClpB (residues 1-147) . Circular dichroism of the isolated N-terminal region showed a high content of alpha-helical structure . Differential scanning calorimetry showed that the N-terminal region of ClpB is thermodynamically stable and contains a single folding domain . The N-terminal domain is monomeric, as determined by gel-filtration chromatography, and the elution profile of the N-terminal domain does not change in the presence of the N-terminally truncated ClpB (ClpBDeltaN) . This indicates that the N-terminal domain does not form strong contacts with ClpBDeltaN . Consistently, addition of the separated N-terminal domain does not reverse an inhibition of ATPase activity of ClpBDeltaN in the presence of casein . As shown by ELISA measurements, full-length ClpB and ClpBDeltaN bind protein substrates (casein, inactivated luciferase) with similar affinity . We also found that the isolated N-terminal domain of ClpB interacts with heat-inactivated luciferase . Taken together, our results indicate that the N-terminal fragment of ClpB forms a distinct domain that is not strongly associated with the ClpB core and is not required for ClpB interactions with other proteins, but may be involved in recognition of protein substrates.

Protein Sci, 2002 May, 11(5), 1136 - 51
Role of individual disulfide bonds in hen lysozyme early folding steps; Guez V et al.; To probe the role of individual disulfide bonds in the folding kinetics of hen lysozyme, the variants with two mutations, C30A,C115A, C64A,C80A, and C76A,C94A, were constructed . The corresponding proteins, each lacking one disulfide bond, were produced in Escherichia coli as inclusion bodies and solubilized, purified, and renatured/oxidized using original protocols . Their enzymatic, spectral, and hydrodynamic characteristics confirmed that their conformations were very similar to that of native wild-type (WT) lysozyme . Stopped-flow studies on the renaturation of these guanidine-unfolded proteins with their three disulfides intact showed that, for the three variants, the native far-UV ellipticity was regained in a burst phase within the 4-ms instrument dead-time . The transient overshoots of far-UV ellipticity and tryptophan fluorescence that follow the burst phase, as well as the kinetics of transient 8-anilino-1-naphthalene-sulfonic acid (ANS) binding, were diversely affected depending on the variant . Together with previous reports on the folding kinetics of WT lysozyme carboxymethylated on cysteines 6 and 127, detailed analysis of the kinetics showed that (1) none of the disulfide bonds were indispensable for the rapid formation (<4 ms) of the native-like secondary structure; (2) the two intra-alpha-domain disulfides (C6-C127 and C30-C115) must be simultaneously present to generate the trapped intermediate responsible for the slow folding population observed in WT lysozyme; and (3) the intra-beta-domain (C64-C80) and the inter-alphabeta-domains (C76-C94) disulfides do not affect the kinetics of formation of the trapped intermediate but are involved in its stability.

Protein Sci, 2002 May, 11(5), 1129 - 35
Presence of the cofactor speeds up folding of Desulfovibrio desulfuricans flavodoxin; Apiyo D et al.; Flavodoxin is an alpha/beta protein with a noncovalently bound flavin-mononucleotide (FMN) cofactor . The apo-protein adopts a structure identical to that of the holo-form, although there is more dynamics in the FMN-binding loops . The equilibrium unfolding processes of Azotobacter vinelandii apo-flavodoxin, and Desulfovibrio desulfuricans ATCC strain 27774 apo- and holo-flavodoxins involve rather stable intermediates . In contrast, we here show that both holo- and apo-forms of flavodoxin from D . desulfuricans ATCC strain 29577 (75% sequence similarity with the strain 27774 protein) unfold in two-state equilibrium processes . Moreover, the FMN cofactor remains bound to the unfolded holo-protein . The folding and unfolding kinetics for holo-flavodoxin exhibit two-state behavior, albeit an additional slower phase is present at very low denaturant concentrations . The extrapolated folding time in water for holo-flavodoxin, approximately 280 microsec, is in excellent agreement with that predicted from the protein's native-state topology . Unlike the holo-protein behavior, the folding and unfolding reactions for apo-flavodoxin are best described by two kinetic phases, with rates differing approximately 15-fold, suggesting the presence of a kinetic intermediate . Both folding phases for apo-flavodoxin are orders of magnitude slower (40- and 530-fold, respectively) than that for the holo-protein . We conclude that polypeptide-cofactor interactions in the unfolded state of D . desulfuricans strain 29577 flavodoxin alter the kinetic-folding path towards two-state and speed up the folding reaction.

Protein Sci, 2002 May, 11(5), 1117 - 28
Characterization of Ad5 E3-14.7K, an adenoviral inhibitor of apoptosis: structure, oligomeric state, and metal binding; Kim HJ et al.; The adenovirus E3-14.7K protein, expressed early in the life cycle of human adenoviruses to protect the virus from the antiviral response of host cells, inhibits cell death mediated by TNF-alpha and FasL receptors . To better understand its role in cell death inhibition, we have sought to characterize the biophysical properties of the protein from adenovirus serotype 5 (Ad5 E3-14.7K, or simply 14.7K) through a variety of approaches . To obtain sufficient quantities of recombinantly expressed protein for biophysical characterization, we explored the use of various expression constructs and chaperones; fusion to MBP was by far the most effective at generating soluble protein . Using limited proteolysis, mass spectrometry, and protein-protein interaction assays, we demonstrate that the C-terminal two-thirds of the protein, predicted to be composed of five beta-strands and one alpha-helix, is highly structured and binds its putative cellular receptors . Furthermore, using atomic absorption and ultraviolet/visible spectroscopies, we have studied the metal binding properties of the protein, providing insight into the observation that cysteine/serine mutants of 14.7K lack in vivo antiapoptotic activity . Lastly, results from size exclusion chromatography, dynamic light scattering, sucrose gradient sedimentation, chemical crosslinking, and electron microscopy experiments revealed that 14.7K exists in a stable high-order oligomeric state (nonamer) in solution.

Protein Sci, 2002 May, 11(5), 1062 - 73
The role of the conserved Lys68*:Glu265 intersubunit salt bridge in aspartate aminotransferase kinetics: multiple forced covariant amino acid substitutions in natural variants; Deu E et al.; The role of the Lys68*:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase . Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis . Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context . Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters . The differential sensitivity of k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former . Thus, k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) are 133 and 442 mM(-1)s(-1) for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM(-1)s(-1) for K68E) . A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes . The more deeply rooted tree indicates that the Csb variant was the ancestral specie.

Protein Sci, 2002 May, 11(5), 1026 - 35
Timing and structural consideration for the processing of mitochondrial matrix space proteins by the mitochondrial processing peptidase (MPP); Mukhopadhyay A et al.; Most mitochondrial matrix space proteins are synthesized as a precursor protein, and the N-terminal extension of amino acids that served as the leader sequence is removed after import by the action of a metalloprotease called mitochondrial processing peptidase (MPP) . The crystal structure of MPP has been solved very recently, and it has been shown that synthetic leader peptides bind with MPP in an extended conformation . However, it is not known how MPP recognizes hundreds of leader peptides with different primary and secondary structures or when during import the leader is removed . Here we took advantage of the fact that the structure of the leader from rat liver aldehyde dehydrogenase has been determined by 2D-NMR to possess two helical portions separated by a three amino acid (RGP) linker . When the linker was deleted, the leader formed one long continuous helix that can target a protein to the matrix space but is not removed by the action of MPP . Repeats of two and three leaders were fused to the precursor protein to determine the stage of import at which processing occurs, if MPP could function as an endo peptidase, and if it would process if the cleavage site was part of a helix . Native or linker deleted constructs were used . Import into isolated yeast mitochondria or processing with recombinantly expressed MPP was performed . It was concluded that processing did not occur as the precursor was just entering the matrix space, but most likely coincided with the folding of the protein . Further, finding that hydrolysis could not take place if the processing site was part of a stable helix is consistent with the crystal structure of MPP . Lastly, it was found that MPP could function at sites as far as 108 residues from the N terminus of the precursor protein, but its ability to process decreases exponentially as the distance increases.

J Biol Chem, 2002 Jun 28, 277(26), 23321 - 9 Epub 2002 Apr 19.
Structural and enzymatic characterization of Drosophila Dm2-MMP, a membrane-bound matrix metalloproteinase with tissue-specific expression; Llano E et al.; We report the isolation and characterization of a cDNA encoding Dm2-MMP, the second matrix metalloproteinase (MMP) identified in the Drosophila melanogaster genome . The cloned cDNA codes for a polypeptide of 758 residues that displays a domain organization similar to that of other MMPs, including signal peptide, propeptide, catalytic, and hemopexin domains . However, the structure of Dm2-MMP is unique because of the presence of an insertion of 214 amino acids between the catalytic and hemopexin domains that is not present in any of the previously described MMPs . Dm2-MMP also contains a C-terminal extension predicted to form a cleavable glycosylphosphatidylinositol anchor site . Western blot and immunofluorescence analysis of S2 cells transfected with the isolated cDNA confirmed that Dm2-MMP is localized at the cell surface . Production of the catalytic domain of Dm2-MMP in Escherichia coli and analysis of its enzymatic activity revealed that this proteinase cleaves several synthetic peptides used for analysis of vertebrate MMPs . This proteolytic activity was abolished by MMP inhibitors such as BB-94, confirming that the isolated cDNA codes for an enzymatically active metalloproteinase . Reverse transcription-PCR analysis showed that Dm2-MMP is expressed at low levels in all of the developmental stages of Drosophila as well as in adult flies . However, detailed in situ hybridization at the larval stage revealed a strong tissue-specific expression in discrete regions of the brain and eye imaginal discs . According to these results, we propose that Dm2-MMP plays both general proteolytic functions during Drosophila development and in adult tissues and specific roles in eye development and neural tissues through the degradation and remodeling of the extracellular matrix.

Biol Reprod, 2002 May, 66(5), 1498 - 504
Teleost ovarian carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase: potential role in the production of maturation-inducing hormone during final oocyte maturation; Tanaka M et al.; 17alpha,20beta-Dihydroxy-4-pregnen-3-one is the major oocyte maturation-inducing hormone of several teleost species . Gonadotropin-induced increase in ovarian 20beta-hydroxysteroid dehydrogenase activity is essential for the synthesis of maturation-inducing hormone . Cloning and expression studies suggest that ayu (Plecoglossus altivelis) ovarian carbonyl reductase can function as 20beta-hydroxysteroid dehydrogenase . The amino acid sequence deduced from the isolated cDNA had 276 amino acid residues and shared approximately 60% homology with mammalian and teleostean carbonyl reductases . The sequence data search showed that the ayu cDNA clone belongs to the short-chain dehydrogenase/reductase family . The clear lysate prepared from Escherichia coli harboring the cDNA catalyzed the production of maturation-inducing hormone . Its identification was confirmed by two-dimensional, thin-layer chromatography followed by recrystallization . Purification of the E . coli-expressed cDNA product revealed that it possessed both carbonyl reductase and steroid dehydrogenase activities, and 17alpha-hydroxyprogesterone, the endogenous immediate precursor of maturation-inducing hormone, was one of the preferred substrates . Furthermore, Northern blot analysis denoted that the transcripts are present both in fully grown, immature ovarian follicles and at higher levels in mature ovarian follicles . These results demonstrate that the carbonyl reductase of ayu ovary is involved in the production of maturation-inducing hormone, and they provide evidence for a novel physiological role of this enzyme in the final maturation of oocytes . Based on its functional properties, the enzyme can be referred to as carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase.

Scand J Immunol, 2002 Apr, 55(4), 366 - 72
A heat-stable component of Bartonella henselae upregulates intercellular adhesion molecule-1 expression on vascular endothelial cells; Maeno N et al.; Bartonella henselae upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs) . The induction level of ICAM-1 depended on the inoculation bacterial dose . ICAM-1 expression began increasing 4 h after infection and reached a sustained peak beginning at 12 h after B . henselae infection; this time course was similar to that of lipopolysaccharide (LPS) of Escherichia coli . The stimulatory effect was abolished when live B . henselae were separated from HUVECs by a filter membrane . The nonpiliated strain, which is unable to invade endothelial cells, induced ICAM-1 expression to the same extent as the piliated strain . Inactivation of B . henselae by ultraviolet (UV) irradiation, heat (56 degrees C, 30 min), or sonication did not alter its stimulatory activity . Polymyxin B, which strongly inhibited the effect of LPS, did not exert any influence on the stimulatory activity of B . henselae . Furthermore, the effect of sonicated B . henselae was not inhibited even by boiling, which was also the case with LPS . Our data suggest that some heat-stable component of B . henselae binds to the endothelial cell surface, inducing ICAM-1 expression . Though the participation of LPS could not be completely ruled out, we suppose that some unidentified heat-stable proteins, lipids, or polysaccharides may be the stimulatory factor(s) . The ability of B . henselae to enhance the expression of adhesion molecules on endothelial cells may be an important mechanism in the pathogenesis of B . henselae infection.

Mol Microbiol, 2002 Apr, 44(1), 245 - 55
Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation; Brondijk TH et al.; The nap operon of Escherichia coli K-12, encoding a periplasmic nitrate reductase (Nap), encodes seven proteins . The catalytic complex in the periplasm, NapA-NapB, is assumed to receive electrons from the quinol pool via the membrane-bound cytochrome NapC . Like NapA, B and C, a fourth polypeptide, NapD, is also essential for Nap activity . However, none of the remaining three polypeptides, NapF, G and H, which are predicted to encode non-haem, iron-sulphur proteins, are essential for Nap activity, and their function is currently unknown . The relative rates of growth and electron transfer from physiological substrates to Nap have been investigated using strains defective in the two membrane-bound nitrate reductases, and also defective in either ubiquinone or menaquinone biosynthesis . The data reveal that Nap is coupled more effectively to menaquinol oxidation than to ubiquinol oxidation . Conversely, parallel experiments with a second set of mutants revealed that nitrate reductase A couples more effectively with ubiquinol than with menaquinol . Three further sets of strains were constructed with combinations of in frame deletions of ubiCA, menBC, napC, napF and napGH genes . NapF, NapG and NapH were shown to play no role in electron transfer from menaquinol to the NapAB complex but, in the Ubi+Men- background, deletion of napF, napGH or napFGH all resulted in total loss of nitrate-dependent growth . Electron transfer from ubiquinol to NapAB was totally dependent upon NapGH, but not on NapF . NapC was essential for electron transfer from both ubiquinol and menaquinol to NapAB . The results clearly established that NapG and H, but not NapF, are essential for electron transfer from ubiquinol to NapAB . The decreased yield of biomass resulting from loss of NapF in a Ubi+Men+ strain implicates NapF in an energy- conserving role coupled to the oxidation of ubiquinol . We propose that NapG and H form an energy- conserving quinol dehydrogenase functioning as either components of a proton pump or in a Q cycle, as electrons are transferred from ubiquinol to NapC.

Mol Microbiol, 2002 Apr, 44(1), 205 - 16
Selective expression of the beta-subunit of nucleoid-associated protein HU during cold shock in Escherichia coli; Giangrossi M et al.; Expression of Escherichia coli hupA and hupB, the structural genes encoding the most abundant nucleoid-associated proteins HUalpha and HUbeta has been studied during cold shock . This article demonstrates that: (i) transcriptional expression of hupA is blocked following a sudden temperature downshift (from 37 degrees C to 10 degrees C), whereas transcription of hupB from the P2 and P3 promoters is maintained at a constitutive level and is activated de novo from the P4 promoter; (ii) all three hupB mRNAs (transcribed from the three natural promoters P2, P3 and P4) become much more stable than the single hupA transcript; and (iii) the hupB transcripts, unlike that of hupA, are efficiently translated in vivo during cold acclimation and can be actively translated in vitro at low temperature . Taken together, the results indicate that during cold shock the expression of the HUbeta subunit is preferentially stimulated and that of HUalpha repressed, suggesting that an altered HUalpha to HUbeta expression ratio resulting in an increase of HUalpha/HUbeta heterodimers and/or (HUbeta)2 homodimers may play an important role during cold adaptation.

Mol Microbiol, 2002 Apr, 44(1), 131 - 41
Expression of mutant alanine tRNAs increases spontaneous mutagenesis in Escherichia coli; Dorazi R et al.; The expression of mutA, an allele of the glycine tRNA gene glyV, can confer a novel mutator phenotype that correlates with its ability to promote Asp-->Gly mistranslation . Both activities are mediated by a single base change within the anticodon such that the mutant tRNA can decode aspartate codons (GAC/U) instead of the normal glycine codons (GCC/U) . Here, we investigate whether specific Asp-->Gly mistranslation is required for the unexpected mutator phenotype . To address this question, we created and expressed 18 individual alleles of alaV, the gene encoding an alanine tRNA, in which the alanine anticodon was replaced with those specifying other amino acids such that the mutant (alaVX) tRNAs are expected to potentiate X-->Ala mistranslation, where X is one of the other amino acids . Almost all alaVX alleles proved to be mutators in an assay that measured the frequency of rifampicin-resistant mutants, with one allele (alaVGlu) being a stronger mutator than mutA . The alaVGlu mutator phenotype resembles that of mutA in mutational specificity (predominantly transversions), as well as SOS independence, but in a puzzling twist differs from mutA in that it does not require a functional recA gene . Our results suggest that general mistranslation (as opposed to Asp-->Gly alone) can induce a mutator phenotype . Furthermore, these findings predict that a large number of conditions that increase translational errors, such as genetic defects in the translational apparatus, as well as environmental and physiological stimuli (such as amino acid starvation or exposure to antibiotics) are likely to activate a mutator response . Thus, both genetic and epigenetic mechanisms can accelerate the acquisition of mutations.

Mol Microbiol, 2002 Apr, 44(1), 119 - 30
An intrinsic control element for translational initiation in class 1 integrons; Hanau-Bercot B et al.; Integrons are genetic elements able to capture anti-biotic resistance and other genes and to promote their transcription . Here, we have investigated integron-dependent translation of an aminoglycoside 6'-N-acetyltransferase gene (aac(6')-Ib7) inserted at the attI1 site . N-terminal sequencing revealed that translation of this gene was initiated at a GTG codon, which is not part of a plausible translation initiation region (TIR) . A short open reading frame (called ORF-11) overlapping the attI1 site was probed by site-directed mutagenesis for its contribution to aac(6')-Ib7 translation . When ORF-11 and its TIR were deleted en bloc, translational efficiency dropped by over 80%, as determined with an acetyltransferase- luciferase fusion product . Invalidation of the ATG start codon of ORF-11 or its putative Shine-Dalgarno sequence resulted in a decrease of over 60%, whereas the decrease was much less pronounced when the amino acid sequence of the putative ORF-11-encoded peptide was altered or when the distance between ORF-11 and aac(6')-Ib7 was doubled . This demonstrates that aac(6')-Ib7 translation is dependent upon the translation of ORF-11, but almost certainly not upon the corresponding peptide . These results lead us to conclude that an intrinsic short ORF present in the 5'-conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR-deficient anti-biotic resistance genes.

Mol Microbiol, 2002 Apr, 44(1), 89 - 105
Over 1000 genes are involved in the DNA damage response of Escherichia coli; Khil PP et al.; Changes in gene expression after treatment of Escherichia coli cultures with mitomycin C were assessed using gene array technology . Unexpectedly, a large number of genes (nearly 30% of all genes) displayed significant changes in their expression level . Analysis and classification of expression profiles of the corresponding genes allowed us to assign this large number of genes into one or two dozen small clusters of genes with similar expression profiles . This assignment allowed us to describe systematically the changes in the level of gene expression in response to DNA damage . Among the damage-induced genes, more than 100 are novel . From those genes involved in DNA metabolism that have not previously been shown to be induced by DNA damage, the mutS gene involved in mismatch repair is especially noteworthy . In addition to the SOS response, we observed the induction of other stress response pathways, such as those of oxidative stress and osmotic protection . Among the genes that are downregulated in response to DNA damage are numerous protein biosynthesis genes . Analysis of the gene expression data highlighted the essential involvement of sigma(s)-regulated genes and the general stress response network in the response to DNA damage.

Kidney Int, 2002 May, 61(5), 1880 - 6
Acute graft pyelonephritis and long-term kidney allograft outcome; Giral M et al.; BACKGROUND: Long-term graft function is the result of multiple parameters, including both immune and non-immune components, which have a beneficial or detrimental potential . Among these, despite its frequency and theoretical interest (expression of "danger signals" in the graft itself), the effects of acute graft pyelonephritis (AGPN) on immediate and long-term outcome have not been studied in a large series . This article reviews a cohort of 1387 consecutive primary renal transplant recipients . METHODS: The objective of the study was to define the risk factor for AGPN, the risk profile for recurrence, and the impact of AGPN on long-term graft survival . According to a higher risk for AGPN in females during their follow-up, statistical analyses (Cox model, and multiple regression analysis) were performed by recipient sex strata . RESULTS: Multivariate analysis showed that CMV infection was the only risk factor for AGPN occurrence . AGPN occurred in 13% of the graft recipients during their follow-up . Taken as a whole, AGPN was not associated with a significantly poor long-term outcome . However, when assessed in more detail, the outcome of this population was found to be more complex and to depend on several factors . Early AGPN (during the first 3 months) was significantly detrimental for graft outcome, independently of acute rejection episodes . Moreover, E . coli involvement in a first episode was linked to an increased AGPN recurrence . CONCLUSION: This analysis did not support the concept that with current immunosuppression, strong "danger signals" such as those derived from bacteria within an allograft, are instrumental in initiating acute or chronic rejection.

