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| Genetika, 1999 Apr, 35(4), 444 - 9 {Expression and functions of adaptive response genes in Escherichia coli treated with mono- and bifunctional alkylating agents . Interference with SOS response}; Vasil'eva SV et al.; The expression of genes belonging to the Ada regulon of Escherichia coli under the action of mono- and bifunctional alkylating agents--high-efficiency antitumor HMM, ACNU, and BCNU preparations--was studied . The functional specificity of the alkA, alkB, and aidB1 genes concerning both the structure and volume of DNA alkylation and the specificity of cell preadaptation was revealed . Additional experimental evidence for the role of the aidB1 gene as a unique "hazard gene", a component of the E . coli ada operon, was obtained . A phenomenon of positive interference between alternative SOS and Ada responses was observed for the first time upon gene expression. Genetika, 1999 Apr, 35(4), 438 - 43 {Constitutive inhibition of DNA degradation due to the enzyme RecBCD in the radiation-resistant Escherichia coli K-12 mutant Gam(r)444}; Verbenko VN et al.; Exonucleolytic degradation of {3}H-labeled DNA was examined in partially purified fractions of lysates obtained from nonirradiated RecBCD enzyme-containing cells of Escherichia coli and in the radiation-resistant mutant Gamr444 . The degradative activity was shown to be lowered in these cells to the same extent as in the recBC mutant . The efficiency of plating of the mutant phage T4 2-, DNA of which can be degraded by exonuclease V, was 400-fold higher on the strain Gamr444 than on the wild-type strain AB1157 . This value was shown to be only twice as low as that on the recB mutant or on the strain AB1157 carrying plasmid pGam26 with a radiation-resistance allele gam26 cloned from mutant Gamr444 . The data obtained confirmed the hypothesis that the Gamr444 mutant contains a constitutive inhibitor of exonucleolytic activity of the RecBCD enzyme in nonirradiated cells . This inhibitor was shown to be encoded by the gam26 allele that had previously been mapped at 56.8 min of the E . coli chromosome . A possible mechanism of the involvement of this inhibitor in enhanced radiation resistance of the mutant Gamr444 is considered. J Biomed Sci, 1999 Jul-Aug, 6(4), 277 - 84 Probing surface structure of sarcin domain on ribosomes of Escherichia coli by complementary oligo DNAs and ribosome-inactivating protein; Cheung J et al.; The regional structure of the sarcin domain of 23S rRNA of Escherichia coli ribosomes was determined by a combinatory approach of oligo DNA probes and the action of alpha-sarcin . The sarcin domain is protected by a reactive complementary oligo DNA probe against the hydrolytic action of alpha-sarcin . This protective effect is dependent upon the length and the complementary sequence of oligo DNA probes that react to ribosomes . Under UV irradiation and using of the primer extension, nucleotides that contacted by reactive oligo DNA probes were determined . Nucleotides at the 3' side of the domain (positions from G2659 to C2676) were targeted by oligo DNA probes that have their sequences to complement the domain, indicating that the 3' side region was exposed on the surface of ribosomes, whereas nucleotides at the 5' side of stem and extented to two bases at the loop (positions from C2646 to A2654) were not accessible to any oligo DNA probes, implying that the region could be buried in ribosomes . This study also provided evidence that the conformation of the sarcin domain is subjected to alteration if the exposed 3' side of domain is targeted by the reactive DNA probe . The importance of the topological arrangement of the sarcin domain that engages in the translocation event during translation is discussed. Fetal Diagn Ther, 1999 Jul-Aug, 14(4), 240 - 3 Effects of fetal endotoxin administration on plasma prostaglandin f(2alpha) and cortisol levels in late-gestation fetal goats; Kijima K et al.; OBJECTIVE: Fetal plasma prostaglandin F(2alpha) (PGF(2alpha)) and cortisol responses to fetal endotoxin administration were evaluated in late-gestation goats (n = 9) . METHODS: After endotoxin (Escherichia coli, O111:B4 lipopolysaccharide) administration, fetal plasma PGF(2alpha) and cortisol levels, fetal blood gases and pH were measured periodically . RESULTS: After endotoxin administration, fetal plasma cortisol levels increased to 9.8 +/- 1.4 and 9.4 +/- 1 . 2 ng/ml after 1 and 3 h, respectively (p < 0.05) and PGF(2alpha) levels did not change throughout the study . CONCLUSIONS: These results suggest that absent PGF(2alpha) and attenuated cortisol responses to fetal endotoxin administration, relative to the adult, may be a self-protective mechanism which diminishes the likelihood of premature delivery. J Bacteriol, 1999 Aug, 181(15), 4686 - 9 Amino acid residues involved in the functional integrity of Escherichia coli methionine aminopeptidase; Chiu CH et al.; Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated . Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity . However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP . Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function . Both residues are found to be conserved in type I but not type II MAPs. J Bacteriol, 1999 Aug, 181(15), 4680 - 5 Complementation of conjugation functions of Streptomyces lividans plasmid pIJ101 by the related Streptomyces plasmid pSB24.2; Pettis GS et al.; A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24 . 2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1 . The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci . Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems. J Bacteriol, 1999 Aug, 181(15), 4676 - 9 Succinate dehydrogenase (Sdh) from Bradyrhizobium japonicum is closely related to mitochondrial Sdh; Westenberg DJ et al.; The sdhCDAB operon, encoding succinate dehydrogenase, was cloned from the soybean symbiont Bradyrhizobium japonicum . Sdh from B . japonicum is phylogenetically related to Sdh from mitochondria . This is the first example of a mitochondrion-like Sdh functionally expressed in Escherichia coli. J Bacteriol, 1999 Aug, 181(15), 4639 - 43 OxyR and SoxRS regulation of fur; Zheng M et al.; The cytotoxic effects of reactive oxygen species are largely mediated by iron . Hydrogen peroxide reacts with iron to form the extremely reactive and damaging hydroxyl radical via the Fenton reaction . Superoxide anion accelerates this reaction because the dismutation of superoxide leads to increased levels of hydrogen peroxide and because superoxide elevates the intracellular concentration of iron by attacking iron-sulfur proteins . We found that regulators of the Escherichia coli responses to oxidative stress, OxyR and SoxRS, activate the expression of Fur, the global repressor of ferric ion uptake . A transcript encoding Fur was induced by hydrogen peroxide in a wild-type strain but not in a DeltaoxyR strain, and DNase I footprinting assays showed that OxyR binds to the fur promoter . In cells treated with the superoxide-generating compound paraquat, we observed the induction of a longer transcript encompassing both fur and its immediate upstream gene fldA, which encodes a flavodoxin . This polycistronic mRNA is induced by paraquat in a wild-type strain but not in a DeltasoxRS strain, and SoxS was shown to bind to the fldA promoter . These results demonstrate that iron metabolism is coordinately regulated with the oxidative stress defenses. J Bacteriol, 1999 Aug, 181(15), 4561 - 7 The structure of multiple polypeptide domains determines the signal recognition particle targeting requirement of Escherichia coli inner membrane proteins; Newitt JA et al.; The signal recognition particle (SRP) targeting pathway is required for the efficient insertion of many polytopic inner membrane proteins (IMPs) into the Escherichia coli inner membrane, but in the absence of SRP protein export proceeds normally . To define the properties of IMPs that impose SRP dependence, we analyzed the targeting requirements of bitopic IMPs that are structurally intermediate between exported proteins and polytopic IMPs . We found that disruption of the SRP pathway inhibited the insertion of only a subset of bitopic IMPs . Studies on a model bitopic AcrB-alkaline phosphatase fusion protein (AcrB 265-AP) showed that the SRP requirement for efficient insertion correlated with the presence of a large periplasmic domain (P1) . As previously reported, perturbation of the SRP pathway also affected the insertion of a polytopic AcrB-AP fusion . Even exhaustive SRP depletion, however, failed to block the insertion of any AcrB derivative by more than 50% . Taken together, these data suggest that many proteins that are normally targeted by SRP can utilize alternative targeting pathways and that the structure of both hydrophilic and membrane-spanning domains determines the degree to which the biogenesis of a protein is SRP dependent. J Bacteriol, 1999 Aug, 181(15), 4549 - 53 Illegitimate recombination induced by overproduction of DnaB helicase in Escherichia coli; Yamashita T et al.; Illegitimate recombination that usually takes place at a low frequency is greatly enhanced by treatment with DNA-damaging agents . It is thought that DNA double-strand breaks induced by this DNA damage are important for initiation of illegitimate recombination . Here we show that illegitimate recombination is enhanced by overexpression of the DnaB protein in Escherichia coli . The recombination enhanced by DnaB overexpression occurred between short regions of homology . We propose a model for the initiation of illegitimate recombination in which DnaB overexpression may excessively unwind DNA at replication forks and induce double-strand breaks, resulting in illegitimate recombination . The defect in RecQ has a synergistic effect on the increased illegitimate recombination in cells containing the overproduced DnaB protein, implying that DnaB works in the same pathway as RecQ does but that they work at different steps. J Bacteriol, 1999 Aug, 181(15), 4499 - 504 Escherichia coli open reading frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate isomerase; Hahn FM et al.; Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) . In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway . The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions . In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway . An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E . coli chromosome at 65.3 min . ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically . The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway . E . coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3'-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication . FH12/pFMH33 was able to grow at the restrictive temperature of 44 degrees C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene . Although the V(max) of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in K(m)s . The E . coli protein requires Mg(2+) or Mn(2+) for activity . The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases. Protein Expr Purif, 1999 Jul, 16(2), 355 - 8 A modified procedure for fast purification of T7 RNA polymerase; Li Y et al.; The in vitro T7 transcription system allows one to synthesize biochemical amounts of RNA molecules functionally equivalent or similar to those transcripts normally existing at extremely low levels in vivo . In this study we described a modified method for efficient large-scale preparation of pure T7 RNA polymerase free of RNase activity from the recombinant Escherichia coli strain BL21/pAR1219 (4) . The procedure, which used preparative column chromatography on DEAE-Sepharose CL-6B and Blue 3GA, was shown to be simple, rapid, and cost effective in comparison with other methods reported previously . Protein Expr Purif, 1999 Jul, 16(2), 347 - 54 Production of chemokines CTAPIII and NAP/2 by digestion of recombinant ubiquitin-CTAPIII with yeast ubiquitin C-terminal hydrolase and human immunodeficiency virus protease; Mildner AM et al.; Recombinant yeast ubiquitin C-terminal hydrolase (YUH1), which has an N-terminal (His)(6) tag, and an autolysis-resistant mutant of the human immunodeficiency virus-1 protease (HIV-1 Pr) have been used as specific proteases to yield peptides from a ubiquitin conjugate . In the present example, connective tissue-activating peptide (CTAPIII) and neutrophil-activating peptide 2 (NAP/2) were generated by digestion of a ubiquitin-CTAPIII conjugate with YUH1 and HIV Pr, respectively, as indicated below: {see text} YUH1 cleaved at the peptide bond formed by the C-terminal Gly(76) of ubiquitin (Ub) and the N-terminal Asn(1) of the 85-residue peptide CTAPIII . The HIV-1 Pr cleaved between Tyr(15) and Ala(16), the N-terminal Ala of the 70-residue peptide NAP/2 . Both enzymes produced authentic peptides from the Ub fusion protein, with a nearly 100% yield . The liberated CTAPIII and NAP/2 were separated from (His)(6)-Ub, the trace amounts of unreacted (His)(6)-Ub-CTAPIII, HIV-1 Pr, and the (His)(6)-YUH1 by passage over a nickel-chelate column; the final yield was about 10 mg of peptide/liter of cell culture . (His)(6)-YUH1, the HIV Pr mutant, and the (His)(6)-Ub-CTAPIII substrate were all expressed individually in Escherichia coli . (His)(6)-YUH1 and (His)(6)-Ub-CTAPIII were highly expressed in a soluble form, but about 75% of the total (His)(6)-YUH1 was also found in inclusion bodies . Both proteins from the soluble fractions were easily purified in a single step by immobilized metal ion affinity chromatography with a yield of about 27 mg of (His)(6)-Ub-CTAPIII and 13.6 mg of (His)(6)-YUH1 protein/liter of cell culture . Chemotactic factor activity, as assessed by the neutrophil shape change assay, was observed for NAP/2, but not for CTAPIII . This strategy, which employs YUH1 and the HIV-1 Pr as tools for the highly selective cleavage of the chimeric substrate, should be applicable to the large-scale production of a variety of peptides . Protein Expr Purif, 1999 Jul, 16(2), 251 - 60 Heterologously expressed polypeptide from the yeast meiotic gene HOP1 binds preferentially to yeast DNA; Alche JD et al.; HOP1 protein, present in sporulating cells of Saccharomyces cerevisiae and believed to be a component of the synaptonemal complex, has been expressed in Escherichia coli fused to a biotinylated tag protein . Once solubilized from bacterial inclusion bodies, the HOP1 fusion protein was purified by using a combination of avidin-affinity chromatography and gel filtration FPLC and refolded . Sequence comparisons indicate that the HOP1 gene product contains a zinc finger motif, which may confer DNA binding properties, and the recombinant polypeptide was used to assess the putative DNA binding properties of the product of native HOP1 protein using a gel-shift assay . Protein and protein-DNA complexes were detected by exploiting the affinity of streptavidin-alkaline phosphatase for the biotinylated tag protein after Western blotting . The HOP1 fusion protein bound unambiguously to digested genomic yeast DNA . This binding possessed some degree of specificity, was maintained under a wide range of salt concentrations, and was unaffected by the presence of high concentrations of competitor DNA (synthetic poly{dI-dC}.poly{dI-dC}) . In contrast, no shift was detected when the fusion protein was incubated with digested genomic DNA from Arabidopsis, or with lambda/HindIII DNA . Incubation with digested genomic DNA from Lilium produced a small change in the mobility of the protein . The biotinylated tag protein failed to show any DNA binding activity . Scatchard analysis indicated an apparent yeast genomic DNA:HOP1 fusion protein dissociation constant of K(d) = 5 x 10(-7) M . Protein Expr Purif, 1999 Jul, 16(2), 243 - 50 Expression and characterization of human salivary statherin from Escherichia coli using two different fusion constructs; Gilbert M et al.; Saliva is a supersaturated solution with respect to hydroxyapatite, the main inorganic component of tooth enamel . Several acidic phosphoproteins are present in saliva which allow the supersaturated state to be maintained without random crystallization occurring . Statherin is the only salivary protein currently known to inhibit both the primary and secondary precipitation of hydroxyapatite in the supersaturated environment of saliva . To identify the residues of statherin that are necessary to control biomineralization, a recombinant form of human statherin was produced from Escherichia coli using a yeast intein fusion construct . The primary structure of the recombinant statherin was characterized by SDS-PAGE, N-terminus sequencing, MALDI mass spectrometry, and amino acid analysis and found to have the expected values relative to human-derived statherin . The secondary structure of the recombinant statherin was investigated by circular dichroism spectroscopy, which revealed the predominant presence of random coil in phosphate-buffered saline solution, with a higher propensity toward alpha helicity in 100% TFE . This increase in helicity in 100% TFE was also found in statherin that was synthesized by solid-phase synthesis . These results demonstrate that human statherin can be produced in a recombinant form which behaves comparably to the natural form . Protein Expr Purif, 1999 Jul, 16(2), 224 - 30 Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli; Morrow JA et al.; Apolipoprotein (apo) E plays an important role in lipid metabolism, and the major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different metabolic effects . Apolipoprotein E4 is associated with a higher risk of both heart disease and Alzheimer's disease (AD) . Patients homozygous for apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2 may be protective against AD . Structure/function studies have proved to be a useful tool in understanding how the different apoE isoforms result in different pathological consequences . As these studies continue, it is essential to have a reliable method to produce large quantities of apoE and mutants of apoE . We describe here a method of apoE production in Escherichia coli strain BL21(DE3) . The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter and a fusion partner (thioredoxin) . The T7 promoter results in high expression of an easily purified His-tagged fusion protein . A thrombin recognition site was positioned in the expression vector so that only two novel amino acids (Gly-Ser) are added to the amino terminus of apoE following the removal of thioredoxin . Approximately 20 mg of apoE is obtained from a 1-liter culture . The major isoforms of apoE produced with this system were extensively characterized for their ability to bind the low-density lipoprotein (LDL) receptor, for their characteristic lipid association preferences, and for their stability as measured by guanidine denaturation . The recombinant proteins behaved identically to plasma-derived apoE isoforms . J Colloid Interface Sci, 1999 Jul 15, 215(2), 244 - 257 Microstructure of a Biocatalytic Latex Coating Containing Viable Escherichia coli Cells; Thiagarajan VS et al.; Cryogenic scanning electron microscopy (cryo-SEM) was employed to visualize the microstructure of latex coatings in which viable Escherichia coli cells were entrapped for use as biocatalysts . Cryo-SEM examination of surfaces and fracture cross sections of dry and hydrated coatings cast with or without glycerol revealed two different porosities in the films: a macroporosity in which the bacterial cells reside and a microporosity made up of the interstitial voids between partially coalesced latex polymer particles . Polymer particle consolidation and coalescence in the cell-laden coatings were at an earlier stage than in cell-free latex coatings detailed in a companion paper . Coatings cast with glycerol showed a lesser degree of latex particle consolidation and coalescence than those cast without glycerol . However, the effect of glycerol was not as pronounced as in cell-free coatings . We conclude that part of the added glycerol is sequestered inside the bacterial cells and the portion remaining outside the cells retards the latex film formation process and enhances microporosity . Two commercial acrylic acid/vinyl acetate copolymer latexes were examined . Coatings made with the polydisperse latex showed less microporosity and a greater degree of particle welding than those made with the nearly monodisperse latex . Mol Pharmacol, 1999 Aug, 56(2), 272 - 8 Molecular characterization of binding of substrates and inhibitors to DT-diaphorase: combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling; Chen S et al.; The molecular basis of the interaction of DT-diaphorase with a cytotoxic nitrobenzamide CB1954 {5-(aziridin-1-yl)-2, 4-dinitrobenzamide} and five inhibitors was investigated with wild-type DT-diaphorase (human and rat) and five mutants {three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)} . hY155F and hH161Q were generated to evaluate a hypothesis that Tyr155 and His161 participate in the obligatory two-electron transfer reaction of the enzyme . The catalytic properties of hY155F and hH161Q were compared with a naturally occurring mutant, hP187S . Pro187 to Ser mutation disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide prosthetic group . With NADH as the electron donor and menadione as the electron acceptor, the k(cat) values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 +/- 1, 23 +/- 0, 5 +/- 0 and 8 +/- 2 x 10(3) min(-1), respectively . Because hY155F still has significant catalytic activity, the hydroxyl group on Tyr155 may not be as important as proposed . Interestingly, hY155F was found to be 3 . 3 times more active than the human wild-type DT-diaphorase in the reduction of CB1954 . Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128 . Dicoumarol, Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the enzyme with respect to nicotinamide coenzymes . The binding orientations of dicoumarol, flavones, and phenindone in the active site of DT-diaphorase were predicted by results from our inhibitor-binding studies and computer modeling based on published X-ray structures . Our studies generated results that explain why dicoumarol is a potent inhibitor and binds differently from flavones and phenindone in the active site of DT-diaphorase. J Biol Chem, 1999 Jul 30, 274(31), 22002 - 7 Marked instability of the sigma(32) heat shock transcription factor at high temperature . Implications for heat shock regulation; Kanemori M et al.; The heat shock response in Escherichia coli depends on a transient increase in the intracellular level of sigma(32) that results from both increased synthesis and transient stabilization of normally unstable sigma(32) . Although the membrane-bound ATP-dependent protease FtsH (HflB) plays an important role in degradation of sigma(32), our previous results suggested that several cytosolic ATP-dependent proteases including HslVU (ClpQY) are also involved in sigma(32) degradation (Kanemori, M., Nishihara, K., Yanagi, H., and Yura, T . (1997) J . Bacteriol . 179, 7219-7225) . We now report on the ATP-dependent proteolysis of sigma(32) by purified HslVU protease and its unusual dependence on high temperature: sigma(32) was rapidly degraded at 44 degrees C, but with much slower rates ( approximately 15-fold) at 35 degrees C . FtsH-dependent degradation of sigma(32) also gave similar results . In agreement with these results in vitro, the turnover of sigma(32) in normally growing cells at high temperature (42 degrees C) was much faster than at low temperature (30 degrees C) . Taken together with other evidence, these results suggest that the sigma(32) level during normal growth is primarily determined by the stability (susceptibility to proteases) and synthesis rate of sigma(32) set by ambient temperature, whereas fine adjustment such as transient stabilization of sigma(32) observed upon heat shock is brought about through monitoring changes in the cellular state of protein folding. J Biol Chem, 1999 Jul 30, 274(31), 21797 - 803 Characterization of PECI, a novel monofunctional Delta(3), Delta(2)-enoyl-CoA isomerase of mammalian peroxisomes; Geisbrecht BV et al.; We report here the identification and characterization of human and mouse PECI, a novel gene that encodes a monofunctional peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase . Human and mouse PECI were identified on the basis of their sequence similarity to Eci1p, a recently characterized peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae . Cloning and sequencing of the human PECI cDNA revealed the presence of a 1077-base pair open reading frame predicted to encode a 359-amino acid protein with a mass of 39.6 kDa . The corresponding mouse cDNA contains a 1074-base pair open reading frame that encodes a 358-amino acid-long protein with a deduced mass of 39.4 kDa . Northern blot analysis demonstrated human PECI mRNA is expressed in all tissues . A bacterially expressed form of human PECI catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 27 units/mg of protein . The human and mouse PECI proteins contain type-1 peroxisomal targeting signals, and human PECI was localized to peroxisomes by both subcellular fractionation and immunofluorescence microscopy techniques . The potential roles for this monofunctional Delta(3),Delta(2)-enoyl-CoA isomerase in peroxisomal metabolism are discussed. J Biol Chem, 1999 Jul 30, 274(31), 21776 - 82 The aspartyl replacement of the active site histidine in histidine-containing protein, HPr, of the Escherichia coli Phosphoenolpyruvate:Sugar phosphotransferase system can accept and donate a phosphoryl group . Spontaneous dephosphorylation of acyl-phosphate autocatalyzes an internal cyclization; Napper S et al.; The active site residue, His(15), in histidine-containing protein, HPr, can be replaced by aspartate and still act as a phosphoacceptor and phosphodonor with enzyme I and enzyme IIA(glucose), respectively . Other substitutions, including cysteine, glutamate, serine, threonine, and tyrosine, failed to show any activity . Enzyme I K(m) for His(15) --> Asp HPr is increased 10-fold and V(max) is decreased 1000-fold compared with wild type HPr . The phosphorylation of Asp(15) led to a spontaneous internal rearrangement involving the loss of the phosphoryl group and a water molecule, which was confirmed by mass spectrometry . The protein species formed had a higher pI than His(15) --> Asp HPr, which could arise from the formation of a succinimide or an isoimide . Hydrolysis of the isolated high pI form gave only aspartic acid at residue 15, and no isoaspartic acid was detected . This indicates that an isoimide rather than a succinimide is formed . In the absence of phosphorylation, no formation of the high pI form could be found, indicating that phosphorylation catalyzed the formation of the cyclization . The possible involvement of Asn(12) in an internal cyclization with Asp(15) was eliminated by the Asn(12) --> Ala mutation in His(15) --> AspHPr . Asn(12) substitutions of alanine, aspartate, serine, and threonine in wild type HPr indicated a general requirement for residues capable of forming a hydrogen bond with the Nepsilon(2) atom of His(15), but elimination of the hydrogen bond has only a 4-fold decrease in k(cat)/K(m). J Biol Chem, 1999 Jul 30, 274(31), 21769 - 75 The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain; Thelen JJ et al.; The dihydrolipoamide S-acetyltransferase (E2) subunit of the maize mitochondrial pyruvate dehydrogenase complex (PDC) was postulated to contain a single lipoyl domain based upon molecular mass and N-terminal protein sequence (Thelen, J . J., Miernyk, J . A., and Randall, D . D . (1998) Plant Physiol . 116, 1443-1450) . This sequence was used to identify a cDNA from a maize expressed sequence tag data base . The deduced amino acid sequence of the full-length cDNA was greater than 30% identical to other E2s and contained a single lipoyl domain . Mature maize E2 was expressed in Escherichia coli and purified to a specific activity of 191 units mg(-1) . The purified recombinant protein had a native mass of approximately 2.7 MDa and assembled into a 29-nm pentagonal dodecahedron as visualized by electron microscopy . Immunoanalysis of mitochondrial proteins from various plants, using a monoclonal antibody against the maize E2, revealed 50-54-kDa cross-reacting polypeptides in all samples . A larger protein (76 kDa) was also recognized in an enriched pea mitochondrial PDC preparation, indicating two distinct E2s . The presence of a single lipoyl-domain E2 in Arabidopsis thaliana was confirmed by identifying a gene encoding a hypothetical protein with 62% amino acid identity to the maize homologue . These data suggest that all plant mitochondrial PDCs contain an E2 with a single lipoyl domain . Additionally, A . thaliana and other dicots possess a second E2, which contains two lipoyl domains and is only 33% identical at the amino acid level to the smaller isoform . The reason two distinct E2s exist in dicotyledon plants is uncertain, although the variability between these isoforms, particularly within the subunit-binding domain, suggests different roles in assembly and/or function of the plant mitochondrial PDC. J Biol Chem, 1999 Jul 30, 274(31), 21637 - 44 DNA binding and aggregation properties of the vaccinia virus I3L gene product; Tseng M et al.; The vaccinia virus I3L gene encodes a single-stranded DNA-binding protein which may play a role in viral replication and genetic recombination . We have purified native and recombinant forms of gpI3L and characterized both the DNA-binding reaction and the structural properties of DNA-protein complexes . The purified proteins displayed anomalous electrophoretic properties in the presence of sodium dodecyl sulfate, behaving as if they were 4-kDa larger than the true mass . Agarose gel shift analysis was used to monitor the formation of complexes composed of single-stranded DNA plus gpI3L protein . These experiments detected two different DNA binding modes whose formation was dependent upon the protein density . The transition between the two binding modes occurred at a nucleotide to protein ratio of about 31 nucleotides per gpI3L monomer . S1 nuclease protection assay revealed that at saturating protein densities, each gpI3L monomer occludes 9.5 +/- 2.5 nucleotides . In the presence of magnesium, gpI3L promoted the formation of large DNA aggregates from which double-stranded DNA was excluded . Electron microscopy showed that, in the absence of magnesium and at low protein densities, gpI3L forms beaded structures on DNA . At high protein density the complexes display a smoother and less compacted morphology . In the presence of magnesium the complexes contained long fibrous and tangled arrays . These results suggest that gpI3L can form octameric complexes on DNA much like those formed by Escherichia coli single-stranded DNA protein . Moreover, the capacity to aggregate DNA may provide an environment in which hybrid DNA formation could occur during DNA replication. J Biol Chem, 1999 Jul 30, 274(31), 21581 - 8 Analysis of engineered multifunctional peptide synthetases . Enzymatic characterization of surfactin synthetase domains in hybrid bimodular systems; Symmank H et al.; The combinatorial reorganization of distinct modules of multimodular peptide synthetases is of increasing interest for the generation of new peptides with optimized bioactive properties . Each module is at least composed of enzymatic domains responsible for the adenylation, thioester formation, and condensation of an amino acid residue of the final peptide product . We analyzed various possible fusion sites for the recombination of peptide synthetases and evaluated the impact of different recombination strategies on the amino acid adenylation and acyl-thioester formation activities of peptide synthetase modules . Hybrid bimodular peptide synthetases were generated by recombination of the corresponding reading frames encoding for L-glutamic acid- and L-leucine-specific modules of surfactin synthetase SrfA-A at presumed inner- and intradomainic regions . We demonstrate that fusions at a previously postulated hinge region, dividing the amino acid adenylating domains of peptide synthetase modules into two subdomains, and at the highly conserved 4'-phosphopantetheine binding motif in acyl-thioester forming domains resulted in enzymatically active hybrid domains . By contrast, most manipulations in condensation domains like deletions, the complete exchange or the construction of chimeric domains considerably reduced or completely abolished the amino acid adenylation and thioester formation activity of the hybrid module. J Neurosurg Spine, 1999 Jul, 91(1), 124 - 7 Anterior sacral meningocele associated with a rectal fistula . Case report and review of the literature; Fitzpatrick MO et al.; The authors report a case of anterior sacral meningocele associated with a rectal fistula in a patient who had presented 20 years earlier with bacterial meningitis . To their knowledge, this is the first case in which a rectal fistula developed due to an anterior sacral meningocele . The clinical presentation, diagnosis, and treatment of this uncommon lesion is discussed. J Steroid Biochem Mol Biol, 1999 Apr-Jun, 69(1-6), 123 - 30 In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis; Culty M et al.; In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis . Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis . Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport . Molecular modeling of PBR suggested that it might function as a channel for cholesterol . This hypothesis was tested in a bacterial system devoid of PBR and cholesterol . Cholesterol uptake and transport by these cells was induced upon PBR expression . Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence . This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria. Biofizika, 1999 Mar-Apr, 44(2), 216 - 23 {Periodicity in contacts of RNA-polymerase with promotors}; Kutuzova GI et al.; Periodicities in the position of E.coli RNA polymerase promoter contacts on several promoters (lacUV5, T7 A3, tetR, lambda cin, lambda c17, RNA1, and trp S.t.) were found by means of Fourier analysis . The comparison of the Fourier spectrum of core RNA polymerase contacts on the lacUV5 promoter and that of holoenzyme revealed a more prominent 7-periodicity in the Fourier spectrum of holoenzyme contacts . 6-, 7-, and 8-periodicities were found in the primary structure of the majority of E.coli promoters . It is shown that RNA polymerase recognizes specific periodic patterns in the promoter structure. Acta Vet Scand, 1999, 40(1), 35 - 46 Acute phase response in dairy cows with experimentally induced Escherichia coli mastitis; Hirvonen J et al.; Six Finnish Ayrshire cows were challenged intramammarily with 1500 CFU of Escherichia coli (E . coli) into single udder quarters, and the challenge was repeated into contralateral quarters 3 weeks later . All cows received flunixine meglumine once, and 3 of them were also treated with enrofloxacin . At the 2nd challenge, treatments were changed vice versa . The development of mastitis was followed by monitoring of systemic and local clinical signs, and with serial milk and serum samples . Intramammary challenge with E . coli produced clinical mastitis in all cows, the severity of the disease varying greatly between the animals . No significant changes between the 2 treatment regimens or sequent challenges were found for any of the clinical parameters . The response of each cow followed the same pattern after both challenges; three of the cows became mildly and the other 3 either moderately or severely affected . Two severely affected cows had to be euthanized because of severe mastitis . Serum haptoglobin and amyloid-A concentrations peaked 2-3 days after bacterial challenge . Serum haptoglobin did not correlate with the severity of the disease . Serum amyloid-A rose gradually in the severely affected cows, and significant differences were found between severely versus moderately or mildly affected cows at day 4 . Serum tumor necrosis factor alpha concentrations increased only in the severely affected cows . Serum cortisol response was prolonged in the severely diseased animals, and was significantly lower after the second challenge . Serum nitrite/nitrate concentration increased in the severely affected cows . This indicated excess nitric oxide production during acute E . coli mastitis . Strongly decreased milk production, and high bacterial growth in the infected quarters were best predictors for the outcome from acute E . coli mastitis. FEMS Microbiol Lett, 1999 Jul 1, 176(1), 219 - 27 Effects of pre-protein overexpression on SecB synthesis in Escherichia coli; Muller JP; Protein translocation through the cytoplasmic membrane of Escherichia coli involves cytosolic chaperones . The export-dedicated chaperone SecB mediates targeting of a subset of pre-proteins . In this report, synthesis of SecB in response to plasmid-mediated overexpression of pre-proteins was studied . Overexpression of SecB-dependent pre-proteins stimulated synthesis of SecB under conditions where the cellular export capacity was saturated or uncomplexed SecB was trapped . On the contrary, overexpression of SecB-independent pre-beta-lactamase reduced the promoter activity of secB . The results suggest that uncomplexed SecB can be sequestered by synthesis of SecB-dependent pre-proteins . Furthermore, these data demonstrate the distinct action of the SecB- and signal recognition particle-dependent protein targeting pathways. FEMS Microbiol Lett, 1999 Jul 1, 176(1), 39 - 43 Transcription of cspA, the gene for the major cold-shock protein of Escherichia coli, is negatively regulated at 37 degrees C by the 5'-untranslated region of its mRNA; Fang L et al.; The gene for CspA, the major cold-shock protein in Escherichia coli, is tightly regulated at both optimal and low temperatures . While CspA is drastically induced after temperature downshift, it is hardly detectable at 37 degrees C . Here we demonstrate that the deletion of parts of the 5'-untranslated region (5'-UTR) of the cspA mRNA results in constitutive expression of CspA at 37 degrees C . By analyzing the amounts and the stabilities of the mRNAs produced from the deletion constructs, we rule out the possibility that the CspA production is due to the stabilization of the mutant mRNAs . We propose that significant premature termination or pausing occurs during the transcription of the unusually long 5'-UTR of the cspA mRNA at 37 degrees C, which represents a new mechanism that contributes to the tight repression of CspA production at higher temperature. Wound Repair Regen, 1999 May-Jun, 7(3), 172 - 8 In vivo characterization of keratinocyte growth factor-2 as a potential wound healing agent; Soler PM et al.; Human keratinocyte growth factor-2 exerts a proliferative effect on epithelial cells and mediates keratinocyte migration . It has also been shown to increase both deposition of granulation tissue and collagen and maturation of collagen . Because these properties should affect the healing trajectory of wounds, this study set out to investigate the effects of keratinocyte growth factor-2 on the healing of three different types of wounds . Human meshed skin grafts explanted to athymic "nude" rats, surgical incisions in Sprague-Dawley rats, and acute excisional rat wounds inoculated with Escherichia coli were used . Two concentrations of recombinant human keratinocyte growth factor-2 were compared to a vehicle control and keratinocyte growth factor-1 . Keratinocyte growth factor-2 significantly accelerated the rate of epithelialization in the meshed skin graft model and effected a modestly more rapid gain in breaking strength of surgical incisions than keratinocyte growth factor-1 or the vehicle control treatment . Neither keratinocyte growth factors accelerated wound closure by contraction of the excisional wounds . Based on these data, keratinocyte growth factor-2 may be useful in accelerating healing in wounds healing mainly by the process of epithelialization such as venous stasis ulcers, partial thickness burn wounds, and skin graft donor sites . It might also accelerate the gain in incisional wound strength in acute surgical or traumatic wounds. Plant J, 1999 Jul, 19(1), 65 - 73 Gibberellin 2-oxidation and the SLN gene of Pisum sativum; Lester DR et al.; Two cDNAs encoding gibberellin 2-oxidases were isolated from maturing pea seeds . The first, PsGA2ox1, was isolated by activity screening of a Lambda-ZAP cDNA library excised into phagemid form and expressed in Escherichia coli . The second, PsGA2ox2, was obtained initially as a PCR product using degenerate primers designed according to conserved regions of plant 2-oxoglutarate-dependent dioxygenases . E . coli heterologous expression products of PsGA2ox1 and PsGA2ox2 converted GA1 to GA8, as shown by HPLC-radiocounting, and gas chromatography-MS . PsGA2ox1 converted GA20 to GA29, but GA20 was a poor substrate for the PsGA2ox2 expression product . Furthermore, PsGA2ox1 converted GA29 to GA29-catabolite at a low level of efficiency while PsGA2ox2 did not catalyse this step . A cDNA of PsGA2ox1 isolated from plants of genotype sln contained a single base deletion which was predicted to produce a truncated protein and gibberellin 2-oxidase activity could not be demonstrated from this cDNA . A 10 bp size difference between the introns of the SLN and sln PsGA2ox1 genes was used to show co-segregation between the SLN and sln phenotypes and the size of the PCR products . PsGA2ox1 transcripts were more abundant in cotyledons than in shoots, while the reverse was the case for PsGA2ox2 . The expression patterns of the genes, together with the effects of the sln mutation, indicate that PsGA2ox1 plays a major role in GA20 deactivation in both shoots and maturing seeds, while the PsGA2ox2 gene might be important for GA1 deactivation in the shoot. Plant J, 1999 Jun, 18(5), 465 - 75 Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum; Unterlinner B et al.; The narcotic analgesic morphine is the major alkaloid of the opium poppy Papaver somniferum . Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent . Along the morphine biosynthetic pathway in opium poppy, codeinone reductase catalyzes the NADPH-dependent reduction of codeinone to codeine . In this study, we have isolated and characterized four cDNAs encoding codeinone reductase isoforms and have functionally expressed them in Escherichia coli . Heterologously expressed codeinone reductase-calmodulin-binding peptide fusion protein was purified from E . coli using calmodulin affinity column chromatography in a yield of 10 mg enzyme l-1 . These four isoforms demonstrated very similar physical properties and substrate specificity . As least six alleles appear to be present in the poppy genome . A comparison of the translations of the nucleotide sequences indicate that the codeinone reductase isoforms are 53% identical to 6'-deoxychalcone synthase from soybean suggesting an evolutionary although not a functional link between enzymes of phenylpropanoid and alkaloid biosynthesis . By sequence comparison, both codeinone reductase and 6'-deoxy- chalcone synthase belong to the aldo/keto reductase family, a group of structurally and functionally related NADPH-dependent oxidoreductases, and thereby possibly arise from primary metabolism. Mol Microbiol, 1999 Aug, 33(3), 583 - 9 PDZ domains determine the native oligomeric structure of the DegP (HtrA) protease; Sassoon N et al.; DegP (HtrA) is a periplasmic heat shock serine protease of Escherichia coli that degrades misfolded proteins at high temperatures . Biochemical and biophysical experiments have indicated that the purified DegP exists as a hexamer . To examine whether the PDZ domains of DegP were required for oligomerization, we constructed a DegP variant lacking both PDZ domains . This truncated variant, DegPDelta, exhibited no proteolytic activity but exerted a dominant-negative effect on growth at high temperatures by interfering with the functional assembly of oligomeric DegP . Thus, the PDZ domains contain information necessary for proper assembly of the functional hexameric structure of DegP. Mol Microbiol, 1999 Aug, 33(3), 569 - 82 Molecular function of the dual-start motif in the lambda S holin; Graschopf A et al.; The lambda S gene represents the prototype of holin genes with a dual-start motif, which leads to the synthesis of two polypeptides, S105 and S107 . They differ at their N-terminus by only two amino acids, Met-1 and Lys-2, at the beginning of the longer product . Despite the minor difference, the two proteins have opposing functions in lysis, with protein S107 being an inhibitor and protein S105 being an effector of 'hole formation' in the inner membrane . Here, we have studied the molecular mechanism underlying the 'lysis clock' contributed by the dual-start motif . We have used protein fusions in which the secretory signal sequence of the M13 procoat protein VIII has been abutted to the N-terminal Met residues of S105 and S107 respectively . S-dependent 'hole formation' required removal of the signal sequence in both fusion proteins, as both the VIII-S105 and the VIII-S107 fusion proteins were non-functional when leader peptidase cleavage was inhibited . These results strongly supported the hypothesis that functional assembly of S proteins requires translocation of their N-terminus to the periplasm . Using signal sequence cleavage as a measure of translocation, we observed that the translocation kinetics of the N-terminus of the S107 moiety was reduced about threefold when compared with the N-terminus of the S105 moiety . Moreover, depolarization of the membrane resulted in an immediate cleavage of the signal sequence and 'hole formation' exerted by the S107 moiety of the VIII-S107 fusion protein . A model is presented in which S107 with a reversed topology of its N-terminus interacts with S105 and poisons 'hole formation' . Upon depolarization of the membrane, translocation of the N-terminus of S107 to the periplasm results in the functional assembly of S proteins, i.e . 'hole formation'. Mol Microbiol, 1999 Aug, 33(3), 524 - 36 Helicobacter pylori cadA encodes an essential Cd(II)-Zn(II)-Co(II) resistance factor influencing urease activity; Herrmann L et al.; Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H . pylori strains, designated 69A . All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II) . In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits . Gene complementation experiments with plasmid (pY178)-derived H . pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored . Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild-type strain 69A . Heterologous expression of H . pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II) . Expression of cadA in E . coli SE5000 harbouring H . pylori nixA, which encodes a divalent cation importer along with the H . pylori urease gene cluster, led to about a threefold increase in urease activity compared with E . coli control cells lacking the H . pylori cadA gene . These results suggest that H . pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II) . They also point to a possible role of H . pylori CadA in high-level activity of H . pylori urease, an enzyme sensitive to a variety of metal ions. Infect Immun, 1999 Aug, 67(8), 4019 - 26 Characterization of an enterotoxigenic Escherichia coli strain from Africa expressing a putative colonization factor; Khalil SB et al.; An enterotoxigenic Escherichia coli (ETEC) strain of serotype O114:H- that expressed both heat-labile and heat-stable enterotoxins and tested negative for colonization factors (CF) was isolated from a child with diarrhea in Egypt . This strain, WS0115A, induced hemagglutination of bovine erythrocytes and adhered to the enterocyte-like cell line Caco-2, suggesting that it may elaborate novel fimbriae . Surface-expressed antigen purified by differential ammonium sulfate precipitation and column chromatography yielded a single protein band with M(r) 14,800 when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (16% polyacrylamide) . A monoclonal antibody against this putative fimbrial antigen was generated and reacted with strain WS0115A and also with CS1-, CS17-, and CS19-positive strains in a dot blot assay . Reactivity was temperature dependent, with cells displaying reactivity when grown at 37 degrees C but not when grown at 22 degrees C . Immunoblot analysis of a fimbrial preparation from strain WS0115A showed that the monoclonal antibody reacted with a single protein band . Electron microscopy and immunoelectron microscopy revealed fimbria-like structures on the surface of strain WS0115A . These structures were rigid and measured 6.8 to 7.4 nm in diameter . Electrospray mass-spectrometric analysis showed that the mass of the purified fimbria was 14,965 Da . The N-terminal sequence of the fimbria established that it was a member of the CFA/I family, with sequence identity to the amino terminus of CS19, a new CF recently identified in India . Cumulatively, our results suggest that this fimbria is CS19 . Screening of a collection of ETEC strains isolated from children with diarrhea in Egypt found that 4.2% of strains originally reported as CF negative were positive for this CF, suggesting that it is biologically relevant in the pathogenesis of ETEC. Biotechnol Bioeng, 1999 Sep 20, 64(6), 644 - 9 Tolerance of Escherichia coli beta-galactosidase C-terminus to different-sized fusions; Corchero JL et al.; The tolerance of the beta-galactosidase C-terminus to foreign protein fusions has been explored by using different-sized derivatives of the chimeric protein LACVP1 . While the molecular mass of the partner domain shows a minor influence on protein toxicity for the producing E . coli cells, it dramatically affects the proteolytic susceptibility of the whole fusion . Surprisingly, the observed structural modulation of proteolysis is not an all-or-nothing process, but it exhibits a continuous effect concomitantly with the length of the fusion . The conformational effects caused by increasingly sized partners seem to progressively expose cryptic protease target sites, initiating a proteolytic cascade that dramatically reduces the yield of the recombinant protein . Infect Immun, 1999 Aug, 67(8), 4276 - 9 Intranasal immunization of mice with influenza vaccine in combination with the adjuvant LT-R72 induces potent mucosal and serum immunity which is stronger than that with traditional intramuscular immunization; Barackman JD et al.; Immunization of mice by the intranasal route with influenza virus hemagglutinin in combination with the mutant Escherichia coli heat-labile enterotoxin R72 (LT-R72) induced significantly enhanced serum and mucosal antibodies, surpassing, in most cases, responses achieved by traditional intramuscular immunization using inactivated split influenza vaccine . Furthermore, intranasal immunization with LT-R72 induced a potent serum immunoglobulin G2a response, indicating that this adjuvant has Th1 character. Infect Immun, 1999 Aug, 67(8), 4231 - 6 Interleukin-1 and tumor necrosis factor activities partially account for calvarial bone resorption induced by local injection of lipopolysaccharide; Chiang CY et al.; The present study was undertaken to test the hypothesis that tumor necrosis factor (TNF) and/or interleukin-1 (IL-1) activity mediates lipopolysaccharide (LPS)-induced bone resorption in vivo . To test this hypothesis, Escherichia coli LPS or Porphyromonas gingivalis LPS was injected into the subcutaneous tissues overlying mouse calvariae . Histological sections, prepared from the center of the lesion, were stained for tartrate-resistant acid phosphatase, and histomorphometric analysis was performed to quantify the osteoclast number and the area of bone resorption . In time course experiments using normal mice, a peak of bone resorption occurred 5 days after endotoxin stimulation . In dose-response experiments, IL-1 receptor type 1 deletion (IL-1R(-/-)), TNF double-receptor p55/p75 deletion (TNF p55(-/-)/p75(-/-)), combined TNF p55 and IL-1 receptor type 1 deletion (TNF p55(-/-)/IL-1R(-/-)), and IL-1beta-converting enzyme-deficient (ICE(-/-)) mice and the respective wild-type mice were injected with 500, 100, or 20 micrograms of P . gingivalis LPS and sacrificed 5 days after LPS injection . At the highest dose (500 micrograms), significant decreases in osteoclast number occurred in mutant mice compared to wild-type mice: (i) a 64% reduction for the TNF p55(-/-)/IL-1R(-/-) mice, (ii) a 57% reduction for the IL-1R(-/-) mice, (iii) a 41% reduction for the TNF p55(-/-)/p75(-/-) mice, and (iv) a 38% reduction for the ICE(-/-) mice . At the two lower doses, bone resorption was apparent but no significant differences between mutant and wild-type animals were observed . The present data indicate that at higher doses, LPS-induced bone resorption is substantially mediated by IL-1 and TNF receptor signaling . Furthermore, IL-1 receptor signaling appears to be slightly more important than TNF receptor signaling . At lower LPS doses, other pathways leading to osteoclast activity that are independent of TNF and IL-1 are involved. Infect Immun, 1999 Aug, 67(8), 4084 - 91 Identification of a glycoprotein produced by enterotoxigenic Escherichia coli; Lindenthal C et al.; Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro . Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain . These loci direct nonadherent and noninvasive laboratory strains of E . coli to adhere to and invade cultured human intestinal epithelial cells . The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes . TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity . Outer membranes of recombinant E . coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose . The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin . Only TibA could be detected as a glycoprotein . Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407 . Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed . TibA shows homology with AIDA-I from diffuse-adhering E . coli and with pertactin precursor from Bordetella pertussis . Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms . Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter . Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase . The product of this tib locus open reading frame is proposed to be responsible for TibA modification . These results suggest that TibA glycoprotein acts as an adhesin that may participate in the disease process. Infect Immun, 1999 Aug, 67(8), 3998 - 4007 Cloning, expression, and immunological evaluation of two putative secreted serine protease antigens of Mycobacterium tuberculosis; Skeiky YA et al.; Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity against Mycobacterium infection . To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M . tuberculosis genomic expression library in Escherichia coli . In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family . Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP . The predicted molecular masses of the mature, fully processed forms of both antigens are approximately 32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins . Thus, these proteins have been designated MTB32A and MTB32B . Interestingly, and despite 66% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens . The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds . MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon . MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M . tuberculosis complex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested . In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% identity . MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development . Finally, the possible role of MTB32 serine proteases as a virulence factor(s) during Mycobacterium spp . infection is discussed. Infect Immun, 1999 Aug, 67(8), 3989 - 97 Decay-accelerating factor and cytoskeleton redistribution pattern in HeLa cells infected with recombinant Escherichia coli strains expressing Dr family of adhesins; Goluszko P et al.; Escherichia coli strains expressing Dr fimbriae are able to enter epithelial cells by interacting with a complement-regulatory protein, decay-accelerating factor . This model of bacterial internalization, with a well-characterized bacterial ligand and host receptor, provides a unique opportunity to investigate the early stages of invasion . We used immunofluorescence staining techniques to examine the distribution of receptor and cytoskeletal proteins in HeLa cells infected with E . coli recombinant strains that expressed Dr family of adhesins: Dr, Dr-II, F1845, AFA-I, and AFA-III . A major rearrangement of decay-accelerating factor was found at the adherence sites of recombinant strains expressing Dr, Dr-II, and F1845 adhesins . The changes in the distribution of receptor were significantly smaller on HeLa cells infected with E . coli bearing AFA-I or AFA-III afimbrial adhesins . Receptor aggregation was associated with the redistribution of cytoskeleton-associated proteins such as actin, alpha-actinin, ezrin, and occasionally tropomyosin . Purified Dr fimbriae coated on polystyrene beads were capable of triggering clustering of receptor and accumulating actin at the adhesion sites of beads to HeLa cells . Using scanning and transmission electron microscopic techniques, we have shown that beads coated with Dr fimbriae, as opposed to beads coated with bovine serum albumin, were enwrapped by cellular microvilli and ultimately internalized into HeLa cells . This indicates that interaction of Dr fimbriae with decay-accelerating factor is associated with redistribution of receptor and is sufficient to promote bacterial internalization. Infect Immun, 1999 Aug, 67(8), 3757 - 62 Loss of resistance to ingestion and phagocytic killing by O(-) and K(-) mutants of a uropathogenic Escherichia coli O75:K5 strain; Burns SM et al.; To determine the importance of the O75 O antigen and the K5 capsular antigen in resistance to phagocytosis and phagocytic killing, we used previously described O75(-) and K5(-) mutants from an O75(+) K5(+) wild-type uropathogenic Escherichia coli strain in phagocytosis assays with polymorphonuclear leukocytes (PMNs) and monocytes . At a 10-to-1 ratio of bacteria to phagocytes and in the presence of 10% serum, the parental strain GR-12 was resistant to both PMNs and monocytes over a 2-h incubation period . The O75(-) and K5(-) mutants were similar in sensitivity to killing by both PMNs and monocytes, decreasing in viability by 80% in the first hour . Yet, a significant difference in killing between the O75(-) and K5(-) mutants was observed in the first 15 min of incubation . The K5(-) mutant decreased in numbers by almost 60%, while the O75(-) mutant increased in numbers similarly to GR-12 in the first 15 min . The difference in killing was found not to be due to the rate of opsonization . To further determine the mechanism of resistance, a fluorescence assay was used to differentiate attached and internalized bacteria . The K5 capsule hindered the association of both the wild-type strain and the O75(-) mutant in the initial incubation time with PMNs . In conclusion, both the K5 capsule and O75 O antigen play crucial roles in resistance to phagocytosis over time. Infect Immun, 1999 Aug, 67(8), 3727 - 32 Identification of functional domains of Bordetella dermonecrotizing toxin; Kashimoto T et al.; Bordetella dermonecrotizing toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rho . To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutants . Our analyses of the nucleotide and amino acid sequences revealed that the start codon of the DNT gene is a GTG triplet located 39 bp upstream of the reported putative initiation ATG codon; consequently, DNT contains an additional 13 amino acids at its N-terminal end . All of the N-terminally truncated mutants were found to modify Rho . The shortest fragment of DNT possessing the Rho modification activity consists of amino acids from Ile1176 to the C-terminal end . This fragment overlaps the region homologous to Escherichia coli cytotoxic necrotizing factors (CNFs), which show activity similar to that of DNT . The introduction of a mutation at Cys1305 located in the highly conserved region between CNFs and DNT eliminated the activity, indicating that this domain is the catalytic center of DNT . The N-terminal fragment (1 to 531) of DNT failed to modify Rho but reduced the DNT-induced polynucleation in MC3T3-E1 cells when simultaneously added with the holotoxin, suggesting competitive inhibition in the receptor-binding or internalizing step . Our finding that DNT consists of an N-terminal receptor-binding and/or internalizing domain and a C-terminal catalytically active domain may facilitate analysis of the overall action of the toxin on the mammalian target cells. Intensive Care Med, 1999 Jun, 25(6), 612 - 5 GM-CSF increases in vitro the respiratory burst of human neutrophils after liver transplantation; Jaeger K et al.; OBJECTIVE: Superoxide production by polymorphonuclear neutrophils (PMNs) under cyclosporin A (CsA) therapy following kidney transplantation is impaired . We investigated if the respiratory burst of PMNs is similarly depressed in patients undergoing CsA treatment following orthotopic liver transplantation (OLTx) . Additionally, the in vitro influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the superoxide anion production was examined during the respiratory burst . PATIENTS: 10 patients after OLTx and 10 healthy blood donors (control group) . MEASUREMENTS AND RESULTS: PMNs were stimulated with bacteria (Escherichia coli) or a combination of tumour necrosis factor alpha (TNFalpha) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) . The respiratory burst was measured by oxidation of non-fluorescent dihydrorhodamine to the fluorescent rhodamine by means of flow cytometry . No differences in respiratory bursts from OLTx patients compared to those from healthy blood donors could be seen . Under TNFalpha/FMLP stimulation, the respiratory burst was significantly increased after in vitro incubation with GM-CSF (500 U ml(-1)) in patients following OLTx (from 58.2 to 74.5 %) as well as in the control group (from 47.4 to 61.9%) . CONCLUSIONS: Our results demonstrate that superoxide production is not impaired under CsA treatment following OLTx . The respiratory burst of these patients' PMNs can even be augmented by GM-CSF in vitro. Ann N Y Acad Sci, 1999 May 18, 870, 275 - 89 Mechanisms of genome-wide hypermutation in stationary phase; Lombardo MJ et al.; Stationary-phase mutation (a subset of which was previously called adaptive mutation) occurs in apparently nondividing, stationary-phase cells exposed to a nonlethal genetic selection . In one experimental system, stationary-phase reversion of an Escherichia coli F'-borne lac frameshift mutation occurs by a novel molecular mechanism that requires homologous recombination functions of the RecBCD system . Chromosomal mutations at multiple loci are detected more frequently in Lac+ stationary-phase revertants than in cells that were also exposed to selection but did not become Lac+ . Thus, mutating cells represent a subpopulation that experiences hypermutation throughout the genome . This paper summarizes current knowledge regarding stationary-phase mutation in the lac system . Hypotheses for the mechanism of chromosomal hypermutation are discussed, and data are presented that exclude one hypothetical mechanism in which chromosomal mutations result from Hfr formation. Ann N Y Acad Sci, 1999 May 18, 870, 173 - 89 DNA-directed mutations . Leading and lagging strand specificity; Sinden RR et al.; The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation . The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations . These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations . To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation . This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli . Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand . Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand. Ann N Y Acad Sci, 1999 May 18, 870, 133 - 45 Mechanisms of mutation in nondividing cells . Insights from the study of adaptive mutation in Escherichia coli; Foster PL et al.; When populations of cells are subjected to nonlethal selection, mutations arise in the absence of cell division, a phenomenon that has been called "adaptive mutation." In a strain of Escherichia coli that cannot metabolize lactose (Lac-) but that reverts to lactose utilization (Lac+) when lactose is its sole energy and carbon source, the mutational process consists of two components . (1) A highly efficient, recombination-dependent mechanism giving rise to mutations on the F' episome that carries the Lac- allele; and (2) a less efficient, unknown mechanism giving rise to mutations elsewhere in the genome . Both selected and nonselected mutations arise in the Lac- population, but nonselected mutations are enriched in Lac+ mutants, suggesting that some Lac+ cells have passed though a transient period of increased mutation . These results have several evolutionary implications . (1) DNA synthesis initiated by recombination could be an important source of spontaneous mutation, particularly in cells that are not undergoing genomic replication . (2) The highly active mutational mechanism on the episome could be important in the horizontal transfer of variant alleles among species that carry and exchange conjugal plasmids . (3) A sub-population of cells in a state of transient mutation could be a source of multiple variant alleles and could provide a mechanism for rapid adaptive evolution under adverse conditions. Curr Eye Res, 1999 Jul, 19(1), 76 - 85 Murine endotoxin-induced uveitis, but not immune complex-induced uveitis, is dependent on the IL-8 receptor homolog; Brito BE et al.; PURPOSE: To determine the roles of the murine interleukin-8 receptor homolog (mIL-8Rh, neutrophil chemokine CXC receptor 2) and macrophage inflammatory protein-1alpha (MIP-1alpha, a CC chemokine) in two eye inflammation models: endotoxin-induced uveitis (EIU) and immune complex-induced uveitis (reverse passive Arthus reaction (RPAR) uveitis) . METHODS: For the EIU model, 250 ng E.coli endotoxin was injected into the vitreous of mIL-8Rh-/- mice or heterozygous littermate mIL-8Rh+/- controls and into MIP-1alpha-/- mice or congenic MIP-1alpha+/+ controls . Eyes were harvested after 24 h for histologic characterization of infiltrating cells and IL-6 bioassays . For the RPAR model, mouse antiserum against human serum albumin (HSA) was injected into the vitreous of mIL-8Rh-/-, mIL-8Rh+/-, MIP-1alpha-/-, and MIP-1alpha+/+ mice . Twenty-four hours later, animals were challenged with intravenous HSA . Eyes were harvested after 4 h for analysis . RESULTS: RPAR resulted in the deposition of immune complexes at the ciliary area and iris with the subsequent development of uveitis . Genetic deficiency of mIL-8Rh reduced the median number of infiltrating cells in EIU by 63% (p < 0.01) but had no effect on RPAR-induced inflammation . In the EIU model, macrophages comprised a much higher percentage (45%) of infiltrating cells in mice lacking mIL-8Rh than in controls (17%) . Loss of the MIP-1alpha gene had no apparent effect on RPAR uveitis and a 39% reduction of infiltrating cells in EIU that was not statistically significant . IL-6 activity in aqueous humor was much less in mice with RPAR uveitis than in those with EIU . Neither gene deletion had a significant impact on IL-6 levels in either disease model . CONCLUSIONS: Chemokines acting via mIL-8Rh have a significant role in the induction of neutrophil infiltration during EIU but not during RPAR uveitis . MIP-1alpha is not critical for either EIU or RPAR-induced uveitis . The differential dependence on IL-8-like chemokines is in accord with the two forms ofuveitis having different etiologies and, therefore, potentially different optimal therapies. Exp Neurol, 1999 Aug, 158(2), 459 - 68 IL-6 deficiency causes enhanced pathology in Twitcher (globoid cell leukodystrophy) mice; Pedchenko TV et al.; The expression of IL-6 is greatly enhanced in the twitcher mouse (S . M . LeVine and D . C . Brown, 1997, J . Neuroimmunol . 