Kidney Int, 2002 May, 61(5), 1666 - 73
Human antiglomerular basement membrane autoantibody disease in XenoMouse II; Meyers KE et al.; BACKGROUND: Previous studies have identified regions within alpha3(IV) collagen in human antiglomerular basement membrane (anti-GBM) disease, however, information pertaining to the nature of the pathogenic human autoantibodies has been limited by a lack of a relevant disease model . Availability of engineered mice that produce antibodies (that is, XenoMouse II strains) provides an ideal opportunity to examine the human antibody response . METHODS: XenoMouse II mice that produce human IgG2 (gamma2kappa) in response to antigenic challenge were immunized with various forms of alpha3(IV)NC1 GBM collagen, including native bovine alpha3(IV) NCl collagen, E . coli expressed r alpha3(IV)NCl, and mammalian fetal kidney 293 cell expressed r alpha3(IV)NC1 preparations . The mice were evaluated for autoantibody (Ab) production and nephritis . RESULTS: All immunized XenoMouse II animals produced human anti-GBM Ab associated with proliferative glomerulonephritis, linear IgG deposits along the murine GBM and tubular basement membrane (TBM), C3 deposits (weaker) . A fully human mAb (Ig gamma2kappa), produced from a mouse immunized with native bovine alpha3(IV)NCl collagen produced basement membrane deposits, nephritis and proteinuria on transfer to normal XenoMouse II . Furthermore, monoclonal antibodies (mAb) shared idiotypic properties with polyclonal autoantibodies derived from patients with anti-GBM disease, supporting a structural relationship among the antibodies . CONCLUSIONS: The results further support the importance of alpha3(IV)NCl collagen in the pathogenesis of anti-GBM disease . Moreover, to our knowledge this is the first demonstration that experimentally induced, pathogenic human autoantibodies result in disease . This new model of anti-GBM disease, therefore, provides the means and unique reagents to both decipher the molecular basis of the human anti-GBM autoantibody response and the opportunity to test specific therapies aimed at modulation of either B cells producing human autoantibodies or the human pathogenic antibodies themselves, in vivo, prior to trial in patients with the spontaneous form of the disease.

J Appl Microbiol, 2002, 92(4), 649 - 56
Concentration of Cryptosporidium, microsporidia and other water-borne pathogens by continuous separation channel centrifugation; Borchardt MA et al.; AIMS: The aim of this study was to determine the effectiveness of continuous separation channel centrifugation for concentrating water-borne pathogens of various taxa and sizes . METHODS AND RESULTS: Cryptosporidium parvum oocysts, Giardia lamblia cysts, Encephalitozoon intestinalis spores and Escherichia coli were seeded into different water matrices at densities ranging from 5 to 10 000 organisms l(-1) and recovered using continuous separation channel centrifugation . All pathogens were enumerated on membrane filters using microscopy . Recovery efficiencies were usually > 90% . Oocyst recovery did not vary with source water turbidity or with centrifuge flow rate up to 250 ml min(-1) . Based on excystation, this concentration method did not alter oocyst viability . CONCLUSIONS: Continuous separation channel centrifugation is an effective means of concentrating water-borne pathogens . SIGNIFICANCE AND IMPACT OF THE STUDY: Methods are needed for detecting pathogens in drinking water to ensure public health . The first step for any pathogen detection procedure is concentration . However, this step has been problematic because recovery efficiencies of conventional methods, like filtration, are often low and variable, which may lead to false negatives . Continuous separation channel centrifugation can simultaneously concentrate multiple pathogens as small as 1 microm with high and reproducible efficiency in a variety of water matrices.

Aliment Pharmacol Ther, 2002 Apr, 16 Suppl 2, 167 - 73
High molecular protein of Helicobacter pylori responsible for inhibition of ornithine decarboxylase activity of human gastric cultured cells; Takashima T et al.; BACKGROUND: Ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, mediates epithelial cell proliferation and plays a critical role in the optimal repair of gastric mucosal damage . Several studies have shown that Helicobacter pylori inhibits the growth and proliferation of gastric cells in vitro . AIM: To test whether H . pylori extract affects ODC mRNA expression and its enzyme activity in gastric cells and to examine the partial characterization of the molecule responsible for this effect . METHODS: Human gastric cells (MKN-45) were used . Bacterial extracts from various E . coli or H . pylori strains, namely (1) cagA+, vacA+, CagA+, VacA+; (2) cagA+, vacA+, CagA+ VacA-; or (3) cagA-, vacA+, CagA-, VacA- were added to the cells . Cell proliferation was assessed by {3H}-thymidine incorporation, viability by MTT assay and LDH release test, ODC enzyme activity by 14CO2 counts from L-{1(14)C}ornithine, and ODC mRNA by Northern blotting . RESULTS: H . pylori and E . coli extract did not affect viability of gastric cells . H . pylori extract, especially extracts containing a protein greater than 50 kDa, significantly inhibited proliferation and ODC activity of gastric cells while E . coli extract had no effect . Inhibition of ODC activity was found in extracts of all H . pylori strains, irrespective of CagA and VacA protein expression . Serum stimulation induces an increase in ODC mRNA while H . pylori extract did not affect ODC mRNA expression . CONCLUSION: High molecular weight (greater than 50 kDa) proteins of H . pylori extract without CagA or VacA protein inhibited proliferation and ODC activity of human gastric cells, but did not affect ODC mRNA expression, suggesting that inhibition of ODC activity is regulated at the post-transcriptional level.

Comb Chem High Throughput Screen, 2002 May, 5(3), 253 - 9
Parallel synthesis of PNA-peptide conjugate libraries; Awasthi SK et al.; An optimized semi-automatic protocol for parallel synthesis of up to 96 peptide nucleic acids (PNA) or PNA-peptide conjugates using Boc-protection strategy has been developed using a robotic system . The approach is illustrated by synthesizing PNA and PNA-peptide libraries varying between 15 and 27 amino acid units . The peptides (NLS (nuclear localization signal) or Tat-peptide) were attached to N-terminus of the PNA . The method was found to be far superior to that based on the SPOT/Fmoc protocol by which PNA oligomers are synthesized on a modified cellulose membrane . On a 0.5 micromole scale the method typically yielded 2 mg product of 90% purity by HPLC/MALDI-TOF analysis . This approach is suitable for screening of a large number of PNA and/or peptide sequences for biochemical and biological studies.

Medicina (B Aires), 2002, 62(1), 66 - 72
{Therapeutic applications of pore-forming lytic toxins: potential use of Escherichia coli alpha-hemolysin}; Herlax V et al.; Many infectious bacteria export soluble proteins which can damage the plasma membrane of eukaryotic cells . Most often they are directed against leukocytes for the purpose of reducing the immune response of the host . In some cases, these toxins are also hemolytic . It has been proposed that both leukotoxic and hemolytic activities could derive from the pore formation in the membranes of the attacked cells . The study of these molecules is not only important from the point of view of basic studies to determine the mechanism of action, but also for potential application in biotechnology and medicine . These molecules increase the cell susceptibility to chemotherapy and also can be employed to destroy specifically cancer cells . On the other hand, it is possible to incorporate toxin molecules in liposomes, transforming them in to biosensors or as controlled drug delivery systems . This aspect has not been extensively explored in Escherichia coli alpha-hemolysin, in which the presence of different functional and structural domains in this molecule could be taken advantage of.

J Mol Evol, 2002 May, 54(5), 692 - 702
Group I introns from Zygomycota: evolutionary implications for the fungal IC1 intron subgroup; Tanabe Y et al.; The origins of fungal group I introns within nuclear small-subunit (nSSU) rDNA are enigmatic . This is partly because they have never been reported in basal fungal phyla (Zygomycota and Chytridiomycota), which are hypothesized to be ancestral to derived phyla (Ascomycota and Basidiomycota) . Here we report group I introns from the nSSU rDNA of two zygomycete fungi, Zoophagus insidians (Zoopagales) and Coemansia mojavensis (Kickxellales) . Secondary structure analyses predicted that both introns belong to the IC1 subgroup and that they are distantly related to each other, which is also suggested by different insertion sites . Molecular phylogenetic analyses indicated that the IC1 intron of Z . insidians is closely related to the IC1 intron inserted in the LSU rDNA of the basidiomycete fungus Clavicorona taxophila, which strongly suggests interphylum horizontal transfer . The IC1 intron of C . mojavensis has a low phylogenetic affinity to other fungal IC1 introns inserted into site 943 of nSSU rDNA (relative to E . coli 16S rDNA) . It is noteworthy that this intron contains a putative ORF containing a His-Cys box motif in the antisense strand, a hallmark for nuclear-encoded homing endonucleases . Overall, molecular phylogenetic analyses do not support the placement of these two introns in basal fungal IC1 intron lineages . This result leads to the suggestion that fungal IC1 introns might have invaded or been transferred laterally after the divergence of the four major fungal phyla.

Curr Opin Infect Dis, 2001 Oct, 14(5), 567 - 71
Post-infective diarrhoea; Walker-Smith JA; When diarrhoea caused by gastroenteritis persists for more than two weeks it is referred to as persistent diarrhoea in developing countries . Whilst the Control of Diarrhoeal Diseases programme has decreased mortality from acute diarrhoea, mortality from persistent diarrhoea has not been so responsive . A number of factors have been identified which are determinants for the progression of an acute episode to one which persists in developing communities . In one study from west Africa, current infection with Cryptosporidium parvum was the most significant factor . In studies from Brazil and India, continuing infection with enteropathogenic Escherichia coli was identified in 50% of infants with persistent diarrhoea . Persistent small intestinal mucosal damage is of key importance in such children . Management of established cases is complex and difficult . However, there is clear evidence that zinc is involved in the recovery of small intestinal mucosa after injury . Zinc supplementation may indeed significantly reduce the duration of persistent diarrhoea . However, the whole question of public health supplementation with zinc, vitamin A, or other supplements, is contentious at present.

Curr Opin Infect Dis, 2001 Oct, 14(5), 559 - 65
Intimate interactions of enteropathogenic Escherichia coli at the host cell surface; Delahay RM et al.; Unlike many gastrointestinal pathogens, enteropathogenic Escherichia coli orchestrates the modulation of host cellular and immune responses from the exterior of the infected cell, chiefly via the secreted and translocated components of a type III secretion system . Close inspection of these enteropathogenic Escherichia coli proteins and the interactions they mediate provides an increasingly coherent picture of the pathogenic mechanisms that enteropathogenic Escherichia coli uses to exploit its host.

Curr Opin Infect Dis, 2000 Oct, 13(5), 511 - 517
Enteropathogenic and enterohaemorrhagic Escherichia coli and diarrhoea; Roe AJ et al.; Enteropathogenic and enterohaemorrhagic Escherichia coli are important causes of bacterial gastroenteritis with the potential for progression to more serious syndromes, especially in the case of enterohaemorrhagic E . coli . Consequently, recent developments in molecular epidemiology and treatment regimens have focused on enterohaemorrhagic E . coli, while the similar initial pathogenic mechanisms of both enterohaemorrhagic and enteropathogenic E . coli continue to be investigated in detail . The carriage of most E . coli virulence determinants on pathogenicity islands, plasmids or phages allows the rapid evolution of these pathotypes, which need to be monitored closely.

Anesthesiology, 2002 Apr, 96(4), 926 - 33
Radical scavengers protect murine lungs from endotoxin-induced hyporesponsiveness to inhaled nitric oxide; Raveh Y et al.; BACKGROUND: Sepsis is associated with an impaired pulmonary vasodilator response to inhaled nitric oxide (NO) . A combination of NO and other inflammatory mediators appears to be responsible for endotoxin-induced pulmonary vascular hyporesponsiveness to inhaled NO . The authors investigated whether scavengers of reactive oxygen species could preserve inhaled NO responsiveness in endotoxin-challenged mice . METHODS: The vasorelaxation to inhaled NO was studied in isolated, perfused, and ventilated lungs obtained from mice 16 h after an intraperitoneal challenge with saline or 50 mg/kg Escherichia coli lipopolysaccharide . In some mice, challenge with saline or lipopolysaccharide was followed by intraperitoneal administration of N-acetylcysteine, dimethylthiourea, EUK-8, or polyethylene glycol-conjugated catalase . RESULTS: The pulmonary vasodilator response of U46619-preconstricted isolated lungs to ventilation with 0.4, 4, and 40 ppm inhaled NO in lipopolysaccharide-challenged mice was reduced to 32, 43, and 60%, respectively, of that observed in saline-challenged mice (P < 0.0001) . Responsiveness to inhaled NO was partially preserved in lipopolysaccharide-challenged mice treated with a single dose of N-acetylcysteine (150 or 500 mg/kg) or 20 U/g polyethylene glycol-conjugated catalase (all P < 0.05 vs . lipopolysaccharide alone) . Responsiveness to inhaled NO was fully preserved by treatment with either dimethylthiourea, EUK-8, two doses of N-acetylcysteine (150 mg/kg administered 3.5 h apart), or 100 U/g polyethylene glycol-conjugated catalase (all P < 0.01 vs . lipopolysaccharide alone) . CONCLUSIONS: When administered to mice concurrently with lipopolysaccharide challenge, reactive oxygen species scavengers prevent impairment of pulmonary vasodilation to inhaled NO . Therapy with scavengers of reactive oxygen species may provide a means to preserve pulmonary vasodilation to inhaled NO in sepsis-associated acute lung injury.

Science, 2002 Apr 19, 296(5567), 525 - 30
Control of the selectivity of the aquaporin water channel family by global orientational tuning; Tajkhorshid E et al.; Aquaporins are transmembrane channels found in cell membranes of all life forms . We examine their apparently paradoxical property, facilitation of efficient permeation of water while excluding protons, which is of critical importance to preserving the electrochemical potential across the cell membrane . We have determined the structure of the Escherichia coli aquaglyceroporin GlpF with bound water, in native (2.7 angstroms) and in W48F/F200T mutant (2.1 angstroms) forms, and carried out 12-nanosecond molecular dynamics simulations that define the spatial and temporal probability distribution and orientation of a single file of seven to nine water molecules inside the channel . Two conserved asparagines force a central water molecule to serve strictly as a hydrogen bond donor to its neighboring water molecules . Assisted by the electrostatic potential generated by two half-membrane spanning loops, this dictates opposite orientations of water molecules in the two halves of the channel, and thus prevents the formation of a "proton wire," while permitting rapid water diffusion . Both simulations and observations revealed a more regular distribution of channel water and an increased water permeability for the W48F/F200T mutant.

J Biol Chem, 2002 Jul 5, 277(27), 24435 - 41 Epub 2002 Apr 18.
Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading; Kulkarni S et al.; Integrin-induced cell adhesion results in transmission of signals that induce cytoskeletal reorganizations and resulting changes in cell behavior . The cytoskeletal reorganizations are regulated by transient activation and inactivation of Rho GTPases . Previously, we identified mu-calpain as an enzyme that is activated by signaling across beta1 and beta3 integrins . We showed that it mediates cytoskeletal reorganizations in bovine aortic endothelial (BAE) and Chinese hamster ovary (CHO) cells and does so by acting upstream of Rac1 activation . Here we show that mu-calpain is also involved in inactivating RhoA during integrin-induced signaling . Cleavage of RhoA was detectable in BAE cells plated on an integrin substrate; it did not occur in cells plated on poly-l-lysine . Cleavage was inhibited by calpain inhibitors . In vitro, mu-calpain cleaved RhoA generating a fragment of the same size as in intact cells . The cleavage site was identified, an HA-tagged construct expressing calpain-cleaved RhoA generated, and the construct expressed in BAE and CHO cells . Calpain-cleaved RhoA inhibited integrin-induced stress fiber assembly and decreased cell spreading . Together, our data show that calpain cleaves RhoA and generates a form that inhibits integrin-induced stress fiber assembly and cell spreading.

J Biol Chem, 2002 Jun 28, 277(26), 23596 - 603 Epub 2002 Apr 18.
CheA kinase and chemoreceptor interaction surfaces on CheW; Boukhvalova M et al.; Chemotactic responses of Escherichia coli to aspartic acid are initiated by a ternary protein complex composed of Tar (chemoreceptor), CheA (kinase), and CheW (a coupling protein that binds to both Tar and CheA and links their activities) . We used a genetic selection based on the yeast two-hybrid assay to identify nine cheW point mutations that specifically disrupted CheW interaction with CheA but not with Tar . We sequenced these single point mutants and purified four of the mutant CheW proteins for detailed biochemical characterizations that demonstrated the weakened affinity of the mutant CheW proteins for CheA, but not for Tar . In the three-dimensional structure of CheW, the positions affected by these mutations cluster on one face of the protein, defining a potential binding interface for interaction of CheW with CheA . We used a similar two-hybrid approach to identify four mutation sites that disrupted CheW binding to Tar . Mapping of these "Tar-sensitive" mutation sites and those from previous suppressor analysis onto the structure of CheW defined an extended surface on a face of the protein that is adjacent to the CheA-binding surface and that may serve as an interface for CheW binding to Tar.

J Biol Chem, 2002 Jun 28, 277(26), 23308 - 13 Epub 2002 Apr 18.
F1-ATPase, the C-terminal end of subunit gamma is not required for ATP hydrolysis-driven rotation; Muller M et al.; ATP hydrolysis by the isolated F(1)-ATPase drives the rotation of the central shaft, subunit gamma, which is located within a hexagon formed by subunits (alphabeta)(3) . The C-terminal end of gamma forms an alpha-helix which properly fits into the "hydrophobic bearing" provided by loops of subunits alpha and beta . This "bearing" is expected to be essential for the rotary function . We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coli F(1)-ATPase) . The ATP hydrolysis activity of a load-free ensemble of F(1) with 12 residues deleted decreased to 24% of the control . EF(1) with deletions of 15 or 18 residues was inactive, probably because it failed to assemble . The average torque generated by a single molecule of EF(1) when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 +/- 19% of the observation time) . Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF(1) (full-length gamma) to 34 kJ/mol for EF(1)(gamma-12) . These observations imply that the intactness of the C terminus of subunit gamma provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.

J Biol Chem, 2002 Jun 7, 277(23), 20960 - 4 Epub 2002 Apr 18.
Flipping duplex DNA inside out: a double base-flipping reaction mechanism by Escherichia coli MutY adenine glycosylase; Bernards AS et al.; The Escherichia coli MutY adenine glycosylase plays a critical role in repairing mismatches in DNA between adenine and the oxidatively damaged guanine base 8-oxoguanine . Crystallographic studies of the catalytic core domain of MutY show that the scissile adenine is extruded from the DNA helix to be bound in the active site of the enzyme (Guan, Y., Manuel, R . C., Arvai, A . S., Parikh, S . S., Mol, C . D., Miller, J . H., Lloyd, S., and Tainer, J . A . (1998) Nat . Struct . Biol . 5, 1058-1064) . However, the structural and mechanistic bases for the recognition of the 8-oxoguanine remain poorly understood . In experiments using a single-stranded 8-bromoguanine-containing synthetic oligodeoxyribonucleotide alone and in a duplex construct mismatched to an adenine, we observed UV cross-linking between MutY and the 8-bromoguanine probe . We further observed enhanced cross-linking in the single strand experiments, suggesting that neither the duplex context nor the mismatch with adenine is required for recognition of the 8-oxoguanine moiety . Stopped-flow fluorescence studies using 2-aminopurine-containing oligodeoxyribonucleotides further revealed the sequential extrusion of the 8-oxoguanine at 108 s(-1) followed by the adenine at 16 s(-1) . A protein isomerization step following base flipping at 1.9 s(-1) was also observed and is postulated to provide additional stabilization of the extruded adenine thereby facilitating its capture by the active site for excision.

EMBO Rep, 2002 May, 3(5), 451 - 6 Epub 2002 Apr 18.
Regulation of mitochondrial D-loops by transcription factor A and single-stranded DNA-binding protein; Takamatsu C et al.; During replication, mitochondrial DNA (mtDNA) takes on a triple-stranded structure called a D-loop . Although their physiological roles are not understood, D-loops are implicated in replication and transcription of mtDNA . Little is known about the turnover of D-loops . We investigated the effects of mitochondrial transcription factor A (TFAM) and single-stranded DNA-binding protein (mtSSB) on D-loops . In human HeLa cells, TFAM and mtSSB are, respectively, 1700- and 3000-fold more abundant than mtDNA . This level of TFAM is two orders of magnitude higher than reported previously and is sufficient to wrap human mtDNA entirely . TFAM resolves D-loops in vitro if added in similar stoichiometries . mtSSB inhibits the resolution of mtDNA by TFAM but enhances resolution by RecG, a junction-specific helicase from Escherichia coli . Hence, mtSSB functions in both stabilization and resolution . We propose that TFAM and mtSSB are cooperatively involved in stabilizing D-loops and in the maintenance of mtDNA.