73, 47-56), which is an authentic animal model of globoid cell leukodystrophy (Krabbe's disease) . In order to investigate the role of IL-6 in this disease, twitcher/IL-6-deficient mice were generated and the pathology was compared between them and regular twitcher mice . Twitcher/IL-6-deficient mice had a more severe disease than regular twitcher mice: they had an earlier onset day of twitching, a greater number of PAS-positive cells, a greater susceptibility to LPS, an exaggerated gliotic response around some vessels, an elevated level of TNF-alpha, and a compromised blood-brain barrier, which was evaluated by three independent measures . This latter finding indicates that IL-6 plays a role in maintaining the integrity of the BBB, and it raises the possibility that IL-6 functions in a similar manner in other diseases of the CNS . LPS was found to greatly shorten the life of twitcher and twitcher/IL-6-deficient mice compared to genotyped-matched saline-injected mice . This result indicates that a proinflammatory condition can exacerbate an underlying CNS pathology, which could help explain why some leukodystrophy patients display their initial symptoms following a fever or blow to the head . Arch Biochem Biophys, 1999 Aug 1, 368(1), 193 - 7 His(15) of subunit a of the Escherichia coli ATP synthase is important for the structure or assembly of the membrane sector F(o); Patterson AR et al.; Approximately 37 amino acids at the amino-terminus of subunit a of the Escherichia coli ATP synthase are found localized to the periplasm . Results indicate that a single amino acid substitution, H15D, disrupts assembly of subunit a and causes a loss of ATP synthase function . In this study, a conserved region of nine amino acids, 11-19, was initially mutagenized randomly, generating no mutants that could grow on succinate-minimal medium . Subsequent mutagenesis, confined to residues His(14), His(15), and Asn(17), indicated that constructs containing H15D were the most deleterious . Four single mutants were constructed and analyzed: H15A, H14D, H15A, and H15D . Only H15D was significantly impaired, with respect to ATP-driven proton translocation, passive proton permeability through F(o), and sensitivity of membrane-bound ATPase to DCCD . Immunoblot analysis indicated very low levels of subunit a from H15D . Cysteine mutations were constructed at positions 14, 15, 17, and 18 . Residues 14, 15, and 17 were shown to be accessible in the periplasmic space, while residue 18 was not, indicating that this region was stably folded . While both His(14) and His(15) are conserved among a group of bacteria, results presented here indicate that they are not equivalent, and that a specific role for His(15) in the assembly or structure of the ATP synthase is supported . Arch Biochem Biophys, 1999 Aug 1, 368(1), 161 - 71 Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling; Chang CF et al.; PTR1, the gene promoting MTX resistance following gene amplification or DNA transfection in Leishmania tarentolae and selected mutants, has been cloned and heavily overexpressed (>100 mg/liter) in Escherichia coli strain BL21 (DE3) . Protein has been purified, essentially to homogeneity, in two steps, via ammonium sulfate precipitation and chromatography on DEAE-Trisacryl . The active proteins are tetramers and display optimal pteridine reductase activity at pH 6.0 using biopterin as substrate and NADPH as the reduced dinucleotide cofactor . 2,4-Diaminopteridine substrate analogues are strong competitive inhibitors (K(i) approximately 38 --> 3 nM) against the pterin substrate and both NADP(+) and folate are inhibitors although somewhat weaker . Dihydropteridines are poor substrates compared to the fully oxidized pteridine . Kinetic analysis affords the usual Michaelis constants and in addition shows that inhibition by NADP(+) allows the formation of ternary nonproductive complexes with folate . The kinetic results are consistent with a sequential ordered bi-bi kinetic mechanism in which first NADPH and then pteridine bind to the free enzyme . Sequence comparisons suggest that PTR1 belongs to the short-chain dehydrogenase/reductase (SDR) family containing an amino-terminal glycine-rich dinucleotide binding site plus a catalytic Y(Xaa)(3)K motif . In accord with this observation, the mutants K16A, Y37D, and R39A and the double mutants K17A:R39A and Y37D:R39A all show a two- to threefold lower binding affinity for NADPH and exhibit low or zero activity . Two Y(Xaa)(3)K regions are present in wild-type PTR1 at 152 and 194 . Only Y194F gives protein with zero activity . This observation coupled with affinity labeling of PTR1 by oNADP(+) (2', 3'-dialdehyde derivative of NADP(+)) followed by NaBH(4) reduction, V8 protease digestion, and mass spectral analysis suggests that the motif participating in catalysis is that at 194 . The mutation K198Q eliminates inactivation by oNADP(+) supporting the hypothesis that K198 is associated with nucleotide orientation, as has been demonstrated for similar lysine residues in other members of the SDR family . Arch Biochem Biophys, 1999 Aug 1, 368(1), 139 - 46 In vitro processing of the human alkyl-dihydroxyacetonephosphate synthase precursor; Biermann J et al.; Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme involved in the biosynthesis of ether phospholipids, is synthesized with a cleavable N-terminal presequence containing the peroxisomal targeting signal type 2 . The human alkyl-dihydroxyacetonephosphate synthase precursor produced in vitro or expressed in Escherichia coli could be processed to a lower molecular weight protein by incubation at 37 degrees C with a guinea pig liver fraction, enriched in mitochondria, lysosomes, and peroxisomes . This lower molecular weight protein was identified as the mature human alkyl-dihydroxyacetonephosphate synthase by radiosequencing, indicating that the processing protease is present in this organellar fraction . Characterization of the processing protease indicated that it is a cysteine protease with a pH optimum of 6.5 . Furthermore, it was demonstrated that exogenously added pre-alkyl-dihydroxyacetonephosphate synthase was imported and processed in purified peroxisomes in vitro . Processing of alkyl-dihydroxyacetonephosphate synthase did not increase the activity of the enzyme . This indicates that the presence of the presequence does not affect the activity of the enzyme . Arch Biochem Biophys, 1999 Aug 1, 368(1), 105 - 11 Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin; Khaitlina S et al.; In order to investigate the structural basis for functional differences among actin isoforms, we have compared the polymerization properties and conformations of scallop adductor muscle beta-like actin and rabbit skeletal muscle alpha-actin . Polymerization of scallop Ca(2+)-actin was slower than that of skeletal muscle Ca(2+)-actin . Cleavage of the actin polypeptide chain between Gly-42 and Val-43 with Escherichia coli protease ECP 32 impaired the polymerization of scallop Mg(2+)-actin to a greater extent than skeletal muscle Mg(2+)-actin . When monomeric scallop and skeletal muscle Ca(2+)-actins were subjected to limited proteolysis with trypsin, subtilisin, or ECP 32, no differences in the conformation of actin subdomain 2 were detected . At the same time, local differences in the conformations of scallop and skeletal muscle actin subdomains 1 were revealed as intrinsic fluorescence differences . Replacement of tightly bound Ca(2+) with Mg(2+) resulted in more extensive proteolysis of segment 61-69 of scallop actin than in the case of skeletal muscle actin . Furthermore, segment 61-69 was more accessible to proteolysis with subtilisin in polymerized scallop Ca(2+)-actin than in polymerized skeletal muscle Ca(2+)-actin, indicating that, in the polymeric form, the nucleotide-containing cleft is in a more open conformation in beta-like scallop actin than in skeletal muscle alpha-actin . We suggest that this difference between scallop and skeletal muscle actins is due to a less efficient shift of scallop actin subdomain 2 to the position it has in the polymer . The possible consequences of amino acid substitutions in actin subdomain 1 in the allosteric regulation of the actin cleft, and hence in the different stabilities of polymers formed by different actins, are discussed . Anal Biochem, 1999 Aug 1, 272(2), 191 - 8 Measurement of firefly luciferase reporter gene activity from cells and lysates using Escherichia coli arsenite and mercury sensors; Tauriainen S et al.; The structural gene encoding firefly luciferase from Photinus pyralis is a widely used reporter both in traditional monitoring of gene expression and in bacterial sensors . Its activity can be detected from living cells (in vivo) without disruption or from cell-free lysate (in vitro) . We compared the two measurement methods by using an overall toxicity detecting strain Escherichia coli MC1061(pCSS810), a mercury-sensing strain E . coli MC1061(pTOO11), and two new arsenic sensor strains MC1061(pTOO31) and AW3110(pTOO31) which were constructed for this study . Plasmid pTOO31 was constructed by inserting the ars promoter and the arsR gene from plasmid R773 to control firefly luciferase gene expression . Both in vivo and in vitro methods correlated well with the strains tested {correlation coefficients R = 0.99484 and 0.99834} and gave highly comparable results with standard solutions of arsenite or mercury ions and from six environmental water samples spiked with the ions . Use of the in vivo method resulted in lower variation between replicates of the same sample (CVs ranging from 3.9 to 7.2%) and also between different samples (from 8.6 to 25.9%) compared to the in vitro method (CVs ranging from 8.6 to 17.8% for replicates and from 13.1 to 36.3% for different samples) . Mund Kiefer Gesichtschir, 1999 May, 3 Suppl 1, S134 - 9 {EHBMP-2 . Initial BMP analog with osteoinductive properties}; Kubler NR et al.; For the first time a non natural BMP-variant (EHBMP-2) with osteoinductive properties was produced by expression in E . coli through specific mutation of the amino acid sequence . The substitution of 12 N-terminal amino acids by a nonsense sequence results in a neglectible affinity of EHBMP-2 to the extracellular matrix . In vitro EHBMP-2 induces dose-dependent cartilage formation in neonatal muscle tissue . Single intramuscular implantation in mice results in the formation of an ossicle with functional active bone marrow . The size of the ossicle depends on the amount of implanted EHBMP-2 and can significantly be increased by the combination with a collagen carrier . The largest bone formation is observed after injection of EHBMP-2 containing collagen suspensions . In rats a stronger osteoinductive activity can be achieved by coupling of EHBMP-2 to collagen discs than by coupling natural BMP-2 to the same collagen carrier . Critical size defects in rats' mandibular angels can be restored by the combination of granular collagenous bone matrix (ICBM) with EHBMP-2 . Further investigations have to show whether the altered pharmacokinetics of EHBMP-2 has advantages regarding its therapeutical use and tissue-engineering. Microbios, 1999, 97(388), 137 - 44 Appearance in Japan of highly macrolide-resistant Escherichia coli producing macrolide 2'-phosphotransferase II; Taniguchi K et al.; Escherichia coli CU1, a clinical isolate recovered in Japan in 1997, was found to be highly-resistant to both 14-membered and 16-membered ring macrolide antibiotics . A crude extract prepared from strain CU1 inactivated 14-, 15- and 16-membered ring macrolides in the presence of ATP and the Rf value of inactivated oleandomycin was identical to that of oleandomycin 2'-phosphate . This suggested that strain CU1 produced the enzyme macrolide 2'-phosphotransferase {MPH(2')} . Substrate specificity of the crude enzyme from strain CU1 against 14-, 15- and 16-membered ring macrolides was basically similar to that of MPH(2')II from strain BM2506, differing in that the former more effectively inactivated roxithromycin and tylosin . Subsequent attempts were made to clone the novel mph gene encoding for MPH(2') in strain CU1 . The mph gene carried by strain CU1 was located on nontransmissible plasmid DNA, designated pCU001 . Its molecular weight, estimated by agarose electrophoresis, was approximately 57 kD . The DNA sequence of the cloned mph gene from the Japanese isolate CU1 was identical to that of mphB, which until now had only been recovered in France . The variance in the substrate specificity of MPH(2')II from each strain led us to speculate that other factors in the reaction affect the enzymatic inactivation activity. Microbios, 1999, 97(386), 55 - 67 Physiological consequences of mutations in Escherichia coli heat shock dnaK and dnaJ genes; Wolska KI et al.; Mutations in the heat shock genes, dnaK and dnaJ, cause severe defects of several cellular functions . Null dnaJ and dnaKdnaJ mutations can be transduced in a restricted range of temperature . The efficiency of transformation with three unrelated plasmids, viz pACYC184, pBR322 and pSC101, is two times lower in dnaK mutants while the dnaJ mutant is characterized by slightly impaired transformation with pSC101 only . The lack of DnaJ function negatively influences the stability of pSC101 at 42 degrees C, and this plasmid cannot be stably maintained at 30 degrees C in the delta dnaKdnaJ mutant . The double deletion mutant, delta dbaKdnaJ, is characterized by impaired osmoadaptation . The galactokinase content is lower in both mutants tested compared with wild-type strains even at 30 degrees C . The efficient complementation of some of these defects by the wild-type alleles present on low-copy number plasmid was achieved. Biochemistry, 1999 Jul 20, 38(29), 9485 - 94 Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site; Kosmoski JV et al.; A strategy was developed to assemble nucleosomes specifically damaged at only one site and one structural orientation . The most prevalent UV photoproduct, a cis-syn cyclobutane thymine dimer (cs CTD), was chemically synthesized and incorporated into a 30 base oligonucleotide harboring the glucocorticoid hormone response element . This oligonucleotide was assembled into a 165 base pair double stranded DNA molecule with nucleosome positioning elements on each side of the cs CTD-containing insert . Proton NMR verified that the synthetic photoproduct is the cis-syn stereoisomer of the CTD . Moreover, two different pyrimidine dimer-specific endonucleases cut approximately 90% of the dsDNA molecules . This cleavage is completely reversed by photoreactivation with E . coli UV photolyase, further demonstrating the correct stereochemistry of the photoproduct . Nucleosomes were reconstituted by histone octamer exchange from chicken erythocyte core particles, and contained a unique translational and rotational setting of the insert on the histone surface . Hydroxyl radical footprinting demonstrates that the minor groove at the cs CTD is positioned away from the histone surface about 5 bases from the nucleosome dyad . Competitive gel-shift analysis indicates there is a small increase in histone binding energy required for the damaged fragment (DeltaDeltaG approximately 0.15 kcal/mol), which does not prevent complete nucleosome loading under our conditions . Finally, folding of the synthetic DNA into nucleosomes dramatically inhibits cleavage at the cs CTD by T4 endonuclease V and photoreversal by UV photolyase . Thus, specifically damaged nucleosomes can be experimentally designed for in vitro DNA repair studies. Biochemistry, 1999 Jul 20, 38(29), 9417 - 25 Domain mapping of the DNA binding, endonuclease, and ERCC1 binding properties of the human DNA repair protein XPF; McCutchen-Maloney SL et al.; During nucleotide excision repair, one of the two incisions necessary for removal of a broad spectrum of DNA adducts is made by the human XPF/ERCC1 protein complex . To characterize the biochemical function of XPF, we have expressed and purified the independent 104 kDa recombinant XPF protein from E . coli and determined that it is an endonuclease and can bind DNA in the absence of the ERCC1 subunit . Endonuclease activity was also identified in a stable 70 kDa proteolysis fragment of XPF obtained during protein expression, indicating an N-terminal catalytic domain . Sequence homology and secondary structure predictions indicated a second functional domain at the C-terminus of XPF . To investigate the significance of the two predicted domains, a series of XPF deletion fragments spanning the entire protein were designed and examined for DNA binding, endonuclease activity, and ERCC1 subunit binding . Our results indicate that the N-terminal 378 amino acids of XPF are capable of binding and hydrolyzing DNA, while the C-terminal 214 residues are capable of binding specifically to ERCC1 . We propose that the N-terminal domain of XPF contributes to the junction-specific endonuclease activity observed during DNA repair and recombination events . In addition, evidence presented here suggests that the C-terminal domain of XPF is responsible for XPF/ERCC1 complex formation . A working model for the XPF protein is presented illustrating the function of XPF in the nucleotide excision pathway and depicting the two functional domains interacting with DNA and ERCC1. Biochemistry, 1999 Jul 20, 38(29), 9317 - 27 Signaling domain of the aspartate receptor is a helical hairpin with a localized kinase docking surface: cysteine and disulfide scanning studies; Bass RB et al.; Cysteine and disulfide scanning has been employed to probe the signaling domain, a highly conserved motif found in the cytoplasmic region of the aspartate receptor of bacterial chemotaxis and related members of the taxis receptor family . Previous work has characterized the N-terminal section of the signaling domain {Bass, R . B., and Falke, J . J . (1998) J . Biol . Chem . 273, 25006-25014}, while the present study focuses on the C-terminal section and the interactions between these two regions . Engineered cysteine residues are incorporated at positions Gly388 through Ile419 in the signaling domain, thereby generating a library of receptors each containing a single cysteine per receptor subunit . The solvent exposure of each cysteine is ascertained by chemical reactivity measurements, revealing a periodic pattern of buried hydrophobic and exposed polar residues characteristic of an amphipathic alpha-helix, denoted helix alpha8 . The helix begins between positions R392 and Val401, then continues through the last residue scanned, Ile419 . Activity assays carried out both in vivo and in vitro indicate that both the buried and exposed faces of this amphipathic helix are critical for proper receptor function and the buried surface is especially important for kinase downregulation . Patterns of disulfide bond formation suggest that helix alpha8, together with the immediately N-terminal helix alpha7, forms a helical hairpin that associates with a symmetric hairpin from the other subunit of the homodimer, generating an antiparallel four helix bundle containing helices alpha7, alpha7', alpha8, and alpha8' . Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices alpha7 and alpha8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking. Biochemistry, 1999 Jul 13, 38(28), 9115 - 25 Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE; Kaufmann A et al.; Preprotein translocation in Escherichia coli is mediated by translocase, a multimeric membrane protein complex with SecA as the peripheral ATPase and SecYEG as the translocation pore . Unique cysteines were introduced into transmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible sites of interaction between the integral membrane subunits . The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expression vector and functionally overexpressed . Oxidation of the single-Cys pairs revealed periodic contacts between SecY and SecE that are confined to a specific alpha-helical face of TMS 2 and 3, respectively . A Cys at the opposite alpha-helical face of TMS 3 of SecE was found to interact with a neighboring SecE molecule . Formation of this SecE dimer did not affect the high-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked preprotein translocation and thus uncouples the SecA ATPase activity from translocation . Conditions that prevent membrane deinsertion of SecA markedly stimulated the interhelical contact between the SecE molecules . The latter demonstrates a SecA-mediated modulation of the protein translocation channel that is sensed by SecE. Biochemistry, 1999 Jul 13, 38(28), 9084 - 8 Discrimination of tRNALeu isoacceptors by the insertion mutant of Escherichia coli leucyl-tRNA synthetase; Li T et al.; A variant (LeuRS-A) of Escherichia coli leucyl-tRNA synthetase (LeuRS) carrying a 40-residue duplication in its connective peptide 1 (CP1) has a 3-fold lower specificity for than for, whereas wild-type LeuRS has the same specificity for these two isoacceptors . The replacement of the acceptor stem of with yields a chimeric tRNA(Leu) for which wild-type LeuRS has the same specificity as it does for the two normal isoacceptors mentioned, but for which LeuRS-A has a reduced specificity similar to that for, indicating a difference between these two acceptor stems . LeuRS-A is slightly less stable than the native enzyme . Wild-type LeuRS and LeuRS-A have almost same K(d) value for their interaction with as determined by fluorescence quenching . No difference was detected between these two proteins by CD and fluorescence spectroscopy . These results show that LeuRS-A can discriminate between the two isoacceptors of tRNA(Leu). Lupus, 1999, 8(4), 300 - 4 C1q inhibits autoantibody binding to calreticulin; Racila DM et al.; Calreticulin (CR) is a new rheumatic disease-associated autoantigen that is intimately associated with the Ro/SS-A ribonucleoprotein . CR autoantibodies are frequently observed in patients with photosensitive forms of lupus erythematosus (LE) . CR has been shown to be highly homologous to cC1q-R, the cell surface receptor that binds the collagenous domain of the first component of complement, C1q . C1q has also been shown to directly bind to CR . We therefore asked whether the binding of C1q to CR might interfere with the binding of CR autoantibody to CR . Full-length recombinant human CR, an E . coli fusion proteins was used as antigen in a direct enzyme-linked immunosorbent assay (ELISA) . CR autoantibody-containing sera were assayed before and after C1q removal by two different methods: by heating sera at 56 degrees C for 30 min or adding monoclonal anti-C1q antibodies . ELISA optical density (OD) values were found to be consistently higher in sera depleted of C1q by both methods compared to unmodified sera . The addition of purified C1q to C1q-depleted sera resulted in ELISA OD values similar to those of unmodified sera . These results suggest that C1q levels present in human serum can inhibit the binding of CR autoantibody to CR . One can speculate that the failure of C1q to mask CR autoepitopes in individuals with genetic deficiency of C1q could contribute to the high rate of photosensitive LE that occurs in such individuals. Toxicology, 1999 May 3, 134(1), 9 - 17 Are circulating cytokines interleukin-6 and tumor necrosis factor alpha involved in chlorpyrifos-induced fever? Gordon CJ, Rowsey PJ. Oral exposure to chlorpyrifos (CHP) in the rat results in an initial hypothermic response followed by a delayed fever . Fever from infection is mediated by the release of cytokines, including interleukin-6 (IL-6) and tumor necrosis factor (TNF alpha) . This study determined if the CHP-induced fever involves cytokine-mediated mechanisms similar to that of infectious fevers . Long-Evans rats were gavaged with the corn oil vehicle or CHP (10-50 mg/kg) . The rats were euthanized and blood collected at various times that corresponded with the hypothermic and febrile effects of CHP . Plasma IL-6, TNF alpha, cholinesterase activity (ChE), total iron, unsaturated iron binding capacity (UIBC), and zinc were measured . ChE activity was reduced by approximately 50% 4 h after CHP . There was no effect of CHP on IL-6 when measured during the period of CHP-induced hypothermia or fever . TNF alpha levels nearly doubled in female rats 48 h after 25 mg/kg CHP . The changes in plasma cytokine levels following CHP were relatively small when compared to > 1000-fold increase in IL-6 and > 10-fold rise in TNF alpha following lipopolysaccharide (E . coli; 50 microg/kg; i.p.)-induced fever . This does not preclude a role of cytokines in CHP-induced fever . Nonetheless, the data suggest that the delayed fever from CHP is unique, involving mechanisms other than TNF alpha and IL-6 release into the circulation characteristic of infectious fevers. FEBS Lett, 1999 Jul 2, 454(1-2), 169 - 71 Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence-based prediction and 65Zn blotting; Sujatha S et al.; The availability of repeating 'Cys' and/or 'His' units in a particular order prompts the prediction of Zn(II) finger motifs in a protein . Escherichia coli RNA polymerase has two tightly bound Zn(II) per molecule of the enzyme as detected by atomic absorption spectroscopy . One Zn(II) was identified to be at the beta subunit, whereas the other putative Zn(II) binding site has recently been predicted to be at the N-terminal half of the beta' subunit, from primary sequence analysis . We show here that the beta' subunit has no ability to bind 65Zn(II) . On the other hand, the N-terminal domain of the alpha subunit has strong Zn(II) binding ability with no obvious functional implications. FEBS Lett, 1999 Jul 2, 454(1-2), 157 - 64 Phosphorylation of tau protein by recombinant GSK-3beta: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain; Godemann R et al.; Glycogen synthase kinase-3beta (GSK-3beta) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments . However, it has been claimed that GSK-3beta can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules . We have analyzed the activity of recombinant GSK-3beta and of GSK-3beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing . The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT-8, AT-270, etc) . We also show that the non-proline-directed activity at KXGS motifs is not due to GSK-3beta itself, but to kinase contaminations in common GSK-3beta preparations from tissues which are activated upon addition of heparin. FEBS Lett, 1999 Jul 2, 454(1-2), 90 - 4 Novel tetravalent and bispecific IgG-like antibody molecules combining single-chain diabodies with the immunoglobulin gamma1 Fc or CH3 region; Alt M et al.; Although bispecific IgG molecules have been successfully applied for antibody-mediated immunotherapy of tumours, applicability is hampered by the difficulties associated with their generation . In the present study, we have used a bispecific single-chain diabody (scDb) directed against carcinoembryonic antigen and Escherichia coli beta-galactosidase as a model to generate bispecific IgG-like antibody molecules . We show that the fusion of this single-chain diabody to the Fc (scDb-Fc) or CH3 (scDb-CH3) region of the human immunoglobulin gamma1 chain results in the expression of dimeric fusion proteins exhibiting four functional antigen binding sites with increased functional affinity . This strategy represents a new and convenient way to generate IgG-like multivalent and bispecific molecules that are efficiently secreted from mammalian cells. FEBS Lett, 1999 Jul 2, 454(1-2), 85 - 9 Arfaptin 1 forms a complex with ADP-ribosylation factor and inhibits phospholipase D; Williger BT et al.; ADP-ribosylation factors (ARFs) regulate coatomer assembly on the Golgi as well as recruitment of clathrin adapter proteins and are therefore involved in vesicle budding from the Golgi and vesicular transport . They are also regulators of phospholipase D (PLD) activity . Arfaptin 1 is an ARF binding protein that inhibits PLD activation, vesicular trafficking and secretion . In the present report, we show that arfaptin 1 interacts with 'high speed' membranes independently of ARF . However, addition of myristoylated ARF3 (myrARF3) increases the association of arfaptin 1 with the membranes, suggesting that arfaptin 1 and ARF form a complex on the Golgi . Utilizing several deletion mutants of arfaptin 1 it is shown that the association of arfaptin 1 with myrARF3 is mediated via two binding sites on arfaptin 1 . These two domains are needed for arfaptin 1 inhibition of PLD activation by myrARF3 in vitro. FEBS Lett, 1999 Jul 2, 454(1-2), 71 - 4 Interaction with free beta' subunit unmasks DNA-binding domain of RNA polymerase sigma subunit; Kulbachinskiy A et al.; The promoter recognition site on the sigma70 initiation factor is shielded from interaction with DNA unless sigma70 is bound to the core component of RNA polymerase (RNAP) . It is shown that interaction of sigma70 with the isolated beta' subunit of Escherichia coli RNAP is sufficient to induce unshielding of the DNA binding site . Using UV-induced DNA-protein cross-linking we demonstrate that free beta' stimulates specific cross-links between region 2 of the sigma70 polypeptide and a fragment of the non-template promoter strand containing the TATAAT sequence . Thus the sigmabeta' subassembly of RNAP can assume a functionally competent conformation independently of the bulk of the RNAP core. FEBS Lett, 1999 Jul 2, 454(1-2), 7 - 10 The effect of 17beta-estradiol-DNA adducts on the replication of exon # 5 of the human suppressor gene p53; Yu FL et al.; Using a PCR technique, exon # 5 of the human tumor suppressor gene p53 was amplified and ligated into the pCRII vector and transformed into Escherichia coli INV alphaF' competent cells . The cloned exon # 5 was 184 bp long . Evidence is presented to show that after dimethyldioxirane epoxidation, 17beta-estradiol was able to form 17beta-estradiol-DNA adducts and to strongly inhibit the replication of the cloned exon # 5 producing smaller sizes of DNA fragments and introducing errors of incorporation at the 3'-end of the terminating DNAs . The errors occurred mainly at the clusters of the complementary 'G' and 'A' bases on the template strand DNA, presumably, the major sites where the 17beta-estradiol-DNA adducts were formed. FEBS Lett, 1999 Jul 2, 454(1-2), 1 - 6 Efficient expression, purification and crystallisation of two hyperthermostable enzymes of histidine biosynthesis; Thoma R et al.; Enzymes from hyperthermophiles can be efficiently purified after expression in mesophilic hosts and are well-suited for crystallisation attempts . Two enzymes of histidine biosynthesis from Thermotoga maritima, N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase and the cyclase moiety of imidazoleglycerol phosphate synthase, were overexpressed in Escherichia coli, both in their native and seleno-methionine-labelled forms, purified by heat precipitation of host proteins and crystallised . N'-((5'-phosphoribosyl)-formimino)-5-aminoimidazol-4-carb oxamid ribonucleotide isomerase crystallised in four different forms, all suitable for X-ray structure solution, and the cyclase moiety of imidazoleglycerol phosphate synthase yielded one crystal form that diffracted to atomic resolution . The obtained crystals will enable the determination of the first three-dimensional structures of enzymes from the histidine biosynthetic pathway. Mol Biochem Parasitol, 1999 Jun 25, 101(1-2), 13 - 21 Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein; Ruiz de Eguino AD et al.; A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens . The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains . This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies . Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite . The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA) . The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes. Mol Biochem Parasitol, 1999 Jun 25, 101(1-2), 1 - 11 Cloning and kinetic characterization of the Trypanosoma cruzi S-adenosylmethionine decarboxylase; Kinch LN et al.; The gene for S-adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in the biosynthesis of polyamines, has been cloned from a Trypanosoma cruz cDNA library . The cDNA clone contains a 1.1 kb open reading frame predicted to encode a 42 kDa protein that shares 31% sequence identity to the human proenzyme . T . cruzi AdoMetDC expressed and purified from E . coli is auto-catalytically processed into two subunits of 32 kDa (alpha) and 10 kDa (beta) . The catalytic activity of the purified recombinant enzyme is activated by the addition of putrescine to the reaction . To determine the effect of putrescine on the kinetics of the reaction, the velocity data collected at various substrate and putrescine concentrations were fit to the rate equation describing a non-essential activator . In the presence of fully saturating putrescine, k(cat) increases by 9-fold over the unactivated rate to 0.06 s(-1) . The model derived Km for AdoMet is 0.05 mM in the absence of putrescine and the model-derived Kd for putrescine binding to free enzyme is 2.5 mM . The Km for AdoMet increases by alpha 2-fold when the enzyme is fully saturated with putrescine . Unlike human AdoMetDC, cadaverine activates the T . cruzi enzyme to a similar extent as putrescine. Plant Mol Biol, 1999 May, 40(2), 365 - 72 cDNA cloning and heterologous expression of coniferin beta-glucosidase; Dharmawardhana DP et al.; Coniferin beta-glucosidase (CBG) catalyzes the hydrolysis of monolignol glucosides to release the cinnamyl alcohols for oxidative polymerization to lignin . Utilizing the N-terminal amino acid sequence of the purified enzyme, the corresponding full-length cDNA sequence was isolated from a Pinus contorta xylem-specific library . The isolated 1909 nucleotide cDNA was confirmed to be that of CBG on the basis of its high homology to family 1 glycosyl hydrolases, the sequence identity with the N-terminal amino acid residues of the purified enzyme, and the coniferin hydrolytic activity and substrate specificity profile displayed by the recombinant protein when expressed in Escherichia coli . The presence of a 23 amino acid N-terminal signal peptide in the deduced 513 amino acid enzyme suggests that CBG is a secretory protein targeted to the ER . The isolation of CBG cDNA will facilitate the evaluation of the importance of this enzyme in the ultimate stages of lignin biosynthesis and could be a valuable tool in manipulating lignin levels in xylem cell walls. Klin Padiatr, 1999 May-Jun, 211(3), 149 - 53 {Granulocyte function in premature infants before the 34th week of pregnancy and in mature newborn infants}; Habermehl P et al.; BACKGROUND: Preterm and term neonates have an increased risk to develop severe bacterial infections . Impairment of neutrophil function may be responsible for this increased risk . Other diseases related to prematurity like retinopathia of prematurity (ROP) or broncho-pulmonary dysplasia (BPD) on the other hand may be due to poorly controlled O2-radical production . PATIENTS AND METHODS: Blood samples of 112 premature (34 weeks of gestation and older) and term neonates were analysed . Blood samples of 23 healthy adults (18 to 50 years old) served as controls . O2-radical production and phagocytosis of neutrophils were determined by flow cytometry, using a commercial test system . RESULTS: Under the experimental conditions applied, the capacity to produce O2-radicals following vigorous stimulation (E . coli) is comparable between neutrophils of preterm/term neonates and healthy adults . However, unstimulated or weakly stimulated (fMLP) neutrophils of preterm and term neonates show a statistically higher O2-radical production as neutrophils of the control group . The production of oxygen radicals increases during the first 10 days of the life . The capability of neutrophils to phagocytose E . coli is significantly lower in newborns (preterm and term) compared to the adult controls . CONCLUSIONS: The values reported here for phagocytosis and O2-radical production utilizing a commercially available test system may serve as "preliminary normal values" for neonates . No differences were found between the groups of neonates with and without infection . Impaired neutrophil-phagocytosis possibly contributes to the increased risk of preterm and term neonates to acquire bacterial infections . The increased spontaneous O2-radical production, on the other hand, may play a role for the development of so called "free radical diseases" such as ROP or BPD . However, our results cannot add further proof to this hypothesis. J Cell Biochem, 1999 Sep 1, 74(3), 406 - 17 Differential expression of PSP94 in rat prostate lobes as demonstrated by an antibody against recombinant GST-PSP94; Kwong J et al.; Prostate secretory protein (PSP94, 94 amino acids) is one of the most abundant proteins secreted from the prostate . Its biological role is unknown and still controversial, although it is assumed to have the potential to be a biomarker and a suppressor of prostate cancer . In order to establish an animal model to further elucidate its biological role, we expressed the mature form of rat PSP94 in Escherichia coli, using a glutathione S-transferase (GST) fusion expression vector; we generated a polyclonal rabbit antibody against the recombinant protein . The antibody specifically recognized recombinant rat PSP94 and cross-reacted only very weakly with its human homologue . Using the characterized anti-rat PSP94 antibody, we found that PSP94 was located primarily in rat prostate . Furthermore, PSP94 is present at different levels in different lobes of rat prostate, with significant levels detectable only in the lateral lobe (LP) . In addition, the most abundant PSP94 expression was found in the prostate lobe secretions, and PSP94 levels in LP secretions were at least seven times higher than in secretions from the dorsal prostate (DP) . The rat ventral prostate (VP) and other regions of the male accessory glands were found to be almost completely devoid of PSP94 . Since most rat prostate dysplasia induced by steroid hormone treatment occurs only in dorsolateral prostate, prostate tissue-specific expression and the expression of PSP94 in dorsolateral, but not other, lobes of the prostate suggest a potential role in prostate targeting and prostate cancer development . Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8779 - 83 LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide; Christie JM et al.; Phototropism, the bending response of plant organs to or away from a directional light source, is one of the best studied blue light responses in plants . Although phototropism has been studied for more than a century, recent advances have improved our understanding of the underlying signaling mechanisms involved . The NPH1 gene of Arabidopsis thaliana encodes a blue light-dependent autophosphorylating protein kinase with the properties of a photoreceptor for phototropism . NPH1 apoprotein noncovalently binds FMN to form the holoprotein nph1 . The N-terminal region of the protein contains two LOV (light, oxygen, or voltage) domains that share homology with sensor proteins from a diverse group of organisms . These include the bacterial proteins NIFL and AER, both of which bind FAD, and the phy3 photoreceptor from Adiantium capillus-veneris . The LOV domain has therefore been proposed to reflect a flavin-binding site, regulating nph1 kinase activity in response to blue light-induced redox changes . Herein we demonstrate that the LOV domains of two nph1 proteins and phy3 bind stoichiometric amounts of FMN when expressed in Escherichia coli . The spectral properties of the chromopeptides are similar to the action spectrum for phototropism, implying that the LOV domain binds FMN to function as a light sensor . Thus, our findings support the earlier model that nph1 is a dual-chromophoric flavoprotein photoreceptor regulating phototropic responses in higher plants . We therefore propose the name phototropin to designate the nph1 holoprotein. Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8557 - 61 Gene silencing via protein-mediated subcellular localization of DNA; Kim SK et al.; We previously reported that overexpression of SopB, an Escherichia coli F plasmid-encoded partition protein, led to silencing of genes linked to, but well-separated from, a cluster of SopB-binding sites termed sopC . We show here that in this SopB-mediated repression of sopC-linked genes, all but the N-terminal 82 amino acids of SopB can be replaced by the DNA-binding domain of a sequence-specific DNA-binding protein, provided that the sopC locus is also replaced by the recognition sequence of the DNA-binding domain . These results, together with our previous finding that the N-terminal fragment of SopB is responsible for its polar localization in cells, suggest a mechanism of gene silencing: patches of closely packed DNA-binding domains are formed if a sequence-specific DNA-binding protein is localized to specific cellular sites; such a patch can capture a DNA carrying the recognition site of the DNA-binding domain and sequestrate genes adjacent to the recognition site through nonspecific binding of DNA . The generalization of this model to gene silencing in eukaryotes is discussed. Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8540 - 4 Divergent evolution of membrane protein topology: the Escherichia coli RnfA and RnfE homologues; Saaf A et al.; Although the molecular evolution of protein tertiary structure and enzymatic activity has been studied for decades, little attention has been paid to the evolution of membrane protein topology . Here, we show that two closely related polytopic inner membrane proteins from Escherichia coli have evolved opposite orientations in the membrane, which apparently has been achieved by the selective redistribution of positively charged amino acids between the polar segments flanking the transmembrane stretches . This example of divergent evolution of membrane protein topology suggests that a complete inversion of membrane topology is possible with relatively few mutational changes even for proteins with multiple transmembrane segments. Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8414 - 9 A model for the mechanism of strand passage by DNA gyrase; Kampranis SC et al.; The mechanism of type II DNA topoisomerases involves the formation of an enzyme-operated gate in one double-stranded DNA segment and the passage of another segment through this gate . DNA gyrase is the only type II topoisomerase able to introduce negative supercoils into DNA, a feature that requires the enzyme to dictate the directionality of strand passage . Although it is known that this is a consequence of the characteristic wrapping of DNA by gyrase, the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate is largely unknown . We have addressed this mechanism by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle . We propose a model in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA substrate, setting up an equilibrium of the transported segment across the DNA gate . The overall efficiency of strand passage is determined by the position of this equilibrium, which depends on the superhelical density of the DNA substrate . This mechanism is concerted, in that capture of the transported segment by the ATP-operated clamp induces opening of the DNA gate, which in turn stimulates ATP hydrolysis. Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8396 - 401 Crystal structure of ERA: a GTPase-dependent cell cycle regulator containing an RNA binding motif; Chen X et al.; ERA forms a unique family of GTPase . It is widely conserved and essential in bacteria . ERA functions in cell cycle control by coupling cell division with growth rate . ERA homologues also are found in eukaryotes . Here we report the crystal structure of ERA from Escherichia coli . The structure has been determined at 2.4-A resolution . It reveals a two-domain arrangement of the molecule: an N-terminal domain that resembles p21 Ras and a C-terminal domain that is unique . Structure-based topological search of the C domain fails to reveal any meaningful match, although sequence analysis suggests that it contains a KH domain . KH domains are RNA binding motifs that usually occur in tandem repeats and exhibit low sequence similarity except for the well-conserved segment VIGxxGxxIK . We have identified a betaalphaalphabeta fold that contains the VIGxxGxxIK sequence and is shared by the C domain of ERA and the KH domain . We propose that this betaalphaalphabeta fold is the RNA binding motif, the minimum structural requirement for RNA binding . ERA dimerizes in crystal . The dimer formation involves a significantly distorted switch II region, which may shed light on how ERA protein regulates downstream events. Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8390 - 5 Interactions of Escherichia coli sigma(70) within the transcription elongation complex; Daube SS et al.; A functional transcription elongation complex can be formed without passing through a promoter by adding a complementary RNA primer and core Escherichia coli RNA polymerase in trans to an RNA-primed synthetic bubble-duplex DNA framework . This framework consists of a double-stranded DNA sequence with an internal noncomplementary DNA "bubble" containing a hybridized RNA primer . On addition of core polymerase and the requisite NTPs, the RNA primer is extended in a process that manifests most of the properties of in vitro transcription elongation . This synthetic elongation complex can also be assembled by using holo rather than core RNA polymerase, and in this study we examine the interactions and fate of the sigma(70) specificity subunit of the holopolymerase in the assembly process . We show that the addition of holopolymerase to the bubble-duplex construct triggers the dissociation of the sigma factor from some complexes, whereas in others the RNA oligomer is released into solution instead . These results are consistent with an allosteric competition between sigma(70) and the nascent RNA strand within the elongation complex and suggest that both cannot be bound to the core polymerase simultaneously . However, the dissociation of sigma(70) from the complex can also be stimulated by binding of the holopolymerase to the DNA bubble duplex in the absence of a hybridized RNA primer, suggesting that the binding of the core polymerase to the bubble-duplex construct also triggers a conformational change that additionally weakens the sigma-core interaction. Somat Cell Mol Genet, 1998 Jul, 24(4), 249 - 56 Cointegration of DNA molecules introduced into mammalian cells by electroporation; Chen C et al.; Electroporation was used to introduce a mixture of two plasmid-cloned genes into Chinese hamster ovary (CHO) cells, and the location of the two genes was subsequently determined by fluorescence in situ hybridization (FISH) . The 25 kb Chinese hamster gene for dihydrofolate reductase (dhfr) in the form of a cosmid-derived 40 kb BglI fragment and the SV40 promoter-driven E . coli gene for guanine phosphoribosyltransferase (gpt) were co-electroporated and gpt + transfectants selected . Clones that had also integrated a single copy of the dhfr gene were studied by 2-color fluorescence in situ hybridization (FISH) to localize the integration site(s) of the exogenous DNA in metaphase chromosomes . All 9 clones examined showed co-localization of the two transgenes . The chromosomal site of integration was different in each clone . Co-integration was confirmed by co-amplification experiments . We conclude that, even when provided at low concentrations, separate soluble DNA molecules become linked upon gene transfer by electroporation, either by intracellular ligation prior to integration, or by co-integration at a common site in a given recipient cell. Mol Microbiol, 1999 Jul, 33(2), 338 - 49 Heterotrimerization of PII-like signalling proteins: implications for PII-mediated signal transduction systems; Forchhammer K et al.; PII-like signalling molecules are trimeric proteins composed of 12-13 kDa polypeptides encoded by the glnB gene family . Heterologous expression of a cyanobacterial glnB gene in Escherichia coli leads to an inactivation of E . coli's own PII signalling system . In the present work, we show that this effect is caused by the formation of functionally inactive heterotrimers between the cyanobacterial glnB gene product and the E . coli PII paralogues GlnB and GlnK . This led to the discovery that GlnK and GlnB of E . coli also form heterotrimers with each other . The influence of the oligomerization partner on the function of the single subunit was studied using heterotrimerization with the Synechococcus PII protein . Uridylylation of GlnB and GlnK was less efficient but still possible within these heterotrimers . In contrast, the ability of GlnB-UMP to stimulate the adenylyl-removing activity of GlnE (glutamine synthetase adenylyltransferase/removase) was almost completely abolished, confirming that rapid deadenylylation of glutamine synthetase upon nitrogen stepdown requires functional homotrimeric GlnB protein . Remarkably, however, rapid adenylylation of glutamine synthetase upon exposing nitrogen-starved cells to ammonium was shown to occur in the absence of a functional GlnB/GlnK signalling system as efficiently as in its presence. Mol Microbiol, 1999 Jul, 33(2), 284 - 95 TorC apocytochrome negatively autoregulates the trimethylamine N-oxide (TMAO) reductase operon in Escherichia coli; Ansaldi M et al.; The trimethylamine N-oxide (TMAO) anaerobic respiratory system of Escherichia coli comprises a periplasmic terminal TMAO reductase (TorA) and a pentahaem c-type cytochrome (TorC), which is involved in electron transfer to TorA . The structural proteins are encoded by the torCAD operon whose expression is induced in the presence of TMAO through the TorS/TorR two-component system . By using a genomic library cloned into a multicopy plasmid, we identified TorC as a possible negative regulator of the tor operon . Interestingly, in trans overexpression of torC not only decreased the activity of a torA'-'lacZ fusion, but also dramatically reduced the amount of mature TorC cytochrome . This led us to propose that, after translocation, TorC apocytochrome downregulates the tor operon unless it is properly matured . In agreement with this hypothesis, we have shown that mini-Tn10 insertions within genes involved in the c-type cytochrome maturation pathway or haem biosynthesis decreased tor operon expression . Dithiothreitol (DTT), which reduces disulphide bonds and thus prevents the first step in c-type cytochrome formation, also strongly decreases the tor promoter activity . The DTT effect is TorC dependent, as it is abolished when torC is disrupted . In contrast, overexpression of the c-type cytochrome maturation (ccm ) genes relieved the tor operon of the negative control and allowed the bacteria to produce a higher amount of TorC holocytochrome . Therefore, the TorC negative autoregulation probably means that maturation of the c-type cytochrome is a limiting step for Tor system biogenesis . Genetic experiments have provided evidence that TorC control is mediated by the TorS/TorR two-component system and different from the tor anaerobic control . In our working model, TMAO and apoTorC bind to the periplasmic side of TorS, but TMAO activates TorS autophosphorylation, whereas apoTorC inhibits the TorS kinase activity. Mol Microbiol, 1999 Jul, 33(2), 260 - 73 Expression of the phosphotransferase system both mediates and is mediated by Mlc regulation in Escherichia coli; Plumbridge J; The ptsHIcrr operon encodes the cytoplasmic components of the phosphotransferase system (PTS) . It is expressed from two major promoters, of which the upstream promoter has previously been shown to be induced by glucose and to be dependent upon cAMP/CAP . This promoter is now shown to be repressed by Mlc . Mlc is a transcriptional regulator controlling, among others, the gene ptsG, encoding EIICBGlc, the glucose-specific transporter of the PTS . Transcription of ptsH p0 and ptsG are subject to the same regulatory pattern . In addition to induction by glucose and repression by Mlc, mutations in ptsHIcrr, which interrupt the PEP-dependent phosphate transfer through the soluble components of the PTS, lead to high expression of both ptsH and ptsG, while mutations inactivating EIIBCGlc are non-inducible . Mutations in mlc lead to high constitutive expression and are dominant, implying that Mlc is the ultimate regulator of ptsHI and ptsG expression . Growth on other PTS sugars, besides glucose, also induces ptsH and ptsG expression, suggesting that the target of Mlc regulation is the PTS . However, induction by these other sugars is only observed in the presence of ptsG+, thus confirming the importance of glucose and EIICBGlc in the regulation of the PTS . The ptsG22 mutation, although negative for glucose transport, shows a weak positive regulatory phenotype . The mutation has been sequenced and its effect on regulation investigated. Mol Microbiol, 1999 Jul, 33(2), 223 - 34 Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions; Bernales I et al.; A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed . These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans . A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates . When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold . An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity . Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette . Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat . No deletion or inversion of the intervening DNA was observed . These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition. Mol Microbiol, 1999 Jul, 33(1), 188 - 99 The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo; Lopez PJ et al.; RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay . While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth . We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E . This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected . Another mutation (rne105 ) removing the last 469 residues behaves similarly . Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA . A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants . All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization . Artificial mRNAs generated by E . coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth. Mol Microbiol, 1999 Jul, 33(1), 108 - 18 The 5' region of cnf1 harbours a translational regulatory mechanism for CNF1 synthesis and encodes the cell-binding domain of the toxin; Fabbri A et al.; The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) is organized into three functional domains: the N-terminal part containing the cell-binding domain, a putative central membrane-spanning region, and a C-terminal catalytic region . On the basis of competition assays between CNF1 and GST-recombinant proteins containing different N-terminal fragments, and point mutations, we restricted the binding region to the first 190 amino acids . Hydrophilic amino acids 53-75 are strictly necessary to cell receptor recognition . Using different cnf1-lacZ translational fusions, we demonstrated that the mRNA corresponding to the first 48 codons of cnf1 is involved in the translational regulation of CNF1 synthesis . This regulation consists of both a positive and a negative control . The positive control is exerted by codons 6-20, including a putative downstream box that enhances the translational expression of cnf1 . The negative control depends on codons 45-48 . In this region, an anti-Shine-Dalgarno sequence, highly homologous to the core of the internal complementary sequence already reported for growth rate-regulated metabolic genes, has been detected . To some extent, the inner structural organization of CNF1 would thus suggest the compiling of several functions in a single mRNA protein system. Mol Microbiol, 1999 Jul, 33(1), 41 - 51 The extended promoters for two outer membrane lipoprotein genes of Borrelia spp . uniquely include a T-rich region; Sohaskey CD et al.; OspA and B proteins of Borrelia burgdorferi and Vmp proteins of Borrelia hermsii are abundant outer membrane lipoproteins, whose expression varies with the environment . The genes for these proteins have the '-35' and '-10' elements of a sigma70-type promoter . Deletions of the promoters for these genes were analysed with a chloramphenicol acetyltransferase (CAT) reporter gene and plasmid constructs that were stably maintained in Escherichia coli or transiently transfected into B . burgdorferi . Reporter expression was measured as susceptibility of transformed E . coli cells to chloramphenicol and the CAT activity of E . coli and B . burgdorferi lysates in vitro . Presence of the '-10' element was essential for full activity in both B . burgdorferi and E . coli . Upstream of the '-35' elements of the ospAB and vmp promoters were tracts with Ts in 16 of 20 positions for B . burgdorferi and 18 of 20 positions for B . hermsii . Deletion of the T-rich region from the ospAB or vmp promoter caused a greater reduction of CAT activity in B . burgdorferi than in E . coli . The findings indicate that ospAB and vmp promoters are extended promoters with two parts: (i) a core region containing typical '-35' and '-10' elements and (ii) a unique T-rich region. Mol Microbiol, 1999 Jul, 33(1), 18 - 32 Differential fiu-lacZ fusion regulation linked to Escherichia coli colony development; Newman DL et al.; Colonies of strains carrying a stable lambdaplacMu15 translational fusion displayed sharply defined intense staining at the centre on Xgal medium . The fusion was in fiu (ferric ion uptake), encoding an iron-regulated outer membrane protein (IROMP) controlled via four overlapping ferric uptake regulator (Fur) boxes in the sigma70 promoter region . Fiu-LacZ was synthesized in low amounts (< 1% of a transcriptional fiu:lacZ+ fusion), localized to membranes, and underwent processing from a large protein to one that co-migrated with native beta-galactosidase . Intact cells synthesizing Fiu-LacZ often displayed greater enzymatic activity than permeabilized cells . The colony centre was insensitive to iron regulation observed in liquid cultures and at the colony edge . Within colonies grown on 36 microM iron citrate medium, fiu'-'lacZ protein fusion strains displayed 60-fold higher beta-galactosidase activity in the centre, and transcriptional fiu:lacZ+ fusion strains displayed a 10-fold centre/edge difference . On medium without added iron citrate, the centre/edge difference collapsed to < 2.2-fold for both translational and transcriptional fusions because activity at the edge was derepressed . Iron-insensitive fiu'-'lacZ expression in the colony centre occurred during a 6-18 h time window at the start of colony morphogenesis, corresponding to the initiation of multilayer microcolony development . A simple model for differential fiu'-'lacZ regulation is proposed whereby iron accessibility changes during colony morphogenesis. Eur J Biochem, 1999 Jun, 262(3), 832 - 9 Eukaryotic precursor proteins are processed by Escherichia coli outer membrane protein OmpP; Striebel HM et al.; A new specific endopeptidase that cleaves eukaryotic precursor proteins has been found in Escherichia coli K but not in E . coli B strains . After purification, protein sequencing and Western blotting, the endopeptidase was shown to be identical with E . coli outer membrane protein OmpP {Kaufmann, A., Stierhof, Y.-D . & Henning, U . (1994) J . Bacteriol . 176, 359-367} . Further characterization of enzymatic properties of the new peptidase was performed . Comparison of the cleavage specificities of the newly found endopeptidase and that of rat mitochondrial processing peptidase (MPP) showed that patterns of proteolytic cleavage on the investigated precursor proteins by both enzymes are similar . By using three mitochondrial precursor proteins, the specificity assigned to OmpP previously, a cleavage position between two basic amino-acid residues, was extended to a three amino-acid recognition sequence . Positions +1 to +3 of this extended recognition site consist of an amino-acid residue with a small aliphatic side chain such as alanine or serine, a large hydrophobic residue such as leucine or valine followed by an arginine residue . Additionally, structural motifs of the substrate seem to be required for OmpP cleavage. Eur J Biochem, 1999 Jun, 262(3), 652 - 8 Identification of AtPIS, a phosphatidylinositol synthase from Arabidopsis; Collin S et al.; Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium-dependent responses in the cell . Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme . Sequencing the entire cDNA confirmed the homology between the two proteins . Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells . Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP-diacylglycerol and myo-inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction . These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase . A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1. J Pharmacol Exp Ther, 1999 Aug, 290(2), 635 - 40 A novel transversion in the intron 5 donor splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor metabolizer phenotype for the anticonvulsant drug S-mephenytoin; Ibeanu GC et al.; Cytochrome P-450 (CYP) 2C19 is responsible for the metabolism of a number of therapeutic agents such as S-mephenytoin, omeprazole, proguanil, certain barbiturates, diazepam, propranolol, citalopram and imipramine . Genetic polymorphisms in this enzyme are responsible for the poor metabolizers (PM) of mephenytoin, which represent approximately 13-23% of Asians and 3-5% of Caucasians . Several polymorphisms contribute to this phenotype . We have isolated two new allelic variants that contribute to the PM phenotype in Caucasians . CYP2C19*7 contained a single T --> A nucleotide transversion in the invariant GT at the 5' donor splice site of intron 5 . The second PM allele, CYP2C19*8, consisted of a T358C nucleotide transition in exon 3 that results in a Trp120Arg substitution . In a bacterial expression system, CYP2C198 protein exhibited a dramatic (approximately 90% and 70%) reduction in the metabolism of S-mephenytoin and tolbutamide, respectively, when compared with the wild-type CYP2C191B protein . Restriction fragment length polymerase chain reaction tests were developed to identify the new allelic variants. Microbiology, 1999 Jun, 145 ( Pt 6), 1485 - 90 Two-hybrid assay: construction of an Escherichia coli system to quantify homodimerization ability in vivo; Di Lallo G et al.; A hybrid system which takes advantage of the properties of the lambda repressor allows detection of protein-protein interactions . Fusion of the cI N-terminal domain to a heterologous protein will result in a functional lambda repressor, able to strongly bind to its operator and conferring immunity to lambda infection only when the heterologous protein dimerizes efficiently . In this paper, construction of a recombinant plasmid which allows detection of the activity of the lambda chimeric repressor formed by the N-terminal part of cI fused with a heterologous protein is reported . This construct is interesting due to its potential to be integrated in any target gene of the bacterial host, thus permitting this hybrid assay to be performed, not only in Escherichia coli strains, but in every bacterial genus where the reporter gene can be expressed . In addition, because of its modular construction, this plasmid can be easily modified to be exploitable in many experimental situations, such as in the detection of promoter region activity. Microbiology, 1999 Jun, 145 ( Pt 6), 1421 - 9 Characterization of a molybdenum cofactor biosynthetic gene cluster in Rhodobacter capsulatus which is specific for the biogenesis of dimethylsulfoxide reductase; Solomon PS et al.; The DMSO reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor (Moco) and is located in the periplasm . DNA sequence analysis identified four genes involved in the biosynthesis of the Moco (moaA, moaD, moeB and moaC) immediately downstream of the dor (DMSO respiratory) gene cluster . Rhodobacter capsulatus MoaA was expressed in Escherichia coli as a His6-tagged protein . Although, the expressed protein formed inclusion bodies, EPR spectroscopy showed that MoaA contains a {3Fe-4S} cluster . A moaA mutant was constructed and its phenotype indicates that the Moco biosynthetic gene cluster downstream of the dor operon is specific for the biogenesis of DMSO reductase . Two forms of DMSO reductase were purified by immunoaffinity chromatography from the moaA mutant . A mature form of DMSO reductase was located in the periplasm and a precursor form was found in the cytoplasm. Microbiology, 1999 Jun, 145 ( Pt 6), 1369 - 74 A novel protein of Erysipelothrix rhusiopathiae that confers haemolytic activity on Escherichia coli; Makino S et al.; Erysipelothrix rhusiopathiae, the cause of swine erysipelas and human erysipeloid, produces a haemolysin . A recombinant plasmid, pHLY, conferring haemolytic activity on Escherichia coli was isolated from a genomic library of Ery . rhusiopathiae strain Tama-96 . This plasmid was stable in RecA- E . coli, but unstable in a RecA+ strain . A