EMBO Rep, 2002 May, 3(5), 457 - 62 Epub 2002 Apr 18.
APC/Fizzy-Related targets Aurora-A kinase for proteolysis; Castro A et al.; Aurora-A kinase is a mitotic spindle-pole-associated protein that has been implicated in duplication and separation of centrosomes and in spindle assembly . The proper timing and amplitude of Aurora-A expression seems to be important, as elevated levels of this protein have been associated with centrosome abnormalities and aneuploidy in mammalian cells . We show that Aurora-A increases at the G2-M transistion and disappears completely at G1 in XL2 cells . Using Xenopus oocyte extracts, we demonstrate that degradation of Aurora-A is mediated by the anaphase-promoting complex (APC) and is regulated by Fizzy-Related but not by Fizzy . Degradation of Aurora-A depends on a D-Box, but not on its KEN-Box motif, as mutation of its C-terminal D-Box sequence induces stabilization of the protein . Accordingly, addition into the extracts of a cyclin B-type D-Box-motif-containing peptide completely suppresses its degradation . Furthermore, APC/Fizzy-Related ubiquitylates the wild type but not a D-Box mutant form of Aurora-A in vitro . Consistent with these data, ectopic expression of Fizzy-Related in Xenopus oocytes induces complete degradation of endogenous Aurora-A . Aurora-A is thus the first protein, at least in our assay system, that undergoes a D-Box-dependent degradation mediated by APC/Fizzy-Related but not by APC/Fizzy.

Biophys J, 2002 May, 82(5), 2466 - 75
Site-directed mutagenesis of tyrosine 118 within the central constriction site of the LamB (Maltoporin) channel of Escherichia coli . I . Effect on ion transport; Orlik F et al.; The three-dimensional structure of the malto-oligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) is known from x-ray crystallography . The central constriction of the channel formed by the external loop 3 is controlled by a tyrosine residue (Y118) . Y118 was replaced by site-directed mutagenesis by ten other amino acids (alanine, isoleucine, asparagine, serine, cysteine, aspartic acid, arginine, histidine, phenylalanine, and tryptophane) including neutral ones, negatively and positively charged amino acids to study the effect of their size, hydrophobicity, and charge on ion transport through LamB . The mutant proteins were purified to homogeneity . They were reconstituted into lipid bilayer membranes and single-channel conductance and ion selectivity were measured to get insight into the mechanism of ion transport through LamB . The mutation of Y118 to any other nonaromatic amino acid led to a substantial increase of the single-channel conductance by more than a factor of six at maximum . The highest effect was observed for Y118D . Additionally, a nonlinear relationship between the salt concentration in the aqueous phase and the channel conductance was observed for this mutant, indicating strong discrete charge effects on ion conductance . For all other mutants, with the exception of Y118R, linear relationships were found between single-channel conductance and bulk aqueous concentration . The individual hydrophobicity indices of the amino acids introduced inside the central constriction of the LamB channel had a somewhat smaller effect on the single-channel conductance as compared with the effect of their size and charge.

Biophys J, 2002 May, 82(5), 2373 - 82
Radiolysis of lac repressor by gamma-rays and heavy ions: a two-hit model for protein inactivation; Charlier M et al.; Upon gamma-ray or argon ion irradiation of the lac repressor protein, its peptide chain is cleaved and the protein loses its lac operator-binding activity, as shown respectively by polyacrylamide gel electrophoresis and retardation gel electrophoresis . We developed phenomenological models that satisfactorily account for the experimental results: the peptide chain cleavage model considers that the average number of chain breaks per protomer is proportional to the irradiation dose and that the distribution of the number of breaks per protomer obeys Poisson's law . The repressor inactivation model takes into account the quaternary structure (a dimer of dimer) and the organization of the repressor in domains (two DNA binding sites, one per dimer) . A protomer is inactivated by at least two different radiation-induced damages . A dimer is inactivated when at least one of the two protomers is inactivated . A tetramer is inactivated when both dimers are inactivated . From the combination of both models, we can deduce that chain cleavage cannot account for the protein inactivation, which should mainly result from oxidation of amino acid side chains . Indeed, particularly oxidizable and accessible amino acids (Tyr, His) are involved in the DNA binding process.

Biochem J, 2002 May 1, 363(Pt 3), 785 - 92
Mutation of conserved active-site threonine residues in creatine kinase affects autophosphorylation and enzyme kinetics; Stolz M et al.; Muscle-type creatine kinase (MM-CK) is a member of an isoenzyme family with key functions in cellular energetics . It has become a matter of debate whether the enzyme is autophosphorylated, as reported earlier {Hemmer, Furter-Graves, Frank, Wallimann and Furter (1995) Biochim . Biophys . Acta 1251, 81-90}, or exclusively nucleotidylated . In the present paper, we demonstrate unambiguously that CK is indeed autophosphorylated . However, this autophosphorylation is not solely responsible for the observed microheterogeneity of MM-CK on two-dimensional isoelectric focusing gels . Using phosphoamino-acid analysis of (32)P-labelled CK isoforms, phosphothreonine (P-Thr) residues were identified as the only product of autophosphorylation for all CK isoenzymes . The phosphorylated residues in chicken MM-CK were allocated to a region in the vicinity of the active site, where five putative phosphorylation sites were identified . Site-directed threonine-valine-replacement mutants reveal that autophosphorylation is not specific for one particular residue but occurs at all examined threonine residues . The enzyme kinetic parameters indicate that the autophosphorylation of CK exerts a modulatory effect on substrate binding and the equilibrium constant, rather than on the catalytic mechanism itself.

Biochem J, 2002 May 1, 363(Pt 3), 707 - 15
Molecular and immunological characterization and IgE epitope mapping of Pen n 18, a major allergen of Penicillium notatum; Yu CJ et al.; The mould genus, Penicillium, is a significant source of environmental aero-allergens . A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization-time-of-flight MS analysis of the peptide digest . Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence . The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences . The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi . The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni(2+)-chelate affinity chromatography . On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P . citrinum, Pen c 18 . When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified . Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.

Biochem J, 2002 May 1, 363(Pt 3), 547 - 52
Bid induces cytochrome c-impermeable Bax channels in liposomes; Roucou X et al.; Bax is a proapoptotic member of the Bcl-2 family of proteins . The Bax protein is dormant in the cytosol of normal cells and is activated upon induction of apoptosis . In apoptotic cells, Bax gets translocated to mitochondria, inserts into the outer membrane, oligomerizes and triggers the release of cytochrome c, possibly by channel formation . The BH3 domain-only protein Bid induces a conformational change in Bax before its insertion into the outer membrane . The mechanism by which Bid promotes Bax activation is not understood, and whether Bid is the only protein required for Bax activation is unclear . Here we report that recombinant full-length Bax (Bax(FL)) does not form channels in lipid bilayers when purified as a monomer . In contrast, in the presence of Bid cut with caspase 8 (cut Bid), Bax forms ionic channels in liposomes and planar bilayers . This channel-forming activity requires an interaction between cut Bid and Bax, and is inhibited by Bcl-x(L) . Moreover, in the absence of the putative transmembrane C-terminal domain, Bax does not form ionic channels in the presence of cut Bid . Cut Bid does not induce Bax oligomerization in liposomes and the Bax channels formed in the presence of cut Bid are not large enough to permeabilize vesicles to cytochrome c . In conclusion, our results suggest that monomeric Bax(FL) can form channels only in the presence of cut Bid . Cut Bid by itself is unable to induce Bax oligomerization in lipid membranes . It is suggested that another factor that might be present in mitochondria is required for Bax oligomerization.

Biochem J, 2002 May 1, 363(Pt 3), 503 - 13
A novel TATA-box-binding factor from the silk glands of the mulberry silkworm, Bombyx mori; Srinivasan L et al.; The presence of one or more TATATAA motifs in the flanking sequences of individual members of a multi-gene tRNA(Gly)(1) family from the mulberry silkworm, Bombyx mori, negatively modulated the transcription of the gene copies . Characterization of proteins from posterior silk gland nuclear extracts, binding to the TATATAA motif, identified a novel 43 kD protein, designated here as P43 TATA-box-binding factor (TBF) . The protein was purified to homogeneity . P43 TBF binding was highly sequence-specific and showed a 100-fold-higher affinity for binding than the TATA-box-binding protein (TBP) . The protein also showed binding to the TATAAA sequence of the actin5C promoter . P43 TBF inhibited transcription of all the tRNA genes examined, as well as RNA polymerase II transcription from the actin5C promoter . The amino acid sequence of eleven peptides generated from P43 TBF did not share homology with proteins that bind the TATA box, such as TBP, TRF (TBP-related factor) or TLFs (TBP-like factors) reported from other sources . Inhibition of transcription of tRNA genes by P43 TBF could not be reversed by TBP . The inhibitory effect appeared to be exerted through sequestration of the associated transcription factors.

Biochem J, 2002 May 1, 363(Pt 3), 493 - 501
Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme; Schlick P et al.; The leader proteinase (L(pro)) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase . Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus . It also possesses a unique electrostatic environment around the active site, essentially due to Asp(163), which orients the catalytic histidine residue, and Asp(164); the equivalent residues in papain are Asn(175) and Ser(176) . The importance of these residues for L(pro) activity was examined by site-directed mutagenesis . Replacement of Asp(163) with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates . However, no effect on the cleavage of the only known cellular substrate of L(pro), eukaryotic initiation factor 4GI (eIF4GI), was observed . In contrast, replacement of Asp(164) by either alanine, asparagine or lysine abrogated activity towards the hexapeptide . Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme . The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine . The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8 min required by the wild-type enzyme . Hence, we propose that the electrostatic charge of Asp(164), and to a lesser extent that of Asp(163), is extremely important for L(pro) to attain full activity upon synthesis.

Cell Mol Life Sci, 2002 Mar, 59(3), 552 - 9
Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2; Stromberg P et al.; The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes . A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis . Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation . Thermostability was assayed by activity measurements and circular dichroism spectroscopy . The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage . Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308 . Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance . In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.

J Soc Gynecol Investig, 2002 Mar-Apr, 9(2), 80 - 5
Fetal responses to intra-amniotic endotoxin in sheep; Nitsos I et al.; OBJECTIVE: Our aim was to determine the acute physiologic effects of intra-amniotic endotoxin administration in fetal sheep, and in particular, to determine whether intra-amniotic endotoxin causes an increase in fetal cortisol that could underlie the functional maturation of the fetal lungs previously reported in this model . METHODS: As in our previous experiments, ewes were randomly assigned to receive a single intra-amniotic injection of either endotoxin (20 mg, Escherichia coli {055:B5}, n = 5) or saline (n = 5) . Between 0.5 hours before endotoxin and 168 hours after its administration, we measured maternal and fetal arterial pressures and heart rates; fetal and maternal blood samples were collected for measurement of blood gases, electrolytes, glucose and lactate concentrations, white cell counts (total and differential), and plasma cortisol . RESULTS: Fetal arterial carbon dioxide tension and lactate concentration were significantly elevated 6 and 12 hours after endotoxin but returned to pre-endotoxin levels by 24 hours . Fetal plasma cortisol concentrations were significantly elevated at 4 hours and peaked 24 hours after endotoxin, returning to control levels by 2 days . Fetal white cell counts initially decreased (4 hours) and then increased (after 24 hours), becoming significantly elevated 6 days after treatment . Other fetal variables, and all measured maternal variables, were unaffected . CONCLUSIONS: Our data suggest that fetal sheep respond to intra-amniotic endotoxin with transient, mild physiologic alterations that follow a time course similar to inflammatory responses reported previously . The elevation in fetal cortisol is insufficient to be the cause of preterm lung maturation shown previously with this treatment.

Life Sci Space Res, 1977, 15, 273 - 8
Chronic acceleration in plants; Edwards BF et al.; Since this subject was last reviewed (at the Symposium on Gravity and the Organism, 1967), relatively little new work on hypergravity effects on plants has appeared . Centrifugation has been used widely for separation of cellular components, but only occasionally as a primary environmental condition . With increasing magnitude of accelerative forces, the following effects have been reported by a number of authors working with many different plants . In the range 2-25 g, auxin transport and geotropic response in coleoptiles are increased and growth is stimulated . From 25 to 500 g, coleoptile growth is reduced and some morphological changes may be seen . At 1000-2500 g, root formation in willow cuttings increases . From 1000 g upward, cytoplasmic stratification occurs and seed germination decreases . Between 200 and 15000 g, chromosome damage has been observed . Algal cell polarity may be reversed at 5000 to 20000 g . Above 30000 g, the response of some cells to gibberellic acid is halted . Permanent morphologic changes in Escherichia coli are produced at 110000 g . Some plant cells have survived 176000 g for 20 hr.

Bioorg Khim, 2002 Mar-Apr, 28(2), 126 - 34
{The production of mini antibodies against human granulocyte colony-stimulating factor using murine scFv combinatorial library}; Laman AG et al.; To develop a phage display of single-chain antibodies (scFv), fractions of total cell DNA and RNA were obtained from splenocytes of naive mice . The DNA fragments encoding variable regions of light and heavy immunoglobulin chains were amplified and isolated using primers specific to the conservative regions of these genes . The construction of the library was based on the principle of stochastic combining of the DNA fragments encoding the light and heavy antibody chains with the DNA linker, whose structure corresponded to the (Gly4Ser)3 sequence . The scFv library was constructed using the E . coli TG1 strain and the phagemid vector pHEN1 . The repertoire of the library exceeded 5 x 10(7) independent recombinant clones . The clones producing antibodies to the granulocyte colony-stimulating human factor were isolated . The affinity constants of the resulting scFv were in the range of 2 x 10(4) to 1.8 x 10(7) M-1.

Cancer Gene Ther, 2002 May, 9(5), 443 - 52
Transcription-targeted gene therapy for androgen-independent prostate cancer; Martiniello-Wilks R et al.; The Escherichia coli enzyme (purine nucleoside phosphorylase, PNP) gene is delivered directly into PC3 tumors by one injection of replication-deficient human type-5 adenovirus (Ad5) . Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death . We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen-dependent, prostate-specific rat probasin (Pb) gene . To increase its activity, the promoter was combined with the SV40 enhancer (SVPb) . Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels . Plasmids expressed approximately 20-fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen-independent and retained substantial prostate specificity . Killing by Ad5-SVPb-PNP vector of cell lines cultured with 6MPDR for 6 days was 5- to 10-fold greater in prostate cancer than in liver or lung cells . In vivo, a single intratumoral injection of Ad5-SVPb-PNP (4 x 10(8) pfu), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals . Thus, the androgen-independent, prostate-targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo . This first example of an androgen-independent vector points the way toward treatment of emerging androgen-independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low.

J Gen Virol, 2002 May, 83(Pt 5), 1069 - 74
Identification and localization of a prawn white spot syndrome virus gene that encodes an envelope protein; Zhang X et al.; Among the important challenges to shrimp aquaculture worldwide are the diseases caused by viruses, in particular by white spot syndrome virus (WSSV), which has a genome estimated to contain 305 kb . By analysis and comparison of the WSSV genomic DNA and cDNA libraries, an ORF (vp28 gene) was identified . The gene, encoding a novel 204-amino-acid protein, was expressed in Escherichia coli and purified . A specific antibody was raised using the purified VP28 protein . After inoculation of healthy adult Penaeus monodon shrimp with WSSV, the gene transcript and VP28 protein were first detected at low levels at 6 and 18 h post-infection, respectively . These experiments suggest that it might be a late gene . Immuno-electron microscopy with gold-labelled antibody revealed that the gold particles were distributed in the outer envelope of WSSV virions and showed that vp28 encodes a virus envelope protein.

Mol Pharmacol, 2002 May, 61(5), 1140 - 5
Lack of susceptibility of bicyclic nucleoside analogs, highly potent inhibitors of varicella-zoster virus, to the catabolic action of thymidine phosphorylase and dihydropyrimidine dehydrogenase; Balzarini J et al.; The susceptibility of the bicyclic nucleoside analogs (BCNAs), highly potent and selective inhibitors of varicella-zoster virus (VZV), to the enzymes involved in nucleoside/nucleobase catabolism has been investigated in comparison with the established anti-VZV agent (E)-5-(2-bromovinyl)-2'-deoxyuridine {BVDU; brivudine (Zostex)} . Whereas human and bacterial thymidine phosphorylases (TPases) efficiently converted BVDU to its antivirally inactive free base (E)-5-(2-bromovinyl)uracil (BVU), BCNAs showed no evidence of conversion to the free base in the presence of these enzymes . The lack of substrate affinity of TPase for the BCNAs could be rationalized by computer-assisted molecular modeling of the BCNAs in the TPase active site . Moreover, in contrast with BVU, which is a potent and selective inhibitor of dihydropyrimidine dehydrogenase (DPD) (50% inhibitory concentration; 10 microM in the presence of a 25 microM concentration of the natural substrate thymine), the free base (Cf 1381; 6-octyl-2,3-dihydrofuro{2,3-d}pyrimidin-2-one) of BCNA (Cf 1368; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro{2,3-d}pyrimidin-2-one) and the free base Cf 2200 {6-(4-n-pentylphenyl)-2,3-dihydrofuro{2,3-d}pyrimidin-2-one} of BCNA (Cf 1743; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro{2,3-d}pyrimidin-2-one) did not inhibit the DPD-catalyzed catabolic reaction of pyrimidine bases (i.e., thymine) and pyrimidine base analogs {i.e., 5-fluorouracil (FU)} at a concentration of 250 microM . Consequently, whereas BVU caused a dramatic rise of FU levels in FU-treated mice, the BCNAs did not affect FU levels in such mice . From our data it is evident that BCNAs represent highly stable anti-VZV compounds that are not susceptible to breakdown by nucleoside/nucleobase catabolic enzymes and are not expected to interfere with cellular catabolic processes such as those involved in FU catabolism.

Mol Pharmacol, 2002 May, 61(5), 953 - 63
Parthenolide, an inhibitor of the nuclear factor-kappaB pathway, ameliorates cardiovascular derangement and outcome in endotoxic shock in rodents; Sheehan M et al.; Parthenolide is a sesquiterpene lactone used in folk medicine for its anti-inflammatory activity . Recent in vitro studies have shown that this compound inhibits the nuclear factor (NF)-kappaB pathway . This study examines the effect of parthenolide in endotoxic shock in rodents . Endotoxic shock was induced by administration of Escherichia coli endotoxin in rats . Three groups of rats received parthenolide (0.25, 0.5, or 1 mg/kg) 15 min before endotoxin; another group received parthenolide (1 mg/kg) 3 h after endotoxin . In vehicle-treated rats, administration of endotoxin caused severe hypotension, which was associated with a marked hyporeactivity to norepinephrine in ex vivo thoracic aortas . Immunohistochemistry showed positive staining for nitrotyrosine, poly(ADP-ribose) synthetase (PARS) and apoptosis, whereas Northern blot analysis showed increased mRNA expression of inducible nitric-oxide synthase (iNOS) in thoracic aortas . Elevated levels of plasma nitrate/nitrite were also found . Elevated lung levels of myeloperoxidase activity were indicative of infiltration of neutrophils . These inflammatory events were preceded by cytosolic degradation of inhibitor kappaBalpha (IkappaBalpha) and activation of nuclear NF-kappaB in the lung . In vivo pretreatment and post-treatment with parthenolide improved the hemodynamic profile and reduced plasma nitrate/nitrite and lung neutrophil infiltration in a dose-dependent fashion . Vascular hyporeactivity of ex vivo aortas was ameliorated . Treatment with parthenolide also abolished nitrotyrosine formation, PARS expression, and apoptosis and reduced iNOS mRNA content in thoracic aortas . DNA binding of NF-kappaB was inhibited by parthenolide in the lung, whereas degradation of IkappaBalpha was unchanged . In a separate set of experiments, pretreatment or post-treatment with parthenolide significantly improved survival in mice challenged with endotoxin . We conclude that parthenolide exerts beneficial effects during endotoxic shock through inhibition of NF-kappaB.

J Biol Chem, 2002 Jun 21, 277(25), 22605 - 15 Epub 2002 Apr 17.
Escherichia coli apurinic-apyrimidinic endonucleases enhance the turnover of the adenine glycosylase MutY with G:A substrates; Pope MA et al.; The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) . MutY is a base excision repair (BER) glycosylase that removes misincorporated adenine residues from OG:A mispairs, as well as G:A and C:A mispairs . We have previously shown that, under conditions of low MutY concentrations relative to an OG:A or G:A substrate, the time course of the adenine glycosylase reaction exhibits biphasic kinetic behavior due to slow release of the DNA product by MutY . The dissociation of MutY from its product may require the recruitment of other proteins from the BER pathway, such as an apurinic-apyrimidinic (AP) endonuclease, as turnover-enhancing cofactors . The effect of the AP endonucleases endonuclease IV (Endo IV), exonuclease III (Exo III), and Ape1 on the reaction kinetics of MutY with G:A- and OG:A-containing substrates was investigated . The effect of the glycosylases UDG and MutM and the DNA polymerase pol I was also characterized . Endo IV and Exo III, unlike Ape1, UDG, and pol I, greatly enhance the rate of product release with a G:A substrate, whereas the rate constant for the adenine removal step remains unchanged . Furthermore, the turnover rate with a truncated form of MutY, Stop 225, which lacks 125 amino acids of the C terminus, is unaffected by the presence of Endo IV or Exo III . These results constitute the first evidence of an interaction between the MutY-product DNA complex and Endo IV or Exo III . Furthermore, they suggest a role for the C-terminal domain of MutY in mediating this interaction.

J Biol Chem, 2002 Jun 28, 277(26), 23664 - 9 Epub 2002 Apr 17.
Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli; Poock SR et al.; Nitric oxide is a key element in host defense against invasive pathogens . The periplasmic cytochrome c nitrite reductase (NrfA) of Escherichia coli catalyzes the respiratory reduction of nitrite, but in vitro studies have shown that it can also reduce nitric oxide . The physiological significance of the latter reaction in vivo has never been assessed . In this study the reduction of nitric oxide by Escherichia coli was measured in strains active or deficient in periplasmic nitrite reduction . Nrf(+) cells, harvested from cultures grown anaerobically, possessed a nitric-oxide reductase activity with physiological electron donation of 60 nmol min(-1) x mg dry wt(-1), and an in vivo turnover number of NrfA of 390 NO* s(-1) was calculated . Nitric-oxide reductase activity could not be detected in Nrf(-) strains . Comparison of the anaerobic growth of Nrf(+) and Nrf(-) strains revealed a higher sensitivity to nitric oxide in the NrfA(-) strains . A higher sensitivity to the nitrosating agent S-nitroso-N-acetyl penicillamine (SNAP) was also observed in agar plate disk-diffusion assays . Oxygen respiration by E . coli was also more sensitive to nitric oxide in the Nrf(-) strains compared with the Nrf(+) parent strain . The results demonstrate that active periplasmic cytochrome c nitrite reductase can confer the capacity for nitric oxide reduction and detoxification on E . coli . Genomic analysis of many pathogenic enteric bacteria reveals the presence of nrf genes . The present study raises the possibility that this reflects an important role for the cytochrome c nitrite reductase in nitric oxide management in oxygen-limited environments.

Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5367 - 72
Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1 alpha; Freedman SJ et al.; Adaptation to hypoxia is mediated by transactivation of hypoxia-responsive genes by hypoxia-inducible factor-1 (HIF-1) in complex with the CBP and p300 transcriptional coactivators . We report the solution structure of the cysteine/histidine-rich 1 (CH1) domain of p300 bound to the C-terminal transactivation domain of HIF-1 alpha . CH1 has a triangular geometry composed of four alpha-helices with three intervening Zn(2+)-coordinating centers . CH1 serves as a scaffold for folding of the HIF-1 alpha C-terminal transactivation domain, which forms a vise-like clamp on the CH1 domain that is stabilized by extensive hydrophobic and polar interactions . The structure reveals the mechanism of specific recognition of p300 by HIF-1 alpha, and shows how HIF-1 alpha transactivation is regulated by asparagine hydroxylation.

Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5349 - 54
Unspecific hydrophobic stabilization of folding transition states; Viguera AR et al.; Here we present a method for determining the inference of non-native conformations in the folding of a small domain, alpha-spectrin Src homology 3 domain . This method relies on the preservation of all native interactions after Tyr/Phe exchanges in solvent-exposed, contact-free positions . Minor changes in solvent exposure and free energy of the denatured ensemble are in agreement with the reverse hydrophobic effect, as the Tyr/Phe mutations slightly change the polypeptide hydrophilic/hydrophobic balance . Interestingly, more important Gibbs energy variations are observed in the transition state ensemble (TSE) . Considering the small changes induced by the H/OH replacements, the observed energy variations in the TSE are rather notable, but of a magnitude that would remain undetected under regular mutations that alter the folded structure free energy . Hydrophobic residues outside of the folding nucleus contribute to the stability of the TSE in an unspecific nonlinear manner, producing a significant acceleration of both unfolding and refolding rates, with little effect on stability . These results suggest that sectors of the protein transiently reside in non-native areas of the landscape during folding, with implications in the reading of phi values from protein engineering experiments . Contrary to previous proposals, the principle that emerges is that non-native contacts, or conformations, could be beneficial in evolution and design of some fast folding proteins.

Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5307 - 12
Posttranslational modification of the umuD-encoded subunit of Escherichia coli DNA polymerase V regulates its interactions with the beta processivity clamp; Sutton MD et al.; The Escherichia coli umuDC (pol V) gene products participate in both a DNA damage checkpoint control and translesion DNA synthesis . Interactions of the two umuD gene products, the 139-aa UmuD and the 115-aa UmuD' proteins, with components of the replicative DNA polymerase (pol III), are important for determining which biological role the umuDC gene products will play . Here we report our biochemical characterizations of the interactions of UmuD and UmuD' with the pol III beta processivity clamp . These analyses demonstrate that UmuD possesses a higher affinity for beta than does UmuD' because of the N-terminal arm of UmuD (residues 1-39), much of which is missing in UmuD' . Furthermore, we have identified specific amino acid residues of UmuD that crosslink to beta with p-azidoiodoacetanilide, defining the domain of UmuD important for the interaction . We have recently proposed a model for the solution structure of UmuD(2) in which the N-terminal arm of each protomer makes extensive contacts with the C-terminal globular domain of its intradimer partner, masking part of each surface . Taken together, our findings suggest that UmuD(2) has a higher affinity for the beta-clamp than does UmuD'(2) because of the structures of its N-terminal arms . Viewed in this way, posttranslational modification of UmuD, which entails the removal of its N-terminal 24 residues to yield UmuD', acts in part to attenuate the affinity of the umuD gene product for the beta-clamp . Implications of these structure-function analyses for the checkpoint and translesion DNA synthesis functions of the umuDC gene products are discussed.

Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5283 - 8
Protein deamidation; Robinson NE; A completely automatic computerized technique for the quantitative estimation of the deamidation rates of any protein for which the three-dimensional structure is known has been developed . Calculations of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out . The calculated values have good quantitative reliability when compared with experimental measurements . These rates demonstrate that deamidation may be a biologically relevant phenomenon in a remarkably large percentage of proteins.

J Biol Chem, 2002 Jul 19, 277(29), 26623 - 31 Epub 2002 Apr 16.
Computer-assisted mutagenesis of ecotin to engineer its secondary binding site for urokinase inhibition; Laboissiere MC et al.; Inhibitors of urokinase-type plasminogen activator (uPA) were selected in vitro from two ecotin phage-display libraries to study the effect on binding of amino acid substitutions at critical positions 108, 110, 112, and 113 within the 100s loop (RNKL, respectively, in wild type ecotin) . The first, a focused library, was the result of a computation-assisted approach using the three-dimensional structure of the ecotin-trypsin complex to guide the modeling of amino acid substitutions predicted to increase affinity for uPA . The second, a complete library, allowed for all substitutions at the above identified positions . The consensus sequences selected from the focused, and complete libraries were RRWS and R(R/N)QL, respectively . Inhibition constant determinations showed ecotin variants containing these sequences to be similarly potent (K(i) = 1-2 nm) . These substitutions were combined with previously identified substitutions in another critical region of ecotin . One of these combinations (D70R/M84R/RRQL) is the tightest (K(i) = 50 pm) ecotin variant inhibitor of uPA . The blending of combinatorial methods and computer algorithms designed to predict stronger binders has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target . This technology can be applied to select for enhanced binding interactions at protein-protein interfaces and accelerate the process of protease inhibitor development.

J Biol Chem, 2002 Jul 26, 277(30), 27305 - 11 Epub 2002 Apr 16.
Primary structure and characteristics of a lectin from skin mucus of the Japanese eel Anguilla japonica; Tasumi S et al.; Two types of lactose-binding lectins, AJL-1 and AJL-2, were purified from the skin mucus extract of the Japanese eel Anguilla japonica by lactose affinity chromatography and subsequent gel filtration . The molecular masses of AJL-1 and AJL-2 were 16,091 and 31,743 Da, respectively . Intact AJL-1 was comprised of two identical 16-kDa subunits having blocked N termini and no disulfide bonds . AJL-2 was a homodimer with disulfide bonds . Based on the N-terminal amino acid sequence of the AJL-2 monomer, the nucleotide sequence of cDNA encoding this lectin was determined by 3'- and 5'-rapid amplification of cDNA ends . The deduced amino acid sequence showed approximately 30% homology with C-type lectins, which bind to carbohydrates in a Ca(2+)-dependent manner . In addition, AJL-2 exhibited highly conserved consensus amino acid residues of the C-type carbohydrate recognition domain, although this lectin showed Ca(2+)-independent activity . Gene expression of AJL-2 was detected only in the skin by Northern blot analysis, and this lectin localization was demonstrated in the club cells by immunohistochemistry . These results indicate that AJL-2 is secreted on the body surface and function as a component of skin mucus . AJL-2 agglutinated Escherichia coli and suppressed its growth, suggesting that this lectin is involved in host defense.

Br J Pharmacol, 2002 Apr, 135(8), 2047 - 55
Involvement of purinergic signalling in central mechanisms of body temperature regulation in rats; Gourine AV et al.; 1 . P2 purinoreceptors are present in hypothalamic and brainstem nuclei that are involved in the regulation of body temperature (T(b)) . The role of ATP acting on these P2 receptors in thermoregulation was investigated by studying the effects of the stable ATP analogue alpha,beta-methyleneATP (alpha,beta-meATP) and P2 receptor antagonists suramin and pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on T(b) when injected intracerebroventricularly (i.c.v.) via a pre-implanted cannula in conscious rats at various ambient temperatures and during lipopolysaccharide (LPS)-induced fever . 2 . Depending on ambient temperature, alpha,beta-meATP (0.2 micromol, i.c.v.) induced a fall in T(b) (-3.3 degrees C, P<0.05), no changes in T(b) when compared to pre-injection levels, or an increase in T(b) ( approximately 1.0 degrees C, P<0.05) in rats maintained at 10 degrees C, 25 degrees C and 30 degrees C ambient temperature, respectively . 3 . Suramin (7 nmol, i.c.v.) induced a lasting (up to 6 h) increase in T(b) (on average 1.2 degrees C, P<0.05) in rats kept at 25 degrees C or 30 degrees C, but failed to induce any rise in T(b) in rats at 10 degrees C ambient temperature . An increase in T(b) was also observed in rats (25 degrees C ambient temperature) treated with PPADS (0.2 micromol, i.c.v.) . 4 . alpha,beta-meATP (0.2 micromol) injected i.c.v . or directly into the anterior hypothalamus caused a profound fall in T(b) (by 0.9 degrees C and 1.0 degrees C, respectively; P<0.05) during LPS (E.coli; 50 microg kg(-1))-induced fever in rats at 25 degrees C ambient temperature . Fever was initiated more rapidly in rats treated with suramin (7 nmol) or PPADS (70 nmol), however its late phase was unaffected . Suramin (7 nmol) and PPADS (70 nmol) injected at the time when fever was already developed (2.5 h after LPS injections) did not alter febrile T(b) . 5 . These data indicate that purinergic signalling may play a significant role in central mechanisms of T(b) regulation at various ambient temperatures and during fever.

Antimicrob Agents Chemother, 2002 May, 46(5), 1546 - 9
30S ribosomal subunit assembly is a target for inhibition by aminoglycosides in Escherichia coli; Mehta R et al.; The aminoglycosides paromomycin and neomycin were examined in Escherichia coli cells for an inhibitory effect on 30S ribosomal subunit assembly . Both compounds inhibited the growth rate, viable cell number, and protein synthesis rate with similar 50% inhibitory concentrations . Each drug also showed a concentration-dependent inhibition of 30S subunit formation . The inhibitory effect on 30S particle formation was approximately equivalent to the inhibitory effect on translation for these antibiotics.

Curr Opin Struct Biol, 2002 Apr, 12(2), 231 - 8
Machinery of protein folding and unfolding; Zhang X et al.; During the past two years, a large amount of biochemical, biophysical and low- to high-resolution structural data have provided mechanistic insights into the machinery of protein folding and unfolding . It has emerged that dual functionality in terms of folding and unfolding might exist for some systems . The majority of folding/unfolding machines adopt oligomeric ring structures in a cooperative fashion and utilise the conformational changes induced by ATP binding/hydrolysis for their specific functions.

Curr Opin Struct Biol, 2002 Apr, 12(2), 182 - 9
Theoretical and computational models of ion channels; Roux B; Computational studies can make meaningful contributions to our understanding of biological ion channels . A wide variety of methods, at different levels of approximation, can be used . Over the past few years, progress in the experimental determination of three-dimensional structures has given a fresh impetus to the theorists . Noteworthy progress has been made in carefully constructing realistic models of a number of complex biological channels to address important questions about their function.

FEMS Microbiol Lett, 2002 Mar 5, 208(2), 295 - 301
Inactivation of a Helicobacter pylori DNA methyltransferase alters dnaK operon expression following host-cell adherence; Donahue JP et al.; The Helicobacter pylori hpyIM gene encodes a type II DNA methyltransferase that is highly conserved among strains . To investigate the potential role of M.HpyI methyltransferase activity in controlling gene expression in H . pylori, we analyzed gene transcription profiles in wild-type strain J166 and an isogenic hpyIM mutant strain using gene arrays . This analysis showed that the expression of a majority of genes was unaffected by hpyIM mutation, especially in exponential phase cultures . However, in stationary phase cultures and in cells adherent to AGS gastric epithelial cells in vitro, loss of hpyIM function altered the expression of the stress-responsive dnaK operon . Complementation of the hpyIM mutation using a shuttle plasmid encoding a wild-type copy of the gene re-established the wild-type pattern of dnaK operon expression . These data suggested that hpyIM, encoding a DNA methyltransferase, may have a role in H . pylori physiology that supersedes its original function in a type II restriction-modification system.

FEMS Microbiol Lett, 2002 Mar 5, 208(2), 259 - 62
Colicins produced by the Escherichia fergusonii strains closely resemble colicins encoded by Escherichia coli; Smajs D et al.; Plasmid DNA of six Escherichia fergusonii colicinogenic strains (three producers of colicin E1, two of Ib and one of Ia) was isolated and the colicin-encoding regions of the corresponding Col plasmids were sequenced . Two new variants of colicin E1, one of colicin Ib, and one of colicin Ia were identified as well as new variants of the colicin E1 and colicin Ib immunity proteins and the colicin E1 lysis polypeptide . The recombinant Escherichia coli producer harboring pColE1 from E . fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 colicins produced by two other E . fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid.

FEBS Lett, 2002 Apr 10, 516(1-3), 234 - 8
Der f 16: a novel gelsolin-related molecule identified as an allergen from the house dust mite, Dermatophagoides farinae; Kawamoto S et al.; Allergen from the house dust mite (Dermatophagoides sp.) is a major trigger factor of allergic disorders, and its characterization is crucial for the development of specific diagnosis or immunotherapy . Here we report the identification of a novel dust mite (Dermatophagoides farinae) antigen whose primary structure belongs to the gelsolin family, a group of actin cytoskeleton-regulatory proteins . Isolated mite cDNA, termed Der f 16, encodes 480 amino acids comprising a four-repeated gelsolin-like segmental structure, which is not seen in conventional gelsolin family members . Enzyme immunoassay indicated that recombinant Der f 16 protein, prepared using an Escherichia coli expression system, bound IgE from mite-allergic patients at 47% (8/17) frequency . This is the first evidence that the gelsolin family represents a new class of allergen recognizable by atopic patient IgE.

FEBS Lett, 2002 Apr 10, 516(1-3), 197 - 200
A chimera of a gelatinase inhibitor peptide with streptavidin as a bifunctional tumor targeting reagent; Farlow SJ et al.; A chimeric protein, consisting of streptavidin fused to a cyclic decapeptide with potent inhibitory activity for matrix metalloproteinases (MMP), has been produced in Escherichia coli and purified . The purified chimera formed a tetramer and showed full biotin-binding ability . The chimera was also capable of both binding to MMP-2 and inhibiting its activity . Thus, both the streptavidin moiety and the decapeptide of the chimera are fully functional . This bifunctional nature of the chimera should facilitate the application of the decapeptide since the streptavidin moiety can be used as a specific conjugation site for almost any materials upon biotinylation.

Bioorg Med Chem Lett, 2002 Mar 25, 12(6), 929 - 31
Probing the overlap of chorismate mutase and prephenate dehydrogenase sites in the escherichia coli T-protein: a dehydrogenase-selective inhibitor; Vincent S et al.; An inhibitor of prephenate dehydrogenase has been identified that has no effect on the chorismate mutase activity in the Escherichia coli T-protein, thus supporting the idea of two separate active sites.

Thromb Res, 2002 Jan 15, 105(2), 153 - 60
Trimucytin, a collagen-like snake venom protein, activates platelets independent of I-domain within alpha2 subunit of alpha2beta1 integrin; Liu CZ et al.; Trimucytin is a powerful platelet aggregation inducer isolated from the venom of Taiwan habu snake (Trimeresurus mucrosquamatus) . In this study, we found that the snake venom protein, crovidisin, which prevents collagen-platelet interaction through its high-affinity binding to collagen, inhibited competitively trimucytin-induced aggregation of washed human platelets with a pA(2) value of 6.65 . The ability of trimucytin in triggering platelet aggregation was suppressed by a monoclonal antibody (A2-IIE10) raised against the alpha2 subunit of alpha2beta1 integrin (glycoprotein Ia/IIa), indicating that platelet alpha2beta1 integrin plays a central role in trimucytin's platelet reactivity . Many studies have localized the major reactive site of alpha2beta1 integrin to the I-domain of alpha2 subunit . However, Escherichia coli-produced recombinant alpha2 I-domain (GST-alpha2 fusion protein) blocking collagen-induced platelet aggregation failed to inhibit aggregation of platelets in response to trimucytin . Based on these findings, it is concluded that the platelet reactivity of trimucytin is alpha2beta1 integrin-dependent, while the I-domain present in the alpha2 subunit is not involved . This novel snake venom protein would be useful for mapping the functional domain of alpha2beta1 integrin.

Proc R Soc Lond B Biol Sci, 2002 Apr 22, 269(1493), 761 - 72
Clone mixtures and a pacemaker: new facets of Red-Queen theory and ecology; Sasaki A et al.; Host-parasite antagonistic interaction has been proposed as a potential agent to promote genetic polymorphism and to favour sex against asex, despite its twofold cost in reproduction . However, the host-parasite gene-for-gene dynamics often produce unstable cycles that tend to destroy genetic diversity . Here, we examine such diversity destroying coevolutionary dynamics of host and parasite, which is coupled through local or global migration, or both, between demes in a metapopulation structure . We show that, with global migration in the island model, peculiar out-of-phase islands spontaneously arise in the cluster of islands converging to a global synchrony . Such asynchrony induced by the 'pacemaker islands' serves to restore genetic variation . With increasing fraction of local migration, spots of asynchrony are converted into loci or foci of spiral and target patterns, whose rotating arms then cover the majority of demes . A multi-locus analogue of the model reproduces the same tendency toward asynchrony, and the condition arises for an advantage of asexual clones over their sexual counterpart when enough genetic diversity is maintained through metapopulation storage-migration serves as a cheap alternative to sex.

Arch Virol, 2002 Mar, 147(3), 659 - 63
Importance of thymidine kinase activity for normal growth of lumpy skin disease virus (SA-Neethling); Wallace DB et al.; In order to study the importance of an intact thymidine kinase (TK) gene for the vaccine strain of a southern African capripoxvirus, namely, lumpy skin disease virus (LSDV) (type SA-Neethling), a TK disruption recombinant was generated expressing the Escherichia coli beta-galactosidase (lacZ) reporter gene . A comparative growth study of the recombinant and wild-type (wt) LSDV in TK-positive primary and secondary cells and TK-negative secondary cells was performed . It was found that although recombinant and wt virus both grew in TK-positive cells without selection, the recombinant was unable to grow in TK-negative cells (with or without selection), indicating that TK activity is important, if not essential, for normal growth of LSDV.

Adv Space Res, 1993, 13(7), 251 - 7
Virus protein assembly in microgravity; Chang D et al.; The coat of polyomavirus is composed of three proteins that can self-assemble to form an icosahedral capsid . VP1 represents 75% of the virus capsid protein and the VP1 capsomere subunits are capable of self assembly to form a capsid-like structure . Ground-based and orbiter studies were conducted with VP1 protein cloned in an expression vector and purified to provide ample quantities for capsomere-capsid assembly . Flight studies were conducted on STS-37 on April 5-9, 1991 . Assembly initiated when a VP1 protein solution was interfaced with a Ca+2 buffer solution (pH 5.0) . After four days a second alignment terminated the assembly process and allowed for glutaraldehyde fixation . Flight and ground-based samples were analyzed by electron microscopy . Ground-based experiments revealed the assembly of VP1 into capsid-like structures and a heterogenous size array of capsomere subunits . Samples reacted in microgravity, however, showed capsomeres of a homogenous size, but lack of capsid-like assembly.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jan, 34(1), 77 - 82
{Cloning and expression of Tachypleus tridentatus factor C}; Wang DN et al.; Factor C is an endotoxin-sensitive, intracellular serine protease zymogen which initiates the coagulation cascade system in the horseshoe crab hemolymph . The special lipopolysaccharide (LPS) binding activation of FC makes it a potential drug for anti-LPS treatment and has a high commercial value . Based on the sequence of reported FC from Japan horseshoe crab, two pairs of primers were designed . The total RNA was extracted from amebocytes of Chinese Tachypleus tridentatus and the cDNA was separated into two parts and were cloned using RT-PCR, respectively . FCs from different geographical areas showed high homology in sequence . The whole FC cDNA was cloned into pET-28a (+) containing T7 promoter and recombinant expression plasmid pET-FC was constructed . The recombinant plasmid was transformed into E . coli BL21 (DE3) . Recombinant FC was expressed as inclusion body when the expression strain was induced with 1 mmol/L IPTG . Refolded recombinant FC was confirmed to be active by bacteriostatic assay in vitro . The results of Western blot also suggested the recombinant FC may be able to cleave itself partly and produced an extra immunoblot band.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jan, 34(1), 62 - 6
{Chinese little-fat-tail sheep prion protein gene belongs to PrPARH genotype}; Li YM et al.; The genomic DNA was extracted from lymphocytes of peripheral blood of Chinese little-fat-tail sheep . The prion protein gene(PrPc) was amplified by polymerase chain reaction . The PCR products were cloned into pUC19 plasmid and was sequenced . The results indicated that 771 bp amplified fragment contains the entire sheep PrPc coding sequence . There were 7 base pairs different with the reported sheep PrPc sequence, leading to three amino acids changes in the protein . It is show that its genotype is PrPARH, and these three polymorphisms may be linked to susceptibility or resistance to scrapie infection . Mature sheep prion protein gene was inserted into pET30a and expressed in E . coli, and 23 kD PrP25-231 protein was obtained . Recombinant PrP25-231 protein was purified by inclusion body washing, ion-exchange chromatography, reverse phase chromatography . Results of SDS-PAGE and Western blot showed that the purified PrP25-231 was obtained.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jan, 34(1), 39 - 44
{Cloning, expression and characterization of human vascular endothelial growth factor receptor 1 tyrosine kinase}; Zhuang SF et al.; Vascular endothelial growth factor receptor 1 (Flt1) plays an important role in angiogenesis . It was hypothesized that, upon binding to VEGF, Flt1 tyrosine kinase underwent dimerization and initiated the signal transduction in VEGF/VEGF receptor system . In this report, a soluble active Flt1 tyrosine kinase domain expressed in E . coli was obtained, and its properties were partly characterized . The cDNA of Flt1 tyrosine kinase domain was obtained from the total RNA extracted from human liver cancer tissues by using RT-PCR, and was cloned to vector pGEX-KG . A soluble active GST-fusion protein of Flt1 tyrosine kinase domain (GST-F) was obtained from E . coli BL21 (DE3) pLysS . Although it was reported that GST-F contains no phosphorylation site, it did autophosphorylate in vitro . Mg2+ and Mn2+ were essential for the activity . It was also found that GST-F phosphorylated a synthesized substrate PolyE4Y, but not MBP and Src-related-peptide . The optimal Mg2+ and Mn2+ concentration for polyE4Y phosphorylation was 15 mmol/L and 0.5 mmol/L, respectively . This work is helpful for developing the new anti-cancer drugs.

Appl Microbiol Biotechnol, 2002 Apr, 58(5), 658 - 62 Epub 2002 Feb 12.
Description of a cellulose-binding domain and a linker sequence from Aspergillus fungi; Quentin M et al.; A family I cellulose-binding domain (CBD) and a serine- and threonine-rich linker peptide were cloned from the fungi Aspergillus japonicus and Aspergillus aculeatus . A glutathione S-transferase (GST) fusion protein comprising GST and a peptide linker with the CBD fused to its C-terminus, was expressed in Escherichia coli . The renatured GST-CBD recovered from inclusion bodies had a molecular mass of 36.5 kDa which agrees with the 29 kDa of the GST plus the calculated 7.5 kDa of the linker with the CBD . The isolated GST-CBD protein adsorbed to both bacterial microcrystalline cellulose and carboxymethyl cellulose . Deletion of the linker peptide caused a decrease in cellulose adsorbance and a higher sensitivity to protease digestion.

Int J Oncol, 2002 May, 20(5), 1005 - 11
UDP-GlcNAc2-epimerase regulates cell surface sialylation and cell adhesion to extracellular matrix in Burkitt's lymphoma; Suzuki O et al.; Alteration of cell surface glycoconjugates plays a crucial role in many biological phenomenon, including invasion and metastasis . In the present study, we investigated the relationship between cell surface sialylation and the adhesive properties of two clones (3G3 and 3D2) of a human Burkitt's lymphoma cell line, HBL-8 to extracellular matrix . By flow cytometric analysis we found that the cell surface of 3G3 clone was highly sialylated and that of the 3D2 clone were hyposialylated . Moreover, cell surface sialic acid content was significantly greater in the 3G3 clone than in the 3D2 clone . In vitro adhesion assays showed that cell surface sialylation in the 3G3 clone cells might reduce their attachment to collagen type IV and fibronectin, compared to 3D2 clone . Sialic acid metabolic complementation assays using several precursors of sialic acid suggested that hyposialylation in the 3D2 clone resulted from low activities of uridine diphosphate-N-acetylglucosamine-2-epimerase (UDP-GlcNAc2-epimerase) which is a key enzyme in sialic acid biosynthesis . From RT-PCR analysis the expression of UDP-GlcNAc2-epimerase mRNA was found to be correlated with sialic acid content in the two clones of HBL-8 . Therefore, expression of UDP-GlcNAc2-epimerase mRNA may regulate the expression of sialoglycoconjugates which affect the adhesion of lymphoma cells to collagen type IV and fibronectin.

J Cell Biol, 2002 Apr 15, 157(2), 205 - 10 Epub 2002 Apr 15.
A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid {Delta}pH/Tat translocase; Mori H et al.; The thylakoid DeltapH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane DeltapH as the sole energy source . Three known components of the transport machinery exist in two distinct subcomplexes . A cpTatC-Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition . Here we report that Tha4 assembles with cpTatC-Hcf106 during the translocation step . Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting . cpTatC and Hcf106 were consistently associated under all conditions tested . In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the DeltapH . Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly . The association of all three components was transient and dissipated upon the completion of protein translocation . Such an assembly-disassembly cycle could explain how the DeltapH/Tat system can assemble translocases to accommodate folded proteins of varied size . It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

J Biol Chem, 2002 Jun 21, 277(25), 22534 - 40 Epub 2002 Apr 15.
Importance of the N-terminal region of the phage GA-1 single-stranded DNA-binding protein for its self-interaction ability and functionality; Gascon I et al.; The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages phi 29 and Nf . In this work, the self-interaction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo cross-linking of bacterial cultures infected with phage GA-1 . GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of phi 29 and Nf . Three deletion mutant proteins have been characterized, Delta N19, Delta N26, and Delta N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB . Mutant protein Delta N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins Delta N26 and Delta N33 no longer stimulate viral DNA replication or display helix-destabilizing activity . Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein . Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.

J Biol Chem, 2002 Jun 21, 277(25), 22520 - 7 Epub 2002 Apr 15.
Interchangeable enzyme modules . Functional replacement of the essential linker of the biotinylated subunit of acetyl-CoA carboxylase with a linker from the lipoylated subunit of pyruvate dehydrogenase; Cronan JE Jr; Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase, the enzyme that catalyzes the first committed step of fatty acid synthesis . E . coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin . In most biotinylated proteins, the biotin moiety is attached to a lysine residue located about 35 residues from the carboxyl terminus of the protein, which lies in the center of a strongly conserved sequence that forms a tightly folded anti-parallel beta-barrel structure . Located upstream of the conserved biotinoyl domain sequence are proline/alanine-rich sequences of varying lengths, which have been proposed to act as flexible linkers . In E . coli BCCP, this putative linker extends for about 42 residues with over half of the residues being proline or alanine . I report that deletion of the 30 linker residues located adjacent to the biotinoyl domain resulted in a BCCP species that was defective in function in vivo, although it was efficiently biotinylated . Expression of this BCCP species failed to restore normal growth and fatty acid synthesis to a temperature-sensitive E . coli strain that lacks BCCP when grown at nonpermissive temperatures . In contrast, replacement of the deleted BCCP linker with a linker derived from E . coli pyruvate dehydrogenase gave a chimeric BCCP species that had normal in vivo function . Expression of BCCPs having deletions of various segments of the linker region of the chimeric protein showed that some deletions of up to 24 residues had significant or full biological activity, whereas others had very weak or no activity . The inactive deletion proteins all lacked an APAAAAA sequence located adjacent to the tightly folded biotinyl domain, whereas deletions that removed only upstream linker sequences remained active . Deletions within the linker of the wild type BCCP protein also showed that the residues adjacent to the tightly folded domain play an essential role in protein function, although in this case some proteins with deletions within this region retained activity . Retention of activity was due to fusion of the domain to upstream sequences . These data provide new evidence for the functional and structural similarities of biotinylated and lipoylated proteins and strongly support a common evolutionary origin of these enzyme subunits.

Vet Microbiol, 2002 May 24, 86(4), 303 - 11
DNA sequences coding for the F18 fimbriae and AIDA adhesin are localised on the same plasmid in Escherichia coli isolates from piglets; Mainil JG et al.; The adhesin-involved-in-diffuse-adherence (AIDA) afimbrial adhesin is produced by human, but not by animal, Escherichia coli, with the exception of German porcine verotoxigenic Escherichia coli (VTEC) {Clin . Diagn . Lab . Immunol . 8 (2001) 143} . Presence and localisation of DNA sequences (aidA) coding for and production of an AIDA adhesin were investigated in a collection of Belgian VTEC and non-VTEC E . coli isolated from piglets at weaning time . The 174 isolates were also studied by colony hybridisation for the presence of DNA sequences coding for the Stx2e verocytotoxin and the F18 fimbrial adhesin (fed): 71 were Stx2+F18+AIDA+, 26 were F18+AIDA+, 12 were AIDA+, two were Stx2+AIDA+, and one was Stx2+ only . Fifty-four of the 58 (F18+)AIDA+ isolates tested positive in a western blotting assay with an immune serum raised against the AIDA protein . Hybridisation with the AIDA gene probe on plasmid DNA profiles identified a probe-positive plasmid band in the 10 AIDA+ and in 24 of the 25 F18+AIDA+ isolates studied . Moreover in F18+AIDA+ isolates, only one plasmid band hybridised with both F18 and AIDA probes . These results confirm the presence of aidA-related genes in not only VTEC, but also non-VTEC, isolates from piglets and the production of an antigenically AIDA-related protein by the majority of probe-positive E . coli . Moreover the plasmid DNA hybridisation results suggest a localisation on the same plasmid of the aidA- and fed-related DNA sequences.

Food Chem Toxicol, 2002 May, 40(5), 609 - 16
In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids; Breinholt VM et al.; Human and mouse liver microsomes and membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids . In human and mouse liver microsomes kaempferol, apigenin and naringenin were hydroxylated at the 3'-position to yield their corresponding analogs quercetin, luteolin and eriodictyol, whereas hesperetin and tamarixetin were demethylated at the 4'-position to yield eriodictyol and quercetin, respectively . Microsomal flavonoid metabolism was potently inhibited by the CYP1A2 inhibitors, fluvoxamine and -naphthoflavone . Recombinant CYP1A2 was capable of metabolizing all five investigated flavonoids . CYP3A4 recombinant protein did not catalyze hesperetin demethylation, but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant CYP1A2, although the reaction rates in general were lower as compared to CYP1A2 . CYP2C9 catalyzed the 4'-demethylation of tamarixetin, whereas CYP2D6 did not seem to play any role in the metabolism of the selected flavonoids . The major involvement in flavonoid metabolism of human CYP1A2, which mediates the formation of metabolites with different biochemical properties as compared to the parent compound and furthermore is known to be expressed very differently among individuals, raises the important question of whether individual differences in the CYP enzyme activity might affect the beneficial outcome of dietary flavonoids, rendering some individuals more or less refractory to the health-promoting potential of dietary flavonoids.

Biochem J, 2002 Jul 15, 365(Pt 2), 471 - 9
Aerobic sn-glycerol-3-phosphate dehydrogenase from Escherichia coli binds to the cytoplasmic membrane through an amphipathic alpha-helix; Walz AC et al.; sn-Glycerol-3-phosphate dehydrogenase (GlpD) from Escherichia coli is a peripheral membrane enzyme involved in respiratory electron transfer . For it to display its enzymic activity, binding to the inner membrane is required . The way the enzyme interacts with the membrane and how this controls activity has not been elucidated . In the present study we provide evidence for direct protein-lipid interaction . Using the monolayer technique, we observed insertion of GlpD into lipid monolayers with a clear preference for anionic phospholipids . GlpD variants with point mutations in their predicted amphipathic helices showed a decreased ability to penetrate anionic phospholipid monolayers . From these data we propose that membrane binding of GlpD occurs by insertion of an amphipathic helix into the acyl-chain region of lipids mediated by negatively charged phospholipids.

Biochemistry, 2002 Apr 23, 41(16), 5325 - 32
SecB modulates the nucleotide-bound state of SecA and stimulates ATPase activity; Miller A et al.; In Escherichia coli, the formation of SecA-SecB complexes has a direct effect on SecA ATPase activity . The mechanism of this interaction was evaluated and defined using controlled trypsinolysis, equilibrium dialysis at low temperature, and kinetic analyses of the SecA ATPase reaction . The proteolysis data indicate that SecB and the nonhydrolyzable ATP analogue AMP-P-C-P induce similar conformational changes in SecA which result in a more open or extended structure that is suggestive of the ATP-bound form . The effect is synergistic and concentration-dependent, and requires the occupation of both the high- and low-affinity nucleotide binding sites for maximum effect . The equilibrium dialysis experiments and kinetic data support the observation that the SecB-enhanced SecA ATPase activity is the result of an increased rate of ATP hydrolysis rather than an increase in the affinity of ATP for SecA and that the high-affinity nucleotide binding site is conformationally regulated by SecB . It appears that SecB may function as an intermolecular regulator of ATP hydrolysis by promoting the ATP-bound state of SecA . The inhibition of SecA ATPase activity by sodium azide in the presence of IMVs and a functional signal peptide further indicates that SecB promotes the ATP-bound form of SecA.

Biochemistry, 2002 Apr 23, 41(16), 5266 - 75
Hydrolysis of the 5'-p-nitrophenyl ester of TMP by the proofreading exonuclease (epsilon) subunit of Escherichia coli DNA polymerase III; Hamdan S et al.; The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta) . The epsilon subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (epsilon 186) . A spectrophotometric assay for measurement of epsilon activity is described . Proteins epsilon and epsilon 186 and the epsilon 186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K(M), confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon . On the other hand, formation of the complex with theta stabilized epsilon 186 by approximately 14 degrees C against thermal inactivation . For epsilon 186, k(cat) = 293 min(-)(1) and K(M) = 1.08 mM at pH 8.00 and 25 degrees C, with a Mn(2+) concentration of 1 mM . Hydrolysis of pNP-TMP by epsilon 186 depended absolutely on divalent metal ions, and was inhibited by the product TMP . Dependencies on Mn(2+) and Mg(2+) concentrations were examined, giving a K(Mn) of 0.31 mM and a k(cat) of 334 min(-1) for Mn(2+) and a K(Mg) of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg(2+) . Inhibition by TMP was formally competitive {K(i) = 4.3 microM (with a Mn(2+) concentration of 1 mM)} . The pH dependence of pNP-TMP hydrolysis by epsilon 186, in the pH range of 6.5-9.0, was found to be simple . K(M) was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) of 7.7, approaching limiting values of 50 min(-1) at pH <6.5 and 400 min(-1) at pH >9.0 . These data are used in conjunction with crystal structures of the complex of epsilon 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by epsilon at high and low pH values.

Biochemistry, 2002 Apr 23, 41(16), 5245 - 54
The GTP-dependent restriction enzyme McrBC from Escherichia coli forms high-molecular mass complexes with DNA and produces a cleavage pattern with a characteristic 10-base pair repeat; Pieper U et al.; The GTP-dependent restriction enzyme McrBC consists of two polypeptides: one (McrB) that is responsible for GTP binding and hydrolysis as well as DNA binding and another (McrC) that is responsible for DNA cleavage . It recognizes two methylated or hemimethylated RC sites (R(m)C) at a distance of approximately 30 to more than 2000 base pairs and cleaves the DNA close to one of the two R(m)C sites . This process is strictly coupled to GTP hydrolysis and involves the formation of high-molecular mass complexes . We show here using footprinting techniques, surface plasmon resonance, and scanning force microscopy experiments that in the absence of McrC, McrB binds to a single R(m)C site . If a second R(m)C site is present on the DNA, it is occupied independently by McrB . Whereas the DNA-binding domain of McrB forms 1:1 complexes with each R(m)C site and shows a clear footprint on both R(m)C sites, full-length McrB forms complexes with a stoichiometry of at least 4:1 at each R(m)C site, resulting in a slightly more extended footprint . In the presence of McrC, McrB forms high-molecular mass complexes of unknown stoichiometry, which are considerably larger than the complexes formed with McrB alone . In these complexes and when GTP is present, the DNA is cleaved next to one of the R(m)C sites at distances differing by one to five helical turns, suggesting that in the McrBC-DNA complex only a few topologically well-defined phosphodiester bonds of the DNA are accessible for the nucleolytic center of McrC.

Biochemistry, 2002 Apr 23, 41(16), 5236 - 44
A mutational analysis of the PD...D/EXK motif suggests that McrC harbors the catalytic center for DNA cleavage by the GTP-dependent restriction enzyme McrBC from Escherichia coli; Pieper U et al.; McrBC is a unique restriction enzyme which binds specifically to the bipartite recognition sequence R(m)CN( approximately )(30)(-)( approximately )(2000)R(m)C and in the presence of GTP translocates the DNA and cleaves both strands at multiple positions within the two R(m)C "half-sites" . It is known that McrBC is composed of two subunits: McrB which binds and hydrolyzes GTP and specifically interacts with DNA and McrC whose function is not clear but which has been suspected to harbor the catalytic center for DNA cleavage . A multiple-sequence alignment of the amino acid sequence of Escherichia coli McrC and of six presumably homologous open reading frames from various bacterial species shows that a sequence motif found in many restriction enzymes, but also in other nucleases, the PD.D/EXK motif, is conserved among these sequences . A mutational analysis, in which the carboxylates (aspartic acid in McrC) of this motif were substituted with alanine or asparagine and lysine was substituted with alanine or arginine, strongly suggests that Asp244, Asp257, and Lys259 represent the catalytic center of E . coli McrC . Whereas the variants D244A (or -N), D257A (or -N), and K259A are inactive in DNA cleavage (K259R has residual DNA cleavage activity), they interact with McrB like wild-type McrC, as can be deduced from the finding that they stimulate the McrB-catalyzed GTP hydrolysis to the same extent as wild-type McrC . Thus, whereas McrC variants defective in DNA cleavage can stimulate the GTPase activity of McrB, the DNase activity of McrC is not supported by McrB variants defective in GTP hydrolysis.

Biochemistry, 2002 Apr 23, 41(16), 5213 - 21
Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution; Arjunan P et al.; The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A . The E . coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit . Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds . The functional dimer contains two catalytic centers located at the interface between subunits . The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold . The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts . Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates . These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes . This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species . It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes.

Biochemistry, 2002 Apr 23, 41(16), 5168 - 76
Role of hydrogen bonds in the reaction mechanism of chalcone isomerase; Jez JM et al.; In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit . The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis . The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules . The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190) . Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts . For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K(m) with 4,2',4'-trihydroxychalcone as a substrate . Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction . Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants . The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site . Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule . Hydrogen bonds contributed by Asn113 and Thr190 provide additional stabilization in the transition state . Conservation of these residues in CHIs from other plant species implies a common reaction mechanism for enzyme-catalyzed flavanone formation in all plants.

Biochemistry, 2002 Apr 23, 41(16), 5112 - 9
Solution structure of a monoheme ferrocytochrome c from Shewanella putrefaciens and structural analysis of sequence-similar proteins: functional implications; Bartalesi I et al.; Within the frame of the characterization of the structure and function of cytochromes c, an 81-amino acid cytochrome c was identified in the genome of Shewanella putrefaciens . Because of the scarce information about bacterial cytochromes of this type and the large variability in sequences and possibly function, we decided to proceed to its structural characterization . This protein was expressed in Escherichia coli and purified . The oxidized species is largely high spin, with a detached methionine, whereas the reduced species has the classical His/Met axial ligation to iron . The NMR solution structure of the reduced form was determined on a (15)N-labeled sample, for which 99% of all non-proline backbone (1)H and (15)N resonances have been assigned . One thousand three hundred two meaningful NOEs, out of 1775 NOEs, together with 66 dihedral angles provide a structure with rmsd values from the mean of 0.50 and 0.96 A for backbone and all heavy atoms, respectively . A search of gene banks allowed us to locate 10 different cytochromes c, the sequences of which are more than 30% identical to that of the S . putrefacienscytochrome . For two of them, the structures are known . The structures of the others have been modeled by using the available templates and internally validated . Structural similarities in terms of surface properties account for their biophysical features and provide hints about the function.

J Mol Biol, 2002 Apr 5, 317(4), 601 - 12
Exploring the role of alanine in the structure of the Lac repressor tetramerization domain, a ferritin-like Alacoil; Solan A et al.; We are interested in the determinants that specify the structure of antiparallel coiled coils . Antiparallel coiled coils often contain alanine as an important interfacial packing residue; structures containing alanine at certain well-defined positions in the heptad-repeating unit are referred to as Alacoils . Two types have been identified, containing alanine at either the g position of the heptad repeating unit (defined as the d position in the Richardson nomenclature), referred to as a rop-like Alacoil, or the e position (a position in the Richardson nomenclature), referred to as a ferritin-like Alacoil . The Lac repressor tetramerization domain forms an antiparallel four-chain coiled coil, which falls into the second class of Alacoils based on recent crystal structures . The role of alanine in such structures has not yet been explored experimentally . We test the importance of alanine at the e positions on the oligomeric state and stability of the isolated coiled-coil domain of Lac repressor by testing the effect of mutations at this position . We find that mutation to leucine is tolerated and its moderately stabilizing effect is most likely a consequence of plasticity of this motif . The effects on stability of the mutations to either serine or glutamine can be largely accounted for by helix propensity differences between these residues and alanine . The ability of the helices to adjust to such mutations, while maintaining the basic fold of this coiled coil, was further tested by making the same changes at the more highly exposed g position . Leucine at the g positions also causes an increase in stability, presumably by subtle rearrangement of the helices to allow partial desolvation of this side-chain .

J Mol Biol, 2002 Apr 5, 317(4), 507 - 21
The linkage between magnesium binding and RNA folding; Misra VK et al.; Understanding the linkage between Mg(2+) binding and RNA folding requires a proper theoretical model describing the energetics of Mg(2+) binding to the folded and unfolded states of RNA . Our current understanding of Mg(2+) binding to these different RNA states derives from empirical thermodynamic models that depend on a number of unjustified assumptions . We present a rigorous theoretical model describing the linkage between RNA folding and magnesium ion binding . In this model, based on the non-linear Poisson-Boltzmann (NLPB) equation, the stabilization of RNA by Mg(2+) arises from two distinct binding modes, diffuse binding and site binding . Diffusely bound Mg(2+) are described as an ensemble of hydrated ions that are attracted to the negative charge of the RNA . Site-bound Mg(2+) are partially desolvated ions that are attracted to electronegative pockets on the RNA surface . We explore two systems, yeast tRNA(Phe) and a 58-nucleotide rRNA fragment, with different Mg(2+) binding properties . The NLPB equation accurately describes both the stoichiometric and energetic linkage between Mg(2+) binding and RNA folding for both of these systems without requiring any fitted parameters in the calculation . Moreover, the NLPB model presents a well-defined physical description of how Mg(2+) binding helps fold an RNA . For both of the molecules studied here, the relevant unfolded state is a disordered intermediate state (I) that contains stable helical secondary structure without any tertiary contacts . Diffusely bound Mg(2+) interact with these secondary structure elements to stabilize the I state . The secondary structural elements of the I state fold into a compact, native tertiary structure (the N state) . Diffuse binding plays a dominant role in stabilizing the N state for both RNAs studied . However, for the rRNA fragment, site-binding to a location with extraordinarily high electrostatic potential is also coupled to folding . Our results suggest that much experimental data measuring the linkage between Mg(2+) binding and RNA folding must be reinterpreted .

Shock, 2002 Apr, 17(4), 308 - 11
Taurolidine attenuates the hemodynamic and respiratory changes associated with endotoxemia; Egan BM et al.; The purpose of this study was to determine if prereatment with taurolidine, a known anti-endotoxin agent, would attenuate the hemodynamic and respiratory responses associated with endotoxin induced lung injury in a large animal model in a randomized controlled study under license from the Department of Health . All animals underwent a general anesthetic . Vascular catheters were placed in the femoral artery and in the femoral vein . A Swan-Ganz Catheter was inserted for measurement of pulmonary artery pressure . Animals were randomized into three groups: Control, with measurements taken at baseline and half hourly up to 90 min; Endotoxin, receiving 5microg/Kg E . coli endotoxin intravenously after baseline measurements; and Endotoxin + Taurolidine, receiving 5g of taurolidine via intraperitoneal infusion 1 h before endotoxin administration . Main outcome measures were mean systemic arterial pressure (MAP), mean pulmonary arterial pressure (MPAP), arterial oxygen tension (pO2), serum endotoxin concentration, and pulmonary myeloperoxidase . Endotoxin induced a significant lung injury characterized by an increase in pulmonary artery pressure, hypoxia, and systemic hypotension . Pretreatment with intraperitoneal taurolidine significantly attenuated these hemodynamic and respiratory changes . Serum endotoxin concentration was also significantly reduced as was lung myeloperoxidase . The data suggest that taurolidine may have a therapeutic role in preventing the lung injury seen in endotoxemia.

Shock, 2002 Apr, 17(4), 300 - 3
Characterization of a murine model of endotoxin-induced acute lung injury; Kabir K et al.; Endotoxin-induced microvascular lung injury in mice is a commonly used experimental model of the acute respiratory distress syndrome (ARDS) . The present paper aimed to characterize this popular model in a comprehensive and systematic fashion . Male C57bl/6 mice (n = 5) were administered an LD55 dose of E . coli endotoxin (15 mg/kg, i.p.), and lungs were harvested at several time points and evaluated for injury as well as for expression of a variety of inflammatory mediators . Endotoxin induced many features characteristic of acute microvascular lung injury . These included early (1-2 h) expression of inflammatory mediators (IL-1alpha, IL-1beta, IL-4, IL-6, IL-10, TNF-alpha, interferon-alpha, interferon gamma, and MCP-1) and leukocyte accumulation in lung tissue (lung myeloperoxidase activity 18.5 +/- 7.8 U/g tissue, P < 0.05), followed by pulmonary edema (lung water content index 17.4% +/- 2.5%, P < 0.05) and mortality . Histopathological evaluation of lung tissue was compatible with these findings . The characterization of this murine model of endotoxin-induced microvascular injury will facilitate its utilization in ARDS research.

Appl Microbiol Biotechnol, 2002 Mar, 58(4), 454 - 60 Epub 2002 Jan 30.
Transformation of the Pseudonocardiaceae amycolatopsis sp . strain HR167 is highly dependent on the physiological state of the cells; Priefert H et al.; The Pseudonocardiaceae Amycolatopsis sp . strain HR167 is used in a biotransformation process to produce vanillin from ferulic acid . To make this strain accessible for genetic engineering, a direct mycelium transformation system developed for Amycolatopsis mediterranei {Madon and Hotter (1991) J Bacteriol 173: 6325-6331} was applied and optimized for Amycolatopsis sp . strain HR167 . The physiological state of the cells had a major influence on the transformation rate . The highest transformation rate of about 7x10(5) transformants per microgram of DNA was obtained with mycelium harvested 6.5-7.5 h after the culture has reached the stationary growth phase . When cells were harvested outside of this time slot, the transformation rate drastically decreased . The density of the mycelium suspensions used in the transformation mixture and the methylation state of the plasmid DNA used for the transformation were also crucial parameters . With plasmid DNA isolated from Escherichia coli ET12567, transformation rates were 3,500-fold higher than those obtained with DNA isolated from E . coli XL1-Blue.

Arthritis Rheum, 2002 Apr, 46(4), 929 - 33
Interaction between heat-shock protein 73 and HLA-DRB1 alleles associated or not with rheumatoid arthritis; Auger I et al.; OBJECTIVE: HLA-DRB1 alleles whose third hypervariable region contains a QKRAA/QRRAA/RRRAA motif are associated with rheumatoid arthritis (RA) through unknown mechanisms . We previously demonstrated that the QKRAA motif was also expressed on the Escherichia coli 40-kd heat-shock protein (HSP) DnaJ . The QKRAA motif helps DnaJ bind its partner chaperone, the E coli 70-kd HSP DnaK . Furthermore, we observed that in lymphoblastoid cells, Hsp73, the constitutive 70-kd HSP, associates with HLA-DRB1*0401 (an allele with a QKRAA motif) and targets it to lysosomes . In this study, we sought to classify different HLA-DRB1 alleles according to their ability to bind Hsp73 . METHODS: To evaluate how well different HLA-DRB1 alleles could bind Hsp73, we developed a quantitative precipitation assay and a direct binding assay . RESULTS: Quantitative precipitation assay from total cellular proteins and from lysosomal extracts demonstrated that RA-associated HLA-DRB1 alleles bound Hsp73 better than did HLA-DRB1 alleles that were not associated with RA . HLA-DRB1*0401 was the best Hsp73 binder . These findings were confirmed by direct binding assay between purified proteins . CONCLUSION: HLA-DRB1*0401 was the best Hsp73 binder among the 8 different HLA-DRB1 alleles that were tested.

Saudi Med J, 2002 Apr, 23(4), 467 - 70
Diabetes and infarcted papillary thyroid cancer; Haddad FH et al.; A young black Jordanian lady who has type one diabetes, chronic diabetic complication and ischemic heart disease, presented with a picture of diabetic keto-acidosis, precipitated by an acute neck swelling . This was suggestive of acute suppurative thyroiditis with abscess formation causing compressive symptoms . This unfortunate patient had an eventful course despite aggressive treatment by antibiotics and surgery and then succumbed of an acute cardiac event . The operative tissue biopsy revealed an abscess in an infarcted papillary thyroid cancer . We believe this is a rare presentation of such an association with a fatal outcome.

Nat Struct Biol, 2002 May, 9(5), 353 - 8
Structure of the 16S rRNA pseudouridine synthase RsuA bound to uracil and UMP; Sivaraman J et al.; In Escherichia coli, the pseudouridine synthase RsuA catalyzes formation of pseudouridine (psi) at position 516 in 16S rRNA during assembly of the 30S ribosomal subunit . We have determined the crystal structure of RsuA bound to uracil at 2.0 A resolution and to uridine 5'-monophosphate (UMP) at 2.65 A resolution . RsuA consists of an N-terminal domain connected by an extended linker to the central and C-terminal domains . Uracil and UMP bind in a cleft between the central and C-terminal domains near the catalytic residue Asp 102 . The N-terminal domain shows structural similarity to the ribosomal protein S4 . Despite only 15% amino acid identity, the other two domains are structurally similar to those of the tRNA-specific psi-synthase TruA, including the position of the catalytic Asp . Our results suggest that all four families of pseudouridine synthases share the same fold of their catalytic domain(s) and uracil-binding site.

J Biol Chem, 2002 Jul 19, 277(29), 26652 - 61 Epub 2002 Apr 12.
Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli; De Wulf P et al.; In Escherichia coli, the two-component Cpx system comprising the CpxA sensor kinase and the CpxR response regulator modulates gene expression in response to a variety of stresses including membrane-protein damage, starvation, and high osmolarity . To date, the few known CpxR-P target operons were mostly identified by genetic screens . To facilitate the discovery of all target operons, we derived a 15-bp weighted matrix for CpxR-P recognition that takes into account the relative base frequency at each nucleotide position . This matrix essentially consists of two tandem 5'-GTAAA-3' motifs separated by a 5-bp linker . All of the 15-bp stretches on both strands of the E . coli MG1655 genome were then scored for their degree of matching with the matrix and classified in statistical deviation groups . The effectiveness of this screening is indicated by the identification of eight new target operons (ung, ompC, psd, mviA, aroK, rpoErseABC, secA, and aer) among eleven candidates tested . Moreover, the matrix score correlates with the likelihood that a site is a true target and with the relative site affinity for CpxR-P in vitro . Our data indicate that some 100 operons are under direct CpxR-P control and that the signal transduction pathway interacts with several other control circuits in manners hitherto unanticipated.

J Biol Chem, 2002 Jun 28, 277(26), 23898 - 908 Epub 2002 Apr 12.
PurT-encoded glycinamide ribonucleotide transformylase . Accommodation of adenosine nucleotide analogs within the active site; Thoden JB et al.; PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate . From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains . In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding . Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP . Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket . This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.

J Biol Chem, 2002 Jul 5, 277(27), 24522 - 9 Epub 2002 Apr 12.
Three-dimensional structure of human gamma -glutamyl hydrolase . A class I glatamine amidotransferase adapted for a complex substate; Li H et al.; gamma-Glutamyl hydrolase catalyzes the cleavage of the gamma-glutamyl chain of folylpoly-gamma-glutamyl substrates and is a central enzyme in folyl and antifolyl poly-gamma-glutamate metabolism . The crystal structure of human gamma-glutamyl hydrolase, determined at 1.6-A resolution, reveals that the protein is a homodimer . The overall structure of human gamma-glutamyl hydrolase contains 11 alpha-helices and 14 beta-strands, with a fold in which a central eight-stranded beta-sheet is sandwiched by three and five alpha-helices on each side . The topology is very similar to that of the class I glutamine amidotransferase domains, with the only major differences consisting of extensions in four loops and at the C terminus . These insertions are important for defining the substrate binding cleft and/or the dimer interface . Two sequence motifs are found in common between human gamma-glutamyl hydrolase and the class I glutamine amidotransferase family and include the catalytically essential residues, Cys-110 and His-220 . These residues are located in the center of a large l-shaped cleft that is closed at one end and open at the other . Several conserved residues, including Glu-114, His-171, Gln-218, and Lys-223, may be important for substrate binding . Modeling of a methotrexate thioester intermediate, based on the corresponding complex of the glutamate thioester intermediate of Escherichia coli carbamoyl-phosphate synthetase, indicates that the substrate binds in an orientation with the pteroyl group toward the open end of the cleft.

Infect Immun, 2002 May, 70(5), 2700 - 3
Identification and functional analysis of an immunoreactive DsbA-like thio-disulfide oxidoreductase of Ehrlichia spp; McBride JW et al.; Novel homologous DsbA-like disulfide bond formation (Dsb) proteins of Ehrlichia chaffeensis and Ehrlichia canis were identified which restored DsbA activity in complemented Escherichia coli dsbA mutants . Recombinant Ehrlichia Dsb (eDsb) proteins were recognized by sera from E . canis-infected dogs but not from E . chaffeensis-infected patients . The eDsb proteins were observed primarily in the periplasm of E . chaffeensis and E . canis.

Infect Immun, 2002 May, 70(5), 2681 - 9
Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli; Keller R et al.; O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries . Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5 . ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA) . Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa . An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E . coli . Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures . This is the first characterization and demonstration of an Afa adhesin associated with EPEC.

Infect Immun, 2002 May, 70(5), 2622 - 9
Construction and characterization of an acapsular mutant of Mannheimia haemolytica A1; McKerral LJ et al.; The nmaA and nmaB genes, which code for UDP-GlcNAc-2-epimerase and UDP-ManNAc-dehydrogenase, respectively, are involved in capsular polysaccharide biosynthesis in Mannheimia haemolytica A1 . A chloramphenicol resistance (Cm(r)) cassette cloned behind an M . haemolytica A1 promoter, plpcat, was created and used to interrupt nmaA and nmaB . A 1.3-kbp DNA fragment that encompasses part of nmaA and nmaB was replaced by the 1.0-kbp plpcat, resulting in a knockout mutant which is Cm(r) and unable to synthesize N-acetylmannosamine (ManNAc) and N-acetylmannosaminuronic acid (ManNAcA) . The DNA replacement was confirmed by Southern hybridization and PCR analyses of the nmaA and nmaB loci . Electron microscopy examination of the mutant showed the absence of capsular materials compared to the parent strain . The loss of NmaA and NmaB activity was confirmed by analysis of carbohydrate moieties using capillary electrophoresis . Serum sensitivity assays indicated that the acapsular mutant is as resistant as the encapsulated parent to complement-mediated killing by colostrum-deprived calf serum but is more sensitive to killing by immune bovine serum . Analysis of lipopolysaccharide prepared from the acapsular mutant and encapsulated parent confirmed that these strains have long O-polysaccharide chains, possibly conferring resistance to serum-mediated killing.

Infect Immun, 2002 May, 70(5), 2519 - 25
Modulation of mouse endotoxic fever by complement; Li S et al.; It was recently reported that the complement system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneally (i.p.) injected, lipopolysaccharides (LPS) . The present study was designed to identify which component(s) of the complement cascade may be specifically critical . To this end, we used mice with C3, C5, and CR2 gene deletions . To assess preliminarily the suitability of mice for such a study, we replicated our earlier studies with guinea pigs . Thus, to verify initially whether complement is similarly involved in the febrile response of wild-type (C57BL/6J) mice to i.p . LPS (Escherichia coli, 1 microg/mouse), we depleted complement with cobra venom factor (CVF; 7 U/mouse, intravenously {i.v.}) . These animals did not develop fever, whereas the core temperature (T(c)) of CVF vehicle-treated controls rose approximately 1 degrees C by 80 min postinjection and then gradually abated over the following 2.5 h, confirming the involvement of complement in fever production after i.p . LPS injection and the suitability of this species for these studies . C3- and C5-sufficient (C3(+/+) and C5(+/+)) mice also developed 1 degrees C fevers within 80 min after i.p . LPS (1 or 2 microg/mouse) injection . These fevers were totally prevented by CVF (10 U/mouse, i.v.) pretreatment . C3- and C5-deficient (C3(-/-) and C5(-/-)) mice were also unable to develop T(c) rises after i.p . LPS . Both CR2(+/+) and CR2(-/-) mice responded normally to i.p . LPS (1 microg/mouse) . These data indicate that C5, but not C3d acting through CR2, may play a critical role in the febrile response of mice to i.p . LPS.

Infect Immun, 2002 May, 70(5), 2336 - 43
Evaluation of receptor binding specificity of Escherichia coli K88 (F4) fimbrial adhesin variants using porcine serum transferrin and glycosphingolipids as model receptors; Grange PA et al.; Diarrheal disease caused by enterotoxigenic Escherichia coli expressing the K88 (F4) fimbrial adhesin (K88 ETEC) is a significant source of mortality and morbidity among newborn and weaned piglets . K88 fimbrial adhesins are filamentous surface appendages whose lectin (carbohydrate-binding) activity allows K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells . There are three variants of K88 adhesin (K88ab, K88ac, and K88ad), which possess different, yet related, carbohydrate-binding specificities . We used porcine serum transferrin (pSTf) and purified glycosphingolipids (GSL) to begin to define the minimal recognition sequence for K88 adhesin variants . We found that K88ab adhesin binds with high affinity to pSTf (dissociation constant, 75 microM), while neither K88ac nor K88ad adhesin recognizes pSTf . Degradation of the N-glycan on pSTf by extensive metaperiodate treatment abolished its interaction with the K88ab adhesin, indicating that the K88ab adhesin binds to the single N-glycan found on pSTf . Using exoglycosidase digestion of the pSTf glycan, we demonstrated that K88ab adhesin recognizes N-acetylglucosamine (GlcNAc) residues in the core of the N-glycan on pSTf . All three K88 variants were found to bind preferentially to GSL containing a beta-linked N-acetylhexosamine (HexNAc), either GlcNAc or N-acetylgalactosamine, in the terminal position or, alternatively, in the penultimate position with galactose in the terminal position . Considering the results from pSTf and GSL binding studies together, we propose that the minimal recognition sequence for the K88 adhesin variants contains a beta-linked HexNAc . In addition, the presence of a terminal galactose beta-linked to this HexNAc residue enhances K88 adhesin binding.

Infect Immun, 2002 May, 70(5), 2304 - 10
Enterohemorrhagic Escherichia coli infection induces interleukin-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-kappaB and AP-1 in T84 cells; Dahan S et al.; Enterohemorrhagic Escherichia coli (EHEC) infections are associated with hemorrhagic colitis and the hemolytic-uremic syndrome (HUS) . In vivo, elevated plasma levels of the proinflammatory cytokine interleukin-8 (IL-8) in EHEC-infected children are correlated with a high risk of developing HUS . As IL-8 gene transcription is regulated by the transcription factors NF-kappaB and AP-1, we analyzed the role of these factors in the regulation of IL-8 production after infection of the epithelial intestinal T84 cell line by EHEC . By 6 h of infection, EHEC had induced significant secretion of IL-8 (35.84 +/- 6.76 ng/ml versus 0.44 +/- 0.04 ng/ml in control cells) . EHEC induced AP-1 and NF-kappaB activation by 3 h of infection . Moreover, the three mitogen-activated protein kinases (MAPK) (ERK1/2, p38, and JNK) were phosphorylated in EHEC-infected T84 cells concomitant with induction of AP-1 DNA binding activity, and IkappaB-alpha was phosphorylated and then degraded concomitant with induction of NF-kappaB DNA binding activity . Pretreatment of cells with the highly specific MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and/or the proteasome inhibitor ALLN led to inhibition of the IL-8 secretion induced in EHEC-infected T84 cells . These findings demonstrate that (i) EHEC can induce in vitro a potent proinflammatory response by secretion of IL-8 and (ii) the secretion of IL-8 is due to the involvement of MAPK, AP-1, and NF-kappaB signaling pathways.

Infect Immun, 2002 May, 70(5), 2271 - 7
A gene from the locus of enterocyte effacement that is required for enteropathogenic Escherichia coli to increase tight-junction permeability encodes a chaperone for EspF; Elliott SJ et al.; Disruption of the barrier properties of the enterocyte tight junction is believed to be important in the pathogenesis of diarrhea caused by enteropathogenic Escherichia coli (EPEC) . This phenotype can be measured in vitro as the ability of EPEC to reduce transepithelial resistance (TER) across enterocyte monolayers and requires the products of the locus of enterocyte effacement (LEE) and, in particular, the type III secreted effector protein EspF . We report a second LEE-encoded gene that is also necessary for EPEC to fully reduce TER . rorf10 is not necessary for EPEC adherence, EspADB secretion, or formation of attaching and effacing lesions . However, rorf10 mutants have a diminished TER phenotype, reduced intracellular levels of EspF, and a reduced ability to translocate EspF into epithelial cells . The product of rorf10 is a 14-kDa intracellular protein rich in alpha-helices that specifically interacts with EspF but not with Tir or other EPEC secreted proteins . These properties are consistent with the hypothesis that rorf10 encodes a type III secretion chaperone for EspF, and we rename this protein CesF, the chaperone for EPEC secreted protein F.

Infect Immun, 2002 May, 70(5), 2264 - 70
Functional substitution of the TibC protein of enterotoxigenic Escherichia coli strains for the autotransporter adhesin heptosyltransferase of the AIDA system; Moormann C et al.; The plasmid-encoded AIDA (adhesin involved in diffuse adherence) autotransporter protein derived from diffuse-adhering clinical Escherichia coli isolate 2787 and the TibA (enterotoxigenic invasion locus B) protein encoded by the chromosomal tib locus of enterotoxigenic E . coli (ETEC) strain H10407 are posttranslationally modified by carbohydrate substituents . Analysis of the AIDA-I adhesin showed that the modification involved heptose residues . AIDA-I is modified by the heptosyltransferase activity of the product of the aah gene, which is located directly upstream of adhesin-encoding gene aidA . The carbohydrate modification of the TibA adhesin/invasin is mediated by the TibC protein but has not been elucidated . Based on the sequence similarities between TibC and AAH (autotransporter adhesin heptosyltransferase) and between the TibA and the AIDA proteins we hypothesized that the AIDA system and the Tib system encoded by the tib locus are structurally and functionally related . Here we show that (i) TibC proteins derived from different ETEC strains appear to be highly conserved, (ii) recombinant TibC proteins can substitute for the AAH heptosyltransferase in introducing the heptosyl modification to AIDA-I, (iii) this modification is functional in restoring the adhesive function of AIDA-I, (iv) a single amino acid substitution at position 358 completely abolishes this activity, and (v) antibodies directed at the functionally active AIDA-I recognize a protein resembling modified TibA in ETEC strains . In summary, we conclude that, like AAH, TibC represents an example of a novel class of heptosyltransferases specifically transferring heptose residues onto multiple sites of a protein backbone . A potential consensus sequence for the modification site is suggested.

EMBO J, 2002 Apr 15, 21(8), 1998 - 2008
Exploring intracellular space: function of the Min system in round-shaped Escherichia coli; Corbin BD et al.; The MinCDE proteins help to select cell division sites in normal cylindrical Escherichia coli by oscillating along the long axis, preventing unwanted polar divisions . To determine how the Min system might function in cells with multiple potential division planes, we investigated its role in a round-cell rodA mutant . Round cells lacking MinCDE were viable, but growth, morphology and positioning of cell division sites were abnormal relative to Min+ cells . In round cells with a long axis, such as those undergoing cell division, green fluorescent protein (GFP) fusions to MinD almost always oscillated parallel to the long axis . However, perfect spheres or irregularly shaped cells exhibited MinD movement to and from multiple sites on the cell surface . A MinE-GFP fusion exhibited similar behavior . These results indicate that the Min proteins can potentially localize anywhere in the cell but tend to move a certain maximum distance from their previous assembly site, thus favoring movement along the cell's long axis . A new model for the spatial control of division planes by the Min system in round cells is proposed.

Zhonghua Yi Xue Za Zhi, 2002 Jan 25, 82(2), 94 - 9
{Cloning of a novel gene, ANGPTL4 and the functional study in angiogenesis}; Zhu H et al.; OBJECTIVE: To clone a novel HCC-related gene and study its functions . METHODS: Using a combination of cell growth based functional screening of human placental cDNA library and RACE, a novel gene, angiopoietin-like 4 (ANGPTL4) was isolated . The gene was mapped by RH-PCR; also it was expressed with E . coli . Effects on angiogenesis was studied by MTT assay, in vitro wound migration assay, in vitro invasion assay, in vitro tube formation assay and in vivo mouse matrigel plug assay . RESULTS: ANGPTL4 was cloned and mapped in 19p13.3 . ANGPTL4 had no significant effect on proliferation of PAEC (P = 0.0504), while it can significantly stimulate migration and invasion of PAEC with the dose of 2 microgram/ml (P < 0.05) and promote in vitro tube formation of PAEC and angiogenesis of mouse in vivo . CONCLUSIONS: ANGPTL4 can stimulate angiogenesis.

Mol Microbiol, 2002 Mar, 43(6), 1651 - 6
Studies on the role of the metK gene product of Escherichia coli K-12; Wei Y et al.; We show here that the metK gene is essential to the growth of Escherichia coli K-12 and can be deleted only in the presence of a rescue plasmid carrying a functional metK gene . When metK expression was limited, genomic DNA methylation decreased and cell division was hampered . Through primer extension, the transcription start site of metK was located at 140 bp upstream of the translation start site . The frequently used metK84 mutant has been shown to carry an A(r)G transition in the -10 region of the metK promoter . This accounts for its low level of S-adenosylmethionine (SAM) synthetase and SAM deficiency.

Mol Microbiol, 2002 Mar, 43(6), 1457 - 70
Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis; Buchanan G et al.; The Escherichia coli Tat apparatus is a membrane-bound protein translocase that serves to export folded proteins synthesized with N-terminal twin-arginine signal peptides . The essential TatC component of the Tat translocase is an integral membrane protein probably containing six transmembrane helices . Sequence analysis identified conserved TatC amino acid residues, and the role of these side-chains was assessed by single alanine substitution . This approach identified three classes of TatC mutants . Class I mutants included F94A, E103A and D211A, which were completely devoid of Tat-dependent protein export activity and thus represented residues essential for TatC function . Cross-complementation experiments with class I mutants showed that co-expression of D211A with either F94A or E103A regenerated an active Tat apparatus . These data suggest that different class I mutants may be blocked at different steps in protein transport and point to the co-existence of at least two TatC molecules within each Tat translocon . Class II mutations identified residues important, but not essential, for Tat activity, the most severely affected being L99A and Y126A . Class III mutants showed no significant defects in protein export . All but three of the essential and important residues are predicted to cluster around the cytoplasmic N-tail and first cytoplasmic loop regions of the TatC protein.

Mol Microbiol, 2002 Mar, 43(6), 1445 - 56
RNase G complementation of rne null mutation identifies functional interrelationships with RNase E in Escherichia coli; Lee K et al.; The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity . Whereas the absence of Rne normally is lethal, we show here that E . coli bacteria that lack the rne gene can be made viable by overexpression of Rng . Rng-complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e . M1 RNA), indicating that normal processing of these Rne-cleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro . Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of RNase E . Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng . However, approximately 100 mRNAs that accumulated in Rne-deficient cells were decreased by rng-complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality . Remarkably prominent in this group were mRNAs implicated in energy-generating pathways or in the synthesis or degradation of macromolecules.

Mol Microbiol, 2002 Mar, 43(6), 1431 - 43
Spc24 interacts with Mps2 and is required for chromosome segregation, but is not implicated in spindle pole body duplication; Le Masson I et al.; Mps2 (monopolar spindle protein) is a coiled-coil protein found at the spindle pole body (SPB) and at the nuclear envelope that is required for insertion of the SPB into the nuclear envelope . We identified three proteins that interact with Mps2 in a two-hybrid screen: Bbp1, Ynl107w and Spc24 . All three proteins contain coiled-coil motifs that appear to be required for their interaction with Mps2 . In this work, we verified the Mps2-Spc24 interaction by co-immunoprecipitation in vivo and by the in vitro interaction of recombinant proteins . Previous two-hybrid screens with Spc24 as bait had identified Spc25 and Ndc80 as putative interacting partners, and we verified these interactions in vivo by purification of TAP-tagged derivatives of Spc24 and Ndc80 . Finally, we found that spc24 thermosensitive mutants had a chromosome segregation defect, but no apparent defect in SPB duplication . These results are consistent with recently published data showing that Spc24, Spc25 and Ndc80 are peripheral kinetochore com-ponents required for chromosome segregation . The Mps2-Spc24 interaction may contribute to the localization of Spc24 and other kinetochore components to the inner plaque of the SPB.

Genes Cells, 2002 Apr, 7(4), 385 - 99
Hyper-processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli; Sugaya Y et al.; BACKGROUND: A strong mutator mutation, dnaE173, leads to a Glu612 --> Lys amino acid change in the alpha subunit of Escherichia coli DNA polymerase III (PolIII) holoenzyme and abolishes the proofreading function of the replicative enzyme without affecting the 3' --> 5' exonuclease activity of the epsilon subunit . The dnaE173 mutator is unique in its ability to induce sequence-substitution mutations, suggesting that an unknown function of the alpha subunit is hampered by the dnaE173 mutation . RESULTS: A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the beta clamp subunit) and the beta subunit showed a strong resistance to replication-pausing on the template DNA and readily promoted strand-displacement DNA synthesis . Unlike wild-type PolIII*, dnaE173 PolIII* was able to catalyse highly processive DNA synthesis without the aid of the beta-clamp subunit . The rate of chain elongation by the dnaE173 holoenzyme was reduced to one-third of that determined for the wild-type enzyme . In contrast, an exonuclease-deficient PolIII holoenzyme was vastly prone to pausing, but had the same rate of chain elongation as the wild-type . CONCLUSIONS: The hyper-processivity and slower DNA chain elongation rate of the dnaE173 holoenzyme are distinct effects caused by the dnaE173 mutation and are likely to be involved in the sequence-substitution mutagenesis . A link between the proofreading and chain elongation processes was suggested.

Genes Cells, 2002 Apr, 7(4), 351 - 63
Gene products encoded in the ninR region of phage lambda participate in Red-mediated recombination; Tarkowski TA et al.; BACKGROUND: The ninR region of phage lambda contains two recombination genes, orf (ninB) and rap (ninG), that were previously shown to have roles when the RecF and RecBCD recombination pathways of E . coli, respectively, operate on phage lambda . RESULTS: When lambda DNA replication is blocked, recombination is focused at the termini of the virion chromosome . Deletion of the ninR region of lambda decreases the sharpness of the focusing without diminishing the overall rate of recombination . The phenotype is accounted for in large part by the deletion of rap and of orf . Mutation of the recJ gene of the host partially suppresses the Rap- phenotype . CONCLUSION: ninR functions Orf and Rap participate in Red recombination, the primary pathway operating when wild-type lambda grows lytically in rec+ cells . The ability of recJ mutation to suppress the Rap- phenotype indicates that RecJ exonuclease can participate in Red-mediated recombination, at least in the absence of Rap function . A model is presented for Red-mediated RecA-dependent recombination that includes these newly identified participants.

Eur J Biochem, 2002 Apr, 269(7), 1942 - 6
NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1); Matthey U et al.; The subunit c from the ATP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/water (4 : 4 : 1) . In this solvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G29 to N82, respectively . On comparing the secondary structure of subunit c from P . modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the alpha helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the prominent interruption of the alpha helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the alpha helix after the sodium ion-binding Glu65 residue . The folding of subunit c of P . modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that subunit c of P . modestum did not adopt a stable hairpin structure like subunit c of E . coli.

Eur J Biochem, 2002 Apr, 269(7), 1854 - 65
Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom; Huys I et al.; A novel peptidyl inhibitor of voltage-gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus . This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called 'alpha-KTx1.1') . PBTx3 is the tenth member (called 'alpha-KTx1.10') of subfamily 1 of K+ channel-blocking peptides known thus far . Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels . The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 microm, 547 nm and 492 nm . A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli . The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3) . After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties . The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel beta sheet and a single alpha-helix, connected by three disulfide bridges . The scaffold of PBTx3 is homologous to most other alpha-KTx scorpion toxins.

Eur J Biochem, 2002 Apr, 269(7), 1806 - 13
Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human alphaB-crystallin; Valdez MM et al.; Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection . MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein aggregation in vitro, and it contains the core 'alpha-crystallin' domain found in all sHsps . In this study we characterized the chaperone activity of recombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human alphaB-crystallin, a well characterized member of the sHsp family . Recombinant MTB HSP 16.3 was expressed in Escherichia coli and purified to apparent homogeneity . Similar to alphaB-crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 mm ATP the chaperone activity was enhanced by twofold . ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPgammaS, a nonhydrolyzable analog of ATP . Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E . coli at 48 degrees C . While the sequence similarity between human alphaB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core 'alpha-crystallin' domain are much closer.

Cell Microbiol, 2002 Apr, 4(4), 235 - 43
Enteropathogenic Escherichia coli induces modification of the focal adhesions of infected host cells; Shifrin Y et al.; Enteropathogenic Escherichia coli (EPEC) is a human-specific pathogen that causes severe diarrhoea in young children . The disease involves intimate interaction between the pathogen and the brush border of enterocytes . During infection, EPEC uses a type III secretion system (TTSS) to inject several proteins into the infected cells, and these effector proteins modify specific processes in the host cell . We show that, upon infection, EPEC induces detachment of the infected host cells from the substratum, modification of focal adhesions (FA) in the infected cells and specific dephosphorylation of focal adhesion kinase (FAK) . We also show that EPEC-induced cell detachment is dependent on FAK expression by the infected cells . Finally, we demonstrate that cell detachment, FA modification and FAK dephosphorylation are dependent on functional TTSS in the infecting EPEC . These results suggest that EPEC is using its TTSS to inject protein(s) into the infected cells, which can induce FAK dephosphorylation, as well as FAK-dependent FA modification and cell detachment . These processes are specific and probably play an important role in EPEC virulence.

Cell Microbiol, 2002 Apr, 4(4), 213 - 22
The EspB protein of enterohaemorrhagic Escherichia coli interacts directly with alpha-catenin; Kodama T et al.; Enterohaemorrhagic Escherichia coli (EHEC) belongs to a family of pathogens that cause attaching and effacing (A/E) lesion on target cells . The EspB protein of EHEC is translocated both to the host cell cytoplasm and to the membrane, and is essential for the signalling events leading to A/E lesion . To determine the actual role of EspB in this process, we tried to identify the EspB binding partner of the host cell protein, using a yeast two-hybrid assay, and obtained a cytoskeletal-associated protein, alpha-catenin . The alpha-catenin bound directly to the N-terminal region of EspB, both in solid (overlay assay) and solution (pull-down assay) phases, and it was recruited to the EHEC adherence site, dependent on EspB . Expression of the N-terminal region of EspB, as well as the whole EspB in host cells, inhibited F-actin accumulation on the adherence site . We conclude that EspB recruits alpha-catenin at the EHEC adherence site by direct interaction, and that the recruitment of alpha-catenin is essential for EHEC-induced A/E lesion formation.

Biochem J, 2002 Aug 1, 365(Pt 3), 833 - 40
An open reading frame in intron seven of the sea urchin DNA-methyltransferase gene codes for a functional AP1 endonuclease; Cioffi AV et al.; Analysis of the genome structure of the Paracentrotus lividus (sea urchin) DNA methyltransferase (DNA MTase) gene showed the presence of an open reading frame, named METEX, in intron 7 of the gene . METEX expression is developmentally regulated, showing no correlation with DNA MTase expression . In fact, DNA MTase transcripts are present at high concentrations in the early developmental stages, while METEX is expressed at late stages of development . Two METEX cDNA clones (Met1 and Met2) that are different in the 3' end have been isolated in a cDNA library screening . The putative translated protein from Met2 cDNA clone showed similarity with Escherichia coli endonuclease III on the basis of sequence and predictive three-dimensional structure . The protein, overexpressed in E . coli and purified, had functional properties similar to the endonuclease specific for apurinic/apyrimidinic (AP) sites on the basis of the lyase activity . Therefore the open reading frame, present in intron 7 of the P . lividus DNA MTase gene, codes for a functional AP endonuclease designated SuAP1.

Chem Res Toxicol, 2002 Apr, 15(4), 490 - 6
Effect of liquid depth on the synthesis and oxidation of nitric oxide in macrophage cultures; Chen B et al.; The effect of liquid depth on the synthesis of NO and O(2)(-) was studied in murine macrophage-like RAW 264.7 cells activated by bacterial lipopolysaccharide and interferon-gamma . Rates of NO(2)(-) and NO(3)(-) accumulation were determined 8-11 h after stimulation . The rate of NO synthesis was computed by using a reaction-diffusion model to correct NO(2)(-) and NO(3)(-) accumulation for physical loss of NO, whereas O(2)(-) synthesis was equated with NO(3)(-) formation . Rates of O(2)(-) synthesis determined by a spectrophotometric (cytochrome c) assay were in good agreement with those from NO(3)(-) accumulation and showed production of O(2)(-) to be detectable immediately, in contrast to the approximately 6 h time lag for NO . The assumption that NO(2)(-) and NO(3)(-) are stable end products of the extracellular oxidation of NO by O(2) and O(2)(-), respectively, was supported by the fact that NO(2)(-) and NO(3)(-) concentrations remained constant in the presence of unstimulated cells or stimulated cells where NO synthesis was inhibited . Data were obtained for media depths ranging from 1 to 4 mm . The physical loss of NO was found to be quite significant, exceeding NO(2)(-) and NO(3)(-) accumulation by an order of magnitude at the smallest depth . The principal finding was that the rates of NO(2)(-) and NO(3)(-) accumulation each remained nearly constant over the 4-fold range of liquid depths . Because greater depths should greatly facilitate the trapping of NO as NO(2)(-), this implies that NO synthesis decreased markedly with increasing depth . In contrast, O(2)(-) synthesis remained approximately constant . Oxygen availability is likely to have affected NO synthesis, in that diffusional limitations will yield the lowest cellular O(2) concentrations when the liquid depth is greatest and NO synthesis is known to decrease when O(2) levels are reduced . Concentrations of NO near the cells were calculated to remain at approximately 1 microM for all conditions examined, suggesting that regulation of NO synthase activity by NO might also have mediated the effect of liquid depth.

Int Rev Cytol, 2002, 215, 75 - 104
Aquaglyceroporins: channel proteins with a conserved core, multiple functions, and variable surfaces; Engel A et al.; Membrane channels for water and small nonionic solutes are required for osmoregulation in bacteria, plants, and animals . Aquaporin-1, the water channel of human erythrocytes, is the first channel demonstrated to conduct water, by expression in Xenopus oocytes . Phylogenetic analyses reveal the existence of two clusters of subfamilies, the aquaporins (AQPs) and glycerol facilitators (GLPs) . Sequence-based structure prediction provided a model comprising six membrane-spanning helices, while sequence analyses suggested strategic residues that are important for structure and function . The surface topography of several AQPs has been mapped by atomic force microscopy, revealing different features that correlate with differences in the loops connecting transmembrane helices . The 3D structures of AQP1 and GlpF have been determined by electron cryomicroscopy . The 3.8-A density map allowed the first atomic model of AQP1 to be built, taking into account data from sequence analyses . This model provides some insight into the permeation of water through a channel that blocks the passage of protons . GIpF has been resolved to 6.9 A, revealing helices that are similar to those of AQP1 . Homology modeling shows the channel region of these distant aquaglyceroporins to be similar, as confirmed by the 2.2-A structure of GlpF from X-ray crystallography.

Mol Plant Microbe Interact, 2002 Mar, 15(3), 216 - 24
Localization of melanin in conidia of Alternaria alternata using phage display antibodies; Carzaniga R et al.; Melanins derived from 1,8-dihydroxynaphthalene (DHN) are important for the pathogenicity and survival of fungi causing disease in both plants and animals . However, precise information on their location within fungal cell walls is lacking . To obtain antibodies for the immunocytochemical localization of melanin, 83 phage antibodies binding to 1,8-DHN were selected from a naive semisynthetic single-chain Fv (scFv) phage display library . Sequence analysis of the heavy chain binding domains of 17 antibodies showed a high frequency of positively charged amino acids . One antibody, designated M1, was characterized in detail . M1 bound specifically to 1,8-DHN in competitive inhibition enzyme-linked immunosorbent assays, showing no cross-reaction with nine structurally related phenolic compounds . Epitope recognition required two hydroxyl groups in a 1,8 configuration . M1 also bound to naturally occurring melanin isolated from mycelia of Alternaria alternata, suggesting that epitopes remain accessible in polymerized melanin . Transmission electron microscopy-immunogold labeling, using M1 in the form of soluble scFv fragments, showed that melanin was located in the septa and outer (primary) walls of wild-type A . alternata conidia, but not those of an albino mutant, AKT88-1 . The M1 antibody provides a new tool for detecting melanized pathogens in plant and animal tissues and for precisely mapping the distribution of the polymer within spores, appressoria, and hyphae.

Mol Cell Biochem, 2002 Jan, 230(1-2), 73 - 83
Structure and function of the heat-stable enterotoxin receptor/guanylyl cyclase C; Vaandrager AB; Guanylyl cyclase C (GC-C) was found to function as the principal receptor for heat-stable enterotoxins (STa), major causative factors in E . coli-induced secretory diarrhea . GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues . The enzyme belongs to the family of receptor guanylyl cyclases, and consists of an extracellular receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain . GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa receptor that is coupled non-covalently to the intracellular domain . So far two endogenous ligands of mammalian GC-C have been identified i.e . the small cysteine-rich peptides guanylin and uroguanylin . The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia . They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts . In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO3 secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger . The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.

J Radiat Res (Tokyo), 2001 Dec, 42(4), 409 - 13
Increased base change mutations at G:C pairs in Escherichia coli deficient in endonuclease III and VIII; Tano K et al.; Various types of mutation induced by oxidative DNA damage, induced by hydrogen peroxide and riboflavin photosensitization, were determined in Escherichia coli (E . coli) mutants deficient in endonuclease III (endo III) and endonuclease VIII (endo VIII) . The majority of hydrogen peroxide-induced and spontaneous mutations consisted of G:C to A:T and to T:A base changes, shown on the mutation assay system by a reversion at a specific site of the lacZ gene . Base changes were also localized at G:C pairs in the mutation of the supF gene, induced by riboflavin photosensitization, which specifically yields 7,8-dihydro-8-oxoguanine (8-oxoG) . G:C to T:A and to C:G transversions dominated in both mutants . These results suggest that endo III and endo VIII are involved in the repair of oxidative lesions of guanine.

Med Sci Monit, 2002 Apr, 8(4), BR123 - 35
Functional maps of the junctions between interglobular contacts and active sites in glycolytic enzymes -- a comparative analysis of the biochemical and structural data; Torshin IY; BACKGROUND: Oligomers and separate subunits of the glycolytic enzymes often have different catalytic properties . However, spectral data show an apparent lack of significant conformational changes during oligomerization . Since the conformation of an enzyme determines its catalytic properties, the structural mechanism(s) influencing the activity is of considerable interest . MATERIAL/METHODS: Analysis of the spatial structures of the junctions between interglobular contacts and binding sites may give a clue to the mechanism(s) of the activation . In this work, the problem was studied using available structural and biochemical data for the oligomeric enzymes of glycolysis . RESULTS: Computational analysis of the structures of the junctions has identified three structurally distinct types of junctions: 1 . interglobular binding site (2 of 8 enzymes); 2 . domain-domain stabilization (5 of 8); and 3 . 'sequence overlap' or a local conformational change (all enzymes) . Thus the catalytic activity may be influenced through the shifts of the modules of protein structure (types 1, 2) and/or due to a slight change in the local structure (type 3) . The more common junctions of types 2 and 3 are well conserved among eukaryotic enzymes, which suggests their biological importance . CONCLUSIONS: The results suggest that a profound and a complex change in conformation in subunits of an oligomeric enzyme may not be necessary for a significant change in the catalytic properties . The analysis maps the residues important for the junctions and thus for the link between the catalytic activity and the oligomeric state of the enzymes.

Plant Physiol, 2002 Apr, 128(4), 1200 - 11
Steroleosin, a sterol-binding dehydrogenase in seed oil bodies; Lin LJ et al.; Besides abundant oleosin, three minor proteins, Sop 1, 2, and 3, are present in sesame (Sesamum indicum) oil bodies . The gene encoding Sop1, named caleosin for its calcium-binding capacity, has recently been cloned . In this study, Sop2 gene was obtained by immunoscreening, and it was subsequently confirmed by amino acid partial sequencing and immunological recognition of its overexpressed protein in Escherichia coli . Immunological cross recognition implies that Sop2 exists in seed oil bodies of diverse species . Along with oleosin and caleosin genes, Sop2 gene was transcribed in maturing seeds where oil bodies are actively assembled . Sequence analysis reveals that Sop2, tentatively named steroleosin, possesses a hydrophobic anchoring segment preceding a soluble domain homologous to sterol-binding dehydrogenases/reductases involved in signal transduction in diverse organisms . Three-dimensional structure of the soluble domain was predicted via homology modeling . The structure forms a seven-stranded parallel beta-sheet with the active site, S-(12X)-Y-(3X)-K, between an NADPH and a sterol-binding subdomain . Sterol-coupling dehydrogenase activity was demonstrated in the overexpressed soluble domain of steroleosin as well as in purified oil bodies . Southern hybridization suggests that one steroleosin gene and certain homologous genes may be present in the sesame genome . Comparably, eight hypothetical steroleosin-like proteins are present in the Arabidopsis genome with a conserved NADPH-binding subdomain, but a divergent sterol-binding subdomain . It is indicated that steroleosin-like proteins may represent a class of dehydrogenases/reductases that are involved in plant signal transduction regulated by various sterols.

Mol Biol Cell, 2002 Apr, 13(4), 1238 - 51
Regulation of Fab1 phosphatidylinositol 3-phosphate 5-kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide phosphatase family member; Gary JD et al.; The Saccharomyces cerevisiae FAB1 gene encodes the sole phosphatidylinositol 3-phosphate {PtdIns(3)P} 5-kinase responsible for synthesis of the polyphosphoinositide PtdIns(3,5)P(2) . VAC7 encodes a 128-kDa transmembrane protein that localizes to vacuolar membranes . Both vac7 and fab1 null mutants have dramatically enlarged vacuoles and cannot grow at elevated temperatures . Additionally, vac7Delta mutants have nearly undetectable levels of PtdIns(3,5)P(2), suggesting that Vac7 functions to regulate Fab1 kinase activity . To test this hypothesis, we isolated a fab1 mutant allele that bypasses the requirement for Vac7 in PtdIns(3,5)P(2) production . Expression of this fab1 allele in vac7Delta mutant cells suppresses the temperature sensitivity, vacuolar morphology, and PtdIns(3,5)P(2) defects normally exhibited by vac7Delta mutants . We also identified a mutant allele of FIG4, whose gene product contains a Sac1 polyphosphoinositide phosphatase domain, which suppresses vac7Delta mutant phenotypes . Deletion of FIG4 in vac7Delta mutant cells suppresses the temperature sensitivity and vacuolar morphology defects, and dramatically restores PtdIns(3,5)P(2) levels . These results suggest that generation of PtdIns(3,5)P(2) by the Fab1 lipid kinase is regulated by Vac7, whereas turnover of PtdIns(3,5)P(2) is mediated in part by the Sac1 polyphosphoinositide phosphatase family member Fig4.

J Cell Sci, 2002 Apr 15, 115(Pt 8), 1611 - 22
The time course and chromosomal localization of recombination-related proteins at meiosis in the mouse are compatible with models that can resolve the early DNA-DNA interactions without reciprocal recombination; Moens PB et al.; During mouse meiosis, the early prophase RAD51/DMC1 recombination protein sites, which are associated with the chromosome cores and which serve as markers for ongoing DNA-DNA interactions, are in ten-fold excess of the eventual reciprocal recombinant events . Most, if not all, of these early interactions are eliminated as prophase progresses . The manner in which these sites are eliminated is the focus of this investigation . We report that these sites acquire replication protein A, RPA and the Escherichia coli MUTS homologue, MSH4p, and somewhat later the Bloom helicase, BLM, while simultaneously losing the RAD51/DMC1 component . Eventually the RPA component is also lost and BLM sites remain . At that time, the MUTL homologue, MLH1p, which is essential for reciprocal recombination in the mouse, appears in numbers and locations that correspond to the distribution of reciprocal recombination events . However, the MLH1 foci do not appear to coincide with the remaining BLM sites . The MLH1p is specifically localized to electron-microscope-defined recombination nodules . We consider the possibility that the homology-search RAD51/DMC1 complexes are involved in homologous chromosome synapsis but that most of these early DNA-DNA interactions are later resolved by the anti-recombination RPA/MSH4/BLM-topoisomerase complex, thereby preventing the formation of superfluous reciprocal recombinant events.

Life Sci Space Res, 1969, 7, 160 - 70
Characteristics of biological effects of cosmic radiation, model investigations; Parin VV et al.; In view of the probability of the influence of ionizing radiation on crewmen and the appropriate problem of creating adequate anti-radiation protection, it is necessary to investigate the peculiarities of biological effects of cosmic radiation . Under actual space flight conditions, cosmic radiation will affect the human organism in the complex along with other factors . Full imitation of cosmic radiation on the ground is impossible but it can exert influence on the human radiosensibilty . In this connection, the successful solution of the problem of obtaining appropriate information can be made by a reasonable combination of both ground radiobiological and medical-hygienic investigations and those carried out by using artificial earth satellites . The available experience in carrying out such research and its results are given in this report . Information on investigating the peculiarities of biological effects of protons in the wide spectrum of energy is also included . The report contains the data of observing immediate and later effects of radiation influence on higher animals and also on many biological objects arranged in various levels of evolution and biological organizations . The values of the RBE for protons are given.

J Cell Biochem, 2002, 85(2), 243 - 51
Protein nucleation and crystallization by homologous protein thin film template; Pechkova E et al.; A new method of protein nucleation and crystallization based on Langmuir-Blodgett technology is here utilized for the template stimulation of crystal growth of so far non-crystallized proteins . Microcrystals (60-120 microm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method . The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth .

J Pharm Sci, 2002 Apr, 91(4), 914 - 22
Mannitol-sucrose mixtures--versatile formulations for protein lyophilization; Johnson RE et al.; Mixtures of sucrose (a lyoprotectant) and mannitol (a bulking agent) have been investigated as excipients for the lyophilization of proteins . Four proteins under development have been successfully lyophilized in a formulation of 4% mannitol and 1% sucrose using a lyophilization cycle that produces a cake of crystalline mannitol and amorphous sucrose . The crystalline mannitol allows primary drying to be performed with a product temperature of -10 degrees C even though the sucrose is amorphous and, by itself, would have required primary drying below -35 degrees C to avoid cake collapse . Formation of an unstable mannitol hydrate is avoided by conducting secondary drying at 40 degrees C or higher .

Biotechnol Bioeng, 2002 May 20, 78(4), 376 - 84
Isolation of plasmid DNA from cell lysates by aqueous two-phase systems; Ribeiro SC et al.; This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS) . The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000 . The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated . The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG . Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400 . The bottom phase was preferred when higher PEG molecular weights were used . Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation . The recovery yields were found to be proportional to the plasmid concentration in the lysate . The best yields (>67%) were obtained with PEG 1000 . These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed . Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase . The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load . In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold .

World J Surg, 2002 Jul, 26(7), 783 - 9 Epub 2002 Apr 15.
Current progress in suicide gene therapy for cancer; Yazawa K et al.; Standard chemotherapeutic agents and ionizing radiation destroy dividing cells . Because tumor cells divide more rapidly than normal cells, there is a therapeutic index in which damage to the cancer cells is maximized while keeping the toxicity to the normal host cells acceptable . Suicide gene therapy strives to deliver genes to the cancer cells, which convert nontoxic prodrugs into active chemotherapeutic agents . With this strategy, the systemically administered prodrug is converted to the active chemotherapeutic agent only in cancer cells, thereby allowing a maximal therapeutic effect while limiting systemic toxicity . A literature search was conducted using the MEDLINE database from 1990 to 2001 to identify articles related to suicide gene therapy for cancer . A number of suicide gene systems have been identified, including the herpes simplex virus thymidine kinase gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E . coli Deo gene . Various vectors, including liposomes, retroviruses, and adenoviruses, have been used to transfer these suicide genes to tumor cells . These strategies have been effective in cell culture experiments, laboratory animals, and some early clinical trials . Advances in tissue- and cell-specific delivery of suicide genes using specific promoters will improve the clinical utility of suicide gene therapy.

Physiol Genomics, 2002, 9(1), 15 - 26
Physiological genomics of Escherichia coli protein families; Liang P et al.; The well-researched Escherichia coli genome offers the opportunity to explore the value of using protein families within a single organism to enrich functional annotation procedures and to study mechanisms of protein evolution . Having identified multimodular proteins resulting from gene fusion, and treated each module as a separate protein, nonoverlapping sequence-similar families in E . coli could be assembled . Of 3,902 proteins of length 100 residues or more, 2,415 clustered into 609 protein families . The relatedness of function among members of each family was dissected in detail . Data on paralogous protein families provides valuable information in attributing putative function to unknown genes, supplementing existing function annotation . Enzymes, transporters, and regulators represent the three major types of proteins in E . coli . They are shown to have distinctive patterns in gene duplication and divergence and gene fusion, suggesting that details of protein evolution have been different for genes in these categories . Data for the complete list of paralogous protein families and updated functional annotation for E . coli K-12 are accessible in GenProtEC .

J Neurochem, 2002 Mar, 80(5), 928 - 37
Hypoxia induces mitochondrial DNA damage and stimulates expression of a DNA repair enzyme, the Escherichia coli MutY DNA glycosylase homolog (MYH), in vivo, in the rat brain; Lee HM et al.; Hypoxia-associated, acutely reduced blood oxygenation can compromise energy metabolism, alter oxidant/antioxidant balance and damage cellular components, including DNA . We show in vivo, in the rat brain that respiratory hypoxia leads to formation of the oxidative DNA lesion, 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker for oxidative DNA damage and to increased expression of a DNA repair enzyme involved in protection of the genome from the mutagenic consequences of oh8dG . The enzyme is a homolog of the Escherichia coli MutY DNA glycosylase (MYH), which excises adenine residues misincorporated opposite the oxidized base, oh8dG . We have cloned a full-length rat MYH (rMYH) cDNA, which encodes 516 amino acids, and by in situ hybridization analysis obtained expression patterns of rMYH mRNA in hippocampal, cortical and cerebellar regions . Ensuing hypoxia, mitochondrial DNA damage was induced and rMYH expression strongly elevated . This is the first evidence for a regulated expression of a DNA repair enzyme in the context of respiratory hypoxia . Our findings support the premise that oxidative DNA damage is repaired in neurons and the possibility that the hypoxia-induced expression of a DNA repair enzyme in the brain represents an adaptive mechanism for protection of neuronal DNA from injurious consequences of disrupted energy metabolism and oxidant/antioxidant homeostasis.

J Biol Chem, 2002 Jun 14, 277(24), 21624 - 9 Epub 2002 Apr 10.
Purification and characterization of the Escherichia coli exoribonuclease RNase R . Comparison with RNase II; Cheng ZF et al.; Escherichia coli RNase R, a 3' --> 5' exoribonuclease homologous to RNase II, was overexpressed and purified to near homogeneity in its native untagged form by a rapid procedure . The purified enzyme was free of nucleic acid . It migrated upon gel filtration chromatography as a monomer with an apparent molecular mass of approximately 95 kDa, in close agreement with its expected size based on the sequence of the rnr gene . RNase R was most active at pH 7.5-9.5 in the presence of 0.1-0.5 mm Mg(2+) and 50-500 mm KCl . The enzyme shares many catalytic properties with RNase II . Both enzymes are nonspecific processive ribonucleases that release 5'-nucleotide monophosphates and leave a short undigested oligonucleotide core . However, whereas RNase R shortens RNA processively to di- and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches approximately 10 nucleotides, and it leaves an undigested core of 3-5 nucleotides . Both enzymes work on substrates with a 3'-phosphate group . RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ . RNase II is more active on poly(A), whereas RNase R is much more active on rRNAs . Neither RNase R nor RNase II can degrade a complete RNA-RNA or DNA-RNA hybrid or one with a 4-nucleotide 3'-RNA overhang . RNase R differs from RNase II in that it cannot digest DNA oligomers and is not inhibited by such molecules, suggesting that it does not bind DNA . Although the in vivo function of RNase R is not known, its ability to digest certain natural RNAs may explain why it is maintained in E . coli together with RNase II.

J Bacteriol, 2002 May, 184(9), 2552 - 6
ZipA is required for recruitment of FtsK, FtsQ, FtsL, and FtsN to the septal ring in Escherichia coli; Hale CA et al.; The septal ring in Escherichia coli consists of at least nine essential gene products whose order of assembly resembles a mostly linear dependency pathway: FtsA and ZipA directly bind FtsZ polymers at the prospective division site, followed by the sequential addition of FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN . Recruitment of FtsK and all downstream components requires the prior localization of FtsA . Here we show that recruitment of FtsK, FtsQ, FtsL, and FtsN equally requires ZipA . The results imply that association of both FtsA and ZipA with FtsZ polymers is needed for further maturation of the nascent organelle.

J Bacteriol, 2002 May, 184(9), 2447 - 54
Transcriptional interference by a complex formed at the centromere-like partition site of plasmid P1; Sawitzke JA et al.; The partition site, parS, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied . The parS site was inserted into the Escherichia coli chromosome between the promoter and the structural gene for beta-galactosidase, lacZ . There was little interference with lacZ expression when ParA and ParB were supplied in trans . However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of lacZ was shut down . ParAM314I, ParB, and parS appear to form a nucleoprotein complex that blocks transcription . Mutations in parA and parB that relieved the parAM314I-dependent block were found . In addition, new mutations which impose the block were selected . Five of the latter mapped to parA and one to parB; all had a propagation-defective phenotype (Par(PD)) similar to that of parAM314I . Thus, whereas a null par mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid . We propose that ParA protein normally interacts transiently with the ParB-parS complex for partition to proceed but that the mutations block ParA dissociation . This "permanent" ParA-ParB-parS complex acts as a transcription block . Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other . When the transcription block is imposed, regional silencing of nearby genes occurs . However, the requirement for ParA and a mutant parA or parB allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described.

J Bacteriol, 2002 May, 184(9), 2360 - 9
Critical regions of secM that control its translation and secretion and promote secretion-specific secA regulation; Sarker S et al.; SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins . secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions . A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM . The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation . The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon . We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation . In addition, mutational analysis of the 3' end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level . Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.

FEBS Lett, 1970 Jul 29, 9(2), 100 - 102
The inhibition of ribonucleic acid synthesis by the thiol-oxidizing agent, diamide, in Escherichia coli; Zehavi-Willner T et al.; The thiol-oxidizing agent, diamide, has been used to convert glutathione to glutathione disulfide within the cells of a stringent strain of Escherichia coli (CP 78), leading to a cessation of 14C-leucine incorporation (protein synthesis) and 3H-uracil incorporation (RNA synthesis) . Parallel experiments with an isogenic relaxed strain (CP 79) gave similar results, providing evidence that glutathione is closely linked to RNA synthesis indepently of the link previously shown to protein synthesis.

FEBS Lett, 1970 May 1, 7(4), 314 - 316

Lehmann J, Pfeiffer E.
An incubation mixture of TDP-D-glucose and a purified enzyme extract from E . coli B containing TDP-D-glucose 4,6-hydro-lyase was treated with NaB(3)H(4) . Hydrolysis of the separated sugar nucleotide fraction yielded D-galactose-4-(3)H but no 3H-labelled D-glucose . This indicates the presence of TDP-4-keto-D-glucose as an intermediate in the enzymatic conversion of TDP-D-glucose to TDP-L-rhamnose . The stereospecifity of the NaB(3)H(4) reduction can only be explained if TDP-4-keto-D-glucose exists as an enzyme bound complex.

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