spontaneous deletion plasmid, pMini-HLY, also conferring haemolytic activity was derived from pHLY . Two ORFs were detected in pHLY . Analysis of pMini-HLY and other deletion clones established that ORF2 was associated with haemolytic activity . The sequence of ORF1 was highly homologous to those of transposases in the IS30 family . The deletion which generated pMini-HLY was between two short direct repeat (DR) sequences flanking the ORF1 sequence, and there were inverted repeat sequences inside the two DR sequences, suggesting an insertion element . No sequence homology to the deduced amino acid sequence of ORF2 was detected in the databases, but its sequence was characteristic of a surface lipoprotein . Western blot analysis, using antiserum against the 16 kDa protein produced from ORF2, found the protein to be commonly distributed in all Erysipelothrix species. RNA, 1999 Jul, 5(7), 972 - 85 Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli; Goodrich AF et al.; To define basic features of mRNA processing and decay in Escherichia coli, we have examined a set of mRNAs encoded by the filamentous phage f1 that have structures typical of bacterial mRNAs . They bear a stable hairpin stem-loop on the 3' end left from rho-independent termination and are known to undergo processing by RNase E . A small percentage of the f1 mRNAs were found to bear poly(A) tails that were attached to heterogeneous positions near the common 3' end . In a poly(A) polymerase-deficient host, the later-appearing processed mRNAs were stabilized, and a novel small RNA accumulated . This approximately 125-nt RNA proved to arise via RNase E cleavage from the 3'-terminal region of the mRNAs bearing the terminator . Normally ribosomes translating gene VIII appear to protect this cleavage site from RNase E, so that release of the fragment from the mRNAs occurs very slowly . The data presented define additional steps in the f1 mRNA processing and decay pathways and clarify how features of the pathways are used in establishing and maintaining the persistent filamentous phage infection . Although the primary mode of decay is endonucleolytic cleavage generating a characteristic 5' --> 3' wave of products, polyadenylation is involved in part in degradation of the processed mRNAs and is required for turnover of the 125-nt mRNA fragment . The results place polyadenylation at a later rather than an initiating step of decay . They also provide a clear illustration of how stably structured RNA 3' ends act as barriers to 3' --> 5' exonucleolytic mRNA decay. RNA, 1999 Jul, 5(7), 856 - 64 Positions in the 30S ribosomal subunit proximal to the 790 loop as determined by phenanthroline cleavage; Muth GW et al.; Positioning rRNA within the ribosome remains a challenging problem . Such positioning is critical to understanding ribosome function, as various rRNA regions interact to form suitable binding sites for ligands, such as tRNA and mRNA . We have used phenanthroline, a chemical nuclease, as a proximity probe, to help elucidate the regions of rRNA that are near neighbors of the stem-loop structure centering at nt 790 in the 16S rRNA of the Escherichia coli 30S ribosomal subunit . Using phenanthroline covalently attached to a DNA oligomer complementary to nt 787-795, we found that nt 582-584, 693-694, 787-790, and 795-797 were cleaved robustly and must lie within about 15 A of the tethered site at the 5' end of the DNA oligomer, which is adjacent to nt 795 of 16S rRNA. J Immunol Methods, 1999 Jun 24, 226(1-2), 179 - 88 Intracellular and cell surface displayed single-chain diabodies; Kontermann RE et al.; Intracellularly expressed antibody fragments have found various applications in therapy by virtue of their ability to inhibit the function of cellular proteins or interfere with subcellular trafficking . Bivalent antibody fragments might further improve this inhibitory potential by increasing the functional affinity and bispecific antibody fragments may also be useful for the intracellular retargeting of molecules . Here, we have evaluated the functional expression of intracellular diabodies . A previously constructed secreted bispecific single-chain diabody directed against carcinoembryonic antigen and Escherichia coli beta-galactosidase was modified for subcellular targeting to the cell surface membrane, endoplasmic reticulum, mitochondria, cytoplasm, and nucleus . Subcellular localisation was analysed by immunofluorescence, and the assembly of functional antibodies was analysed by binding of beta-galactosidase to the antibody fragment and subsequent substrate conversion . Bispecific single-chain diabodies could be directed to all subcellular compartments analysed . However, functional assembly was only observed for single-chain diabodies retained in the endoplasmic reticulum or displayed in the cell membrane while no antigen binding activity was seen with diabodies directed to the cytoplasm, nucleus, or mitochondria . The results demonstrate the functional expression of bispecific recombinant antibody fragments in the secretory pathway and integration into the plasma membrane of mammalian cells. Annu Rev Biophys Biomol Struct, 1999, 28, 205 - 34 Rotational coupling in the F0F1 ATP synthase; Nakamoto RK et al.; The F0F1 ATP synthase is a large multisubunit complex that couples translocation of protons down an electrochemical gradient to the synthesis of ATP . Recent advances in structural analyses have led to the demonstration that the enzyme utilizes a rotational catalytic mechanism . Kinetic and biochemical evidence is consistent with the expected equal participation of the three catalytic sites in the alpha 3 beta 3 hexamer, which operate in sequential, cooperative reaction pathways . The rotation of the core gamma subunit plays critical roles in establishing the conformation of the sites and the cooperative interactions . Mutational analyses have shown that the rotor subunits are responsible for coupling and in doing so transmit specific conformational information between transport and catalysis. Indian J Biochem Biophys, 1998 Oct, 35(5), 284 - 90 Cell surface alterations induced by methylene blue and direct electric current in Escherichia coli; Holandino C et al.; Cell surface properties, including hydrophobicity, zeta potential, carbohydrate and fatty acid components, were altered on treatment of E . coli K12 with methylene blue (MB) and direct electric current (DC) . The treatment of fimbriated E . coli cells with MB greatly increased the agglutination of yeast cells when compared to untreated bacteria . However, this increased agglutination was markedly reduced when the bacteria were treated with MB plus DC . These results suggest that MB modifies cell surface components in the absence of light and these alterations are more pronounced when cells are treated simultaneously with MB and DC. J Nat Toxins, 1999 Jun, 8(2), 221 - 33 Cobrotoxin: structure and function; Yang CC; Cobrotoxin is the main neurotoxic protein isolated from the venom of Taiwan cobra Naja naja atra . It is a small, basic protein consisting of a single polypeptide chain of 62 amino acids, cross-linked by four disulfide bonds . The disulfide bonds and Tyr-25 which is buried in the molecule form a central core to maintain and stabilize the active conformation of the toxin . Selective and stepwise chemical modifications of cobrotoxin indicate that at least two cationic groups, an epsilon-amino group of Lys-47 and a guanidino group of Arg-33, both of which are common to all known postsynaptic neurotoxins, held at a certain critical distance in the molecule, are functionally important for its neuromuscular blocking activity . The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription polymerase chain reaction . Sequencing several clones containing about 0.5 Kb DNA inserts contained a complete and full-length reading frame of 249 base pairs covering a precursor of cobrotoxin gene with a deduced mature protein sequence of 62 amino acids which are identical to the amino acid sequence of cobrotoxin and a 21 amino acid segment of signal peptide . Expression of cobrotoxin in E . coli vector generated a polypeptide which can cross-react with the antisera against the native cobrotoxin. Exp Mol Med, 1999 Jun 30, 31(2), 101 - 7 Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin; Park EJ et al.; Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals . In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112 . The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT . In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo . Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT . These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself . Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E . coli. Exp Mol Med, 1999 Jun 30, 31(2), 64 - 9 Asp 280 residue is important in the activity of the Escherichia coli leader peptidase; Sung M et al.; Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins . It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space . Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E . coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282 . Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine . All of mutants purified with nickel affinity chromatography were inactive using in vitro assay . It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid . These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket. Biochem Mol Biol Int, 1999 Jun, 47(6), 1009 - 18 Overexpression of human testis antigens in Escherichia coli host cells is influenced by site of expression and the induction temperature; Mburu DN et al.; A panel of twenty human testis cDNA clones were expressed in an Escherichia coli expression system and six clones were found to express identifiable fusion polypeptides . Expression was found to be influenced not only by the site of localization of the polypeptide in the host cells, but also by the temperature used for induction . This emphasized the need for cytoplasmic and periplasmic expression of new antigens of unknown properties, as well as the use of temperatures of 30 degrees C or lower . A majority of the expressed polypeptides were mainly in an insoluble form . By reducing the induction temperature to 30 degrees C production of the soluble fraction was further improved. Mol Cell Biol, 1999 Aug, 19(8), 5823 - 32 A novel 14-base-pair regulatory element is essential for in vivo expression of murine beta4-galactosyltransferase-I in late pachytene spermatocytes and round spermatids; Charron M et al.; During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site . We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N . L . Shaper, A . Harduin-Lepers, and J . H . Shaper, J . Biol . Chem . 269:25165-25171, 1994) . We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences . This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1) . The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors . Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene . Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo. J Biol Chem, 1999 Jul 23, 274(30), 21362 - 8 Biophysical and functional characterization of full-length, recombinant human tissue inhibitor of metalloproteinases-2 (TIMP-2) produced in Escherichia coli . Comparison of wild type and amino-terminal alanine appended variant with implications for the mechanism of TIMP functions; Wingfield PT et al.; Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes . The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases . TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity . TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized . The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala+TIMP-2) was inactive . Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity . This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP . The Ala+TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein . These findings demonstrate that Ala+TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs. J Biol Chem, 1999 Jul 23, 274(30), 21342 - 8 Mutational analysis of the hydrolytic activity of yeast RNA polymerase III; Bobkova EV et al.; For 25 mutant alleles of ret1, encoding the second largest subunit of yeast RNA polymerase III, we have studied the polymerase III nuclease activity, measuring both the total yield and dinucleotide product composition . Mutations affecting amino acids 309-325 gave slightly elevated nuclease activity . In region 367-376, two mutations gave 12-15-fold increased nuclease activity . Our results do not support the catalytic role in nuclease activity proposed for the conserved DDRD motif in this region (Shirai, T., and Go, M . (1991) Proc . Natl . Acad . Sci . U . S . A . 88, 9056-9060) . Mutations centered on a basic region from amino acids 480 to 490, which aligns with Escherichia coli beta-subunit sequences between Rif(r) clusters I and II, produce changes in the relative yields of A- and G-containing dinucleotides . Four such mutant polymerases pause during elongation at GPy sequences and, in addition, have a reduced frequency of termination at T(5) terminator sequences . We propose that the side chains of these mutationally altered amino acids are in direct contact with bases in the RNA-DNA hybrid very near the growing 3'-end . Two mutations in domain I near the C terminus produced very large increases in exonuclease activity and strongly increased termination, suggesting that this region also contacts the nascent RNA in the hybrid region. J Biol Chem, 1999 Jul 23, 274(30), 21326 - 34 Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase; Fisher AB et al.; This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin . Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl- or sn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH . Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM(-1) min(-1)) were k(1) = 1-3 x 10(5) and k(2) = 4-11 x 10(4) . In the presence of 0.36 mM GSH, apparent K(m) was 120-130 microM and apparent V(max) was 1.5-1.6 micromol/min/mg of protein . Assays with H(2)O(2) and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides . Maximal enzymatic activity was at pH 7-8 . Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K(i) 4 miroM . The enzyme had no GSH S-transferase activity . Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases . The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da . These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides . Thus, this enzyme may be an important component of cellular antioxidant defense systems. J Biol Chem, 1999 Jul 23, 274(30), 21251 - 6 On the maximum size of proteins to stay and fold in the cavity of GroEL underneath GroES; Sakikawa C et al.; GroEL encapsulates non-native protein in a folding cage underneath GroES (cis-cavity) . Here we report the maximum size of the non-native protein to stay and fold in the cis-cavity . Using total soluble proteins of Escherichia coli in denatured state as binding substrates and protease resistance as the measure of polypeptide held in the cis-cavity, it was estimated that the cis-cavity can accommodate up to approximately 57-kDa non-native proteins . To know if a protein with nearly the maximum size can complete folding in the cis-cavity, we made a 54-kDa protein in which green fluorescent protein (GFP) and its blue fluorescent variant were fused tandem . This fusion protein was captured in the cis-cavity, and folding occurred there . Fluorescence resonance energy transfer proved that both GFP and blue fluorescent protein moieties of the same fused protein were able to fold into native structures in the cis-cavity . Consistently, simulated packing of crystal structures shows that two native GFPs just fit in the cis-cavity . A fusion protein of three GFPs (82 kDa) was also attempted, but, as expected, it was not captured in the cis-cavity. J Biol Chem, 1999 Jul 23, 274(30), 21186 - 90 Visual arrestin activity may be regulated by self-association; Schubert C et al.; Visual arrestin is the protein responsible for rapid quenching of G-protein-coupled receptor signaling . Arrestin exists as a latent inhibitor which must be 'activated' upon contact with a phosphorylated receptor . X-ray crystal structures of visual arrestin exhibit a tetrameric arrangement wherein an asymmetric dimer with an extensive interface between conformationally different subunits is related to a second asymmetric dimer by a local two-fold rotation axis . To test the biological relevance of this molecular organization in solution, we carried out a sedimentation equilibrium analysis of arrestin at both crystallographic and physiological protein concentrations . While the tetrameric form can exist at the high concentrations used in crystallography experiments, we find that arrestin participates in a monomer/dimer equilibrium at concentrations more likely to be physiologically relevant . Solution interaction analysis of a proteolytically modified, constitutively active form of arrestin shows diminished dimerization . We propose that self-association of arrestin may provide a mechanism for regulation of arrestin activity by (i) ensuring an adequate supply for rapid quenching of the visual signal and (ii) limiting the availability of active monomeric species, thereby preventing inappropriate signal termination. J Biol Chem, 1999 Jul 23, 274(30), 21114 - 20 Nucleoside hydrolase from Leishmania major . Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-Ã¥ crystal structure; Shi W et al.; Protozoan parasites lack the pathway of the de novo synthesis of purines and depend on host-derived nucleosides and nucleotides to salvage purines for DNA and RNA synthesis . Nucleoside hydrolase is a central enzyme in the purine salvage pathway and represents a prime target for the development of anti-parasitic drugs . The full-length cDNA for nucleoside hydrolase from Leishmania major was cloned and sequence analysis revealed that the L . major nucleoside hydrolase shares 78% sequence identity with the nonspecific nucleoside hydrolase from Crithidia fasciculata . The L . major enzyme was overexpressed in Escherichia coli and purified to over 95% homogeneity . The L . major nucleoside hydrolase was identified as a nonspecific nucleoside hydrolase since it demonstrates the characteristics: 1) efficient utilization of p-nitrophenyl beta-D-ribofuranoside as a substrate; 2) recognition of both inosine and uridine nucleosides as favored substrates; and 3) significant activity with all of the naturally occurring purine and pyrimidine nucleosides . The crystal structure of the L . major nucleoside hydrolase revealed a bound Ca(2+) ion in the active site with five oxygen ligands from Asp-10, Asp-15 (bidentate), Thr-126 (carbonyl), and Asp-241 . The structure is similar to the C . fasciculata IU-nucleoside hydrolase apoenzyme . Despite the similarities, the catalytic specificities differ substantially . Relative values of k(cat) for the L . major enzyme with inosine, adenosine, guanosine, uridine, and cytidine as substrates are 100, 0.5, 0.5, 27 and 0.3; while those for the enzyme from C . fasciculata are 100, 15, 14, 510, and 36 for the same substrates . Iminoribitol analogues of the transition state are nanomolar inhibitors . The results provide new information for purine and pyrimidine salvage pathways in Leishmania. J Biol Chem, 1999 Jul 23, 274(30), 21037 - 43 Sequence requirements of the GPNG beta-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening; Wentzel A et al.; The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot . Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure . Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22-25 comprising a beta-turn in native EETI-II is a possible candidate for a folding initiation site . We generated a molecular repertoire of EETI-II variants with variegated 22-25 tetrapeptide sequences and presented these proteins on the outer membrane of Escherichia coli cells via fusion to the Iga(beta) autotransporter . Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting . At least 1-5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides . Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in beta-turns . The folding pathway of two selected variants, EETI-beta(NEDE) and EETI-beta(TNNK), was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate . Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture. J Biol Chem, 1999 Jul 23, 274(30), 21023 - 8 Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus; Tuininga JE et al.; Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases . Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P . furiosus . Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase . The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases . The ADP-dependent phosphofructokinase was purified and characterized . The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits . It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5 . As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent . The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively . The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases . ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively. Chem Res Toxicol, 1999 Jul, 12(7), 630 - 8 Synthesis and biochemical properties of cyanuric acid nucleoside-containing DNA oligomers; Gasparutto D et al.; 1-(2-Deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid (cyanuric acid nucleoside, dY) (1) has been shown to be formed upon exposure of DNA components to ionizing radiation and excited photosensitizers . To investigate the biological and structural significance of dY residue in DNA, the latter modified 2'-deoxynucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides (ODNs) . This was achieved in good yields using the phosphoramidite approach . For this purpose, a convenient glycosylation method involving 3,5-protected 2-deoxyribofuranoside chloride and cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine) was devised . The anomeric mixture of modified 2'-deoxyribonucleosides (1/2 alpha/beta) was resolved by silica gel purification of the 5'-O-dimethoxytritylated derivatives, and then, phosphitylation afforded the desired beta-phosphoramidite monomer (5) . After solid-phase condensation and final deprotection, the purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry . The presence of cyanuric acid nucleoside in a 14-mer was found to have destabilizing effects on the double-stranded DNA fragment as inferred from melting temperature measurements . The piperidine test applied to dY-containing ODNs supported the high stability of cyanuric acid nucleoside inserted into the oligonucleotide chain . Several enzymatic experiments aimed at determining the biological features of such a DNA lesion were carried out . Thus, processing of dY by nuclease P(1), snake venom phosphodiesterase (SVPDE), calf spleen phosphodiesterase (CSPDE), and repair enzymes, including Escherichia coli endonuclease III (endo III) and Fapy glycosylase (Fpg), was investigated . Finally, a 22-mer ODN bearing a cyanuric acid residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of E . coli polymerase I. Chem Res Toxicol, 1999 Jul, 12(7), 617 - 22 Detoxification of methylglyoxal by the nucleophilic bidentate, phenylacylthiazolium bromide; Ferguson GP et al.; Dicarbonyl-containing compounds such as methylglyoxal (MG) are toxic to cells since they can interact with the nucleophilic centers of macromolecules . MG has been found to accumulate during hyperglycemia, and it has been suggested that this reactive dicarbonyl may contribute to the tissue damage and long-term complications of diabetes . A sensitive bacterial assay for investigating the ability of nucleophilic agents to interact with and detoxify MG has been developed . This assay utilizes the sensitivity of exponential phase cells of an Escherichia coli double mutant lacking the KefB and KefC potassium channels toward MG . The bidentate nucleophile, phenylacylthiazolium bromide (PTB), was found to protect and allow the growth of E . coli cells in the presence of either externally added or endogenously produced MG . In the absence of PTB, growth was completely inhibited and rapid cell death occurred under these conditions . PTB protected E . coli against MG almost as well as aminoguanidine, a compound shown previously to be involved in detoxification . The level of protection by PTB against MG was much greater than for the endogenous nucleophile, glutathione . These data suggested that PTB could interact with and detoxify MG . The mechanism of this interaction was characterized by NMR and mass spectroscopy. Hum Mutat, 1999, 13(6), 453 - 7 Four novel mutations in the cystathionine beta-synthase gene: effect of a second linked mutation on the severity of the homocystinuric phenotype; de Franchis R et al.; Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is frequently caused by missense mutations . In this article, we report four novel missense mutations in the CBS gene: 172C-->T (R58W) linked in cis with A114V; 376A-->G (M126V); 904G-->A (E302K); and 1006C-->T (R336C) . The CBS activity of the corresponding mutant enzymes expressed in Escherichia coli was greatly diminished, confirming the pathogenicity of these mutations . Western analysis showed that the R58W+A114V and M126V mutant enzymes were unstable in E . coli, while the E302K subunits were partially degraded to shorter products . Using site-directed mutagenesis we found that CBS containing either the R58W or A114V as the only mutations demonstrated 18% and 46% of normal activity, respectively . Both mutant forms of CBS were stable in E . coli . When these two mutations were expressed in cis, the resultant mutant protein exhibited activity 1.3% that of a control . All these in vitro results were in good agreement with the clinical manifestation in these patients . The Italian patient 2241, an A114V+R58W/M126V compound heterozygote, exhibited severe pyridoxine nonresponsive homocystinuria, while another Italian patient 2242, with an A114V/E302K genotype, responded to pyridoxine treatment and had a much milder phenotype . The third patient 3064, an English compound heterozygote for two severe mutations R336C and G307S, was B6 nonresponsive . This report of a ninth homocystinuric allele carrying two mutations in cis raises the possibility that double mutant alleles may be underestimated in homocystinuric patients . In this context, a search for additional mutations in cis may sometimes be necessary to establish a good genotype-phenotype relationship. Am J Physiol, 1999 Jul, 277(1 Pt 2), H371 - 9 Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries; Aschner JL et al.; We determined the expression and functional correlate of in vitro transfection with a recombinant adenoviral vector encoding the gene for bovine endothelial nitric oxide synthase (AdCMVeNOS) or Escherichia coli beta-galactosidase (AdCMVLacZ) in pulmonary endothelial cells (EC), vascular smooth muscle cells (VSMC), and pulmonary arteries (PA) from newborn piglets . AdCMVeNOS and AdCMVeLacZ vectors, grown in 293-cell monolayers, were purified by double-cesium gradient ultracentrifugation . Cell cultures and PA were incubated with increasing vector titers for 30 or 60 min, followed by incubation in fresh medium for 18 h at 37 degrees C . LacZ expression was assessed by histochemical staining; eNOS expression was evaluated by Western blot analysis . Functional eNOS expression was determined by measurement of cGMP and quantification of the relaxation response to bradykinin (BK) . In PA, LacZ transgene expression was preferentially localized to the adventitia and endothelium . Increased eNOS protein expression was observed in EC and VSMC transfected with AdCMVeNOS . Functional studies revealed increased cGMP abundance in cultured cells and enhanced relaxation to BK in AdCMVeNOS-transfected PA . These studies demonstrate that gene transfer with AdCMVeNOS results in functional expression and altered vasoactive responses in the neonatal pulmonary vasculature . Gene transfer with replication-deficient adenovirus vectors is a useful tool for the study of targeted genes in vascular biology. Eur J Pharmacol, 1999 May 28, 373(1), 41 - 9 Pentoxifylline improves circulatory failure and survival in murine models of endotoxaemia; Wu CC et al.; Pentoxifylline, a methylxanthine derivative, has been widely used to improve erythrocyte deformability and capillary blood circulation in patients with claudication and cerebrovascular disorders as well as in animals with sepsis . Here, we investigate the effects of pentoxifylline on the hypotension, vascular hyporeactivity to noradrenaline, release of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), and inducible NO synthase protein expression in a rat model of circulatory shock induced by bacterial endotoxin (Escherichia coli lipopolysaccharide) . In addition, we have evaluated the effect of pentoxifylline on the 36-h survival rate in a murine model of endotoxaemia . Male Wistar-Kyoto rats were anaesthetised and instrumented for the measurement of mean arterial pressure and heart rate . Injection of lipopolysaccharide (10 mg/kg, i.v.) resulted in a significant fall in mean arterial pressure and an increase of heart rate . In contrast, animals pretreated with pentoxifylline (3 mg/kg, i.v., at 30 min prior to lipopolysaccharide) maintained a significantly higher mean arterial pressure but showed no effect on the tachycardia when compared to rats given only lipopolysaccharide (lipopolysaccharide-rats) . The pressor effect of noradrenaline (1 microg/kg, i.v.) was also significantly reduced after the treatment of rats with lipopolysaccharide . Similarly, rings of thoracic aorta obtained from lipopolysaccharide-rats showed a significant reduction in the contractile responses elicited by noradrenaline (1 microM) . Pretreatment of lipopolysaccharide-rats with pentoxifylline partially, but significantly, prevented this lipopolysaccharide-induced hyporeactivity to noradrenaline in vivo and ex vivo . The injection of lipopolysaccharide resulted in bell-shape changes in plasma TNF-alpha level which reached a peak at 60 min, whereas the effect of lipopolysaccharide on the plasma level of nitrate (an indicator of NO formation) was increased in a time-dependent manner . This increase of both TNF-alpha and nitrate levels induced by lipopolysaccharide was significantly reduced in lipopolysaccharide-rats pretreated with pentoxifylline . Endotoxaemia for 240 min caused a significantly increased protein expression of inducible NO synthase in the lung . In lipopolysaccharide-rats pretreated with pentoxifylline, inducible NO synthase protein expression in lung homogenates was attenuated by 48 +/- 5% . Treatment of conscious mice with a high dose of endotoxin (60 mg/kg, i.p.) resulted in a survival rate of only 10% at 36 h (n = 20) . However, therapeutic application of pentoxifylline (3 mg/kg, i.p . at 0, 6, 15 and 24 h after lipopolysaccharide) increased the 36-h survival to 35% (n = 20) . Thus, pentoxifylline protects against circulatory failure and improves survival in rodents with severe endotoxaemia . These effects may be due to inhibition of the release of TNF-alpha and of the induction of inducible NO synthase. Can J Microbiol, 1999 Mar, 45(3), 191 - 200 Growth properties of a folA null mutant of Escherichia coli K12; Herrington MB et al.; In Escherichia coli, dihydrofolate reductase is required for both the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced during the synthesis of thymidylate . The coding region of the dihydrofolate reductase gene, folA, was replaced with a kanamycin resistance determinant . Unlike earlier deletions, this mutation did not disrupt flanking genes . When the mutation was transferred into a wild-type strain and a thymidine-(thy) requiring strain, the resulting strains were viable but slow growing on rich medium . Both synthesized less folate than their parents, as judged by the incorporation of radioactive para-aminobenzoic acid . The derivative of the wild-type strain did not grow on any defined minimal media tested . In contrast, the derivative of the thy-requiring strain grew slowly on minimal medium with thy but exhibited auxotrophies on some combinations of supplements . These results suggest that when folates are limited, they can be distributed appropriately to folate-dependent biosynthetic reactions only under some conditions. J Inherit Metab Dis, 1999 Jun, 22(4), 414 - 27 4-Aminobutyrate aminotransferase (GABA-transaminase) deficiency; Medina-Kauwe LK et al.; 4-Aminobutyrate aminotransferase (GABA-transaminase, GABA-T, EC 2.6.1.19) deficiency (McKusick 137150), an inborn error of GABA degradation, has until now been documented in only a single Flemish child . Compared to the other defects of GABA degradation, succinic semialdehyde dehydrogenase (SSADH, EC 1.2.1.24) deficiency with > 150 patients (McKusick 271980) and pyridoxine-dependent seizures with > 100 patients ('putative' glutamic acid decarboxylase (GAD, EC 4.1.1.15) deficiency; McKusick 266100), GABA-T deficiency is very rare . We present a summary of the clinical, biochemical, enzymatic and molecular findings on the index proband, and a recently identified second patient, with GABA-T deficiency . The phenotype in both included psychomotor retardation, hypotonia, hyperreflexia, lethargy, refractory seizures and electroencephalographic abnormalities . In an effort to elucidate the molecular basis of GABA-T deficiency, we isolated and characterized a 1.5 kb cDNA encoding human GABA-T, in addition to a 41 kb genomic clone which encompassed the GABA-T coding region . Standard methods of cloning and sequencing revealed an A-to-G transition at nucleotide 754 of the coding region in lymphoblast cDNAs derived from the index proband . This mutation resulted in substitution of an invariant arginine at amino acid 220 by lysine . Expression of the mutant in E . coli, followed by isolation and enzymatic characterization of the recombinant protein, revealed an enzyme whose Vmax was reduced to 25% of wild-type activity . The patient and father were heterozygous for this allele; the second allele in the patient remains unidentified . Genomic Southern analysis revealed that the second proband most likely harbours a deletion in the 3' region of the GABA-T gene. Biotechniques, 1999 Jul, 27(1), 164 - 70, 172, 175 pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis; Boyd AC et al.; A modified bacterial artificial chromosome (BAC) vector, pSURF-2, adapted for the selective subcloning of yeast artificial chromosome (YAC) sequences was constructed . DH10B-U, a pyrF derivative of the highly transformable E . coli strain DH10B was also constructed and used for the detection of Ura+ recombinants carrying DNA linked to YAC right arms . The vector's properties were illustrated in two main ways . (i) An intact 25-kb YAC containing a mouse tyrosinase minigene was cloned into pSURF-2 . Appropriately spliced tyrosinase RNA was detected by reverse transcription (RT)-PCR in extracts of cells transiently lipofected with the cloned YAC . (ii) Cells expressing human cystic fibrosis transmembrane conductance regulator (CFTR) from an integrated pSURF-2 recombinant containing a cDNA expression cassette were selected using the hygromycin-resistance (HyTK) marker of the vector and characterized by RT-PCR and immunoprecipitation . The unique I-SceI site and HyTK marker of pSURF-2 are designed to facilitate subsequent functional studies of cloned DNA. Biotechniques, 1999 Jul, 27(1), 110 - 4, 116, 118-20 Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein; Southworth MW et al.; The Mycobacterium xenopi gyrase A mini-intein has been engineered to yield a controllable N-terminal or C-terminal, single-splice-junction autocleavage element . When combined with an affinity tag, these modified mini-inteins can be used to purify target proteins after a single combined chromatography/cleavage step . Cleavage at the intein N terminus was induced with thiol reagents, while cleavage at the intein C terminus was induced by a temperature shift to 16 degrees-25 degrees C . Different preferences for the residue immediately preceding the intein were observed during thiol-induced, N-terminal splice-junction cleavage of the M . xenopi gyrase A mini-intein vs . the Saccharomyces cerevisiae vacuolar ATPase, subunit A (VMA) intein present in the IMPACT purification system . Furthermore, the M . xenopi gyrase A mini-intein C-terminal autocleavage vector allows isolation of polypeptides with N-terminal cysteine residues that are active in the Intein Mediated Protein Ligation method of protein semisynthesis. Biotechniques, 1999 Jul, 27(1), 86 - 8, 92-4 Application of the green fluorescent protein as a reporter for Ace1-based, two-hybrid studies; Mayer G et al.; The two-hybrid system in Saccharomyces cerevisiae is a genetic approach for the detection of of protein-protein interactions in vivo . This technology relies on the the activity of separated DNA-binding and transactivation domains of specific transcription factors to reconstitute an active transcription factor complex if interacting proteins are fused to these domains . Interactions are consequently detected through the activity of reporter genes . The two-hybrid technology has been successfully applied for the determination of interactions between numerous proteins of several organisms . Conventional reporter systems, such as the beta-galacatosidase from Escherichia coli, suffer from a variety of drawbacks, including the requirement for external substrates . In this report, we describe an alternative version of the two hybrid system using the combined advantages of the copper-inducible transcription factor Acel together with the yeast metallothionein gene CUP1 and the green fluorescence protein from aquatic invertebrates as reporters . This technique allows the copper-dependent monitoring of protein-protein interactions in living yeast cells. Biochim Biophys Acta, 1999 Jul 13, 1432(2), 401 - 5 Effect of redox state on unfolding energetics of heme proteins; Wittung-Stafshede P; Both the enthalpic and entropic contributions to unfolding of three heme proteins, cytochrome b(562), cytochrome c and myoglobin, are larger for the reduced than for the oxidized form . Thus, the higher thermodynamic stability of a reduced, as compared to an oxidized, heme protein is the net result of a large increase of favorable enthalpy and a small increase in unfavorable entropy . Upon comparing the unfolding energetics of the heme proteins to those of other single-domain proteins I find that protein length is the primary determinant of the thermodynamics. Biochim Biophys Acta, 1999 Jul 13, 1432(2), 396 - 400 Fluorimetric detection of enzymatic activity associated with the human tumor suppressor Fhit protein; Asensio AC et al.; The human tumor suppressor Fhit protein exhibits diadenosine triphosphatase activity, hydrolyzing Ap(3)A to AMP and ADP . We report that Fhit protein efficiently cleaves the fluorogenic Ap(3)A analog diethenoadenosine triphosphate giving support to establish a simple fluorimetric assay for quantification of Fhit enzyme . Fluorimetric assays were initially tested to demonstrate that diethyl pyrocarbonate and suramin inhibit Fhit enzyme. Biochim Biophys Acta, 1999 Jul 13, 1432(2), 356 - 70 Ca(2+) and Zn(2+) binding properties of peptide substrates of vertebrate collagenase, MMP-1; Vettakkorumakankav NN et al.; To understand the role of Ca(2+) in vertebrate in the structure and action of collagenase, we have examined peptides that interact with recombinant human fibroblast collagenase for their affinities towards Ca(2+) and Zn(2+) in a non-polar solvent . Two of the peptides, GPQGIAGQ and GNVGLAGA, had sequences in collagen which are, respectively, cleaved and not cleaved by collagenase . A third peptide, PSYFLNAG, had a collagenase-cleaved sequence in ovostatin, a globular protein substrate . Peptides TVGCEECTV and CLPREPGL were derived from TIMP-1; the former competitively inhibits collagenase while the latter does not . The relative rates of hydrolysis of the peptides by collagenase had the order GPQGIAGQ>PSYFLNAG>GNVGLAGA . Circular dichroism spectral data in trifluoroethanol showed that while the TIMP control peptide, CLPREPGL, bound only Zn(2+), the other four peptides bound both Ca(2+) and Zn(2+) with definite stoichiometries . Ca(2+) could displace Zn(2+) in the substrate peptides while Zn(2+) displaced Ca(2+) in the TIMP peptide . GPQGIAGQ, PSYFLNAG and TVGCEECTV formed peptide:Ca(2+):Zn(2+) ternary complexes . Our results suggest that both collagen and globular protein substrates of collagenase may bind Ca(2+) and Zn(2+) in the enzyme's active site . This, in turn, may account for the known importance of the non-catalytic Ca(2+) and Zn(2+) in collagenase activity. Biochim Biophys Acta, 1999 Jul 13, 1432(2), 324 - 32 Structure and stability of recombinant protein depend on the extra N-terminal methionine residue: S6 permutein from direct and fusion expression systems; Uversky VN et al.; Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively . The product of direct expression contained the extra N-terminal methionine residue . The structural properties and conformational stability of these permuteins were compared using 1-D (1)H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding . A pronounced difference in all the parameters studied has been demonstrated . This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence. Biochim Biophys Acta, 1999 Jul 13, 1432(2), 286 - 92 Formation of betaA3/betaB2-crystallin mixed complexes: involvement of N- and C-terminal extensions; Werten PJ et al.; The sequence extensions of the beta-crystallin subunits have been suggested to play an important role in the oligomerization of these eye lens proteins . This, in turn, may contribute to maintaining lens transparency and proper light refraction . In homo-dimers of the betaA3- and betaB2-crystallin subunits, these extensions have been shown by (1)H-NMR spectroscopy to be solvent-exposed and highly flexible . In this study, we show that betaA3- and betaB2-crystallins spontaneously form mixed betaA3/betaB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml(-1)) and tetrameric at higher protein concentrations . (1)H-NMR spectroscopy reveals that in the betaA3/betaB2-crystallin tetramer, the N-terminal extensions of betaA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of betaB2-crystallin lose their flexibility . We conclude that both extensions of betaB2-crystallin are involved in protein-protein interactions in the betaA3/betaB2-crystallin hetero-tetramer . The extensions may stabilize and perhaps promote the formation of this mixed complex. Eur J Biochem, 1999 Jul, 263(2), 526 - 33 Molecular cloning and functional characterization of a snake venom metalloprotease; Jeon OH et al.; A cDNA clone, MT-d, encoding metalloprotease precursor was isolated from snake (Agkistrodon halys brevicaudus) venom gland cDNA library . MT-d-I protein containing both metalloprotease and disintegrin domains, and MT-d-II protein containing the metalloprotease domain only were expressed in Escherichia coli and refolded successfully into their functional forms . Each of the refolded enzyme species exhibited distinct substrate specificity . Proteolytic activity of the MT-d-1 was able to hydrolyse type I gelatin, type-III and V collagens in contrast with the catalytic function of MT-d-II . MT-d-I protein having metalloprotease activity was also able to inhibit platelet aggregation . Functionally active MT-d-I protein underwent autoproteolytic processing in vitro to produce metalloprotease and disintegrin; this processing was accompanied by significant changes in the substrate specificity of the enzyme activity . Experimental evidence strongly suggests that the disintegrin domain in the metalloprotease precursor modulates the catalytic function of the enzyme in hydrolysing extracellular matrix proteins. Eur J Biochem, 1999 Jul, 263(2), 455 - 63 Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins; Emmerich R et al.; Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox-responsive transcriptional activator NifA which is encoded in the fixR-nifA operon . Basal expression of this operon depends on the response regulator RegR and a DNA element located around position -68 in the fixR-nifA promoter region . To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity-purified RegR and a truncated soluble variant of RegS (RegS(C)), both as N-terminally His(6)-tagged proteins . RegS(C) autophosphorylated when incubated with {gamma-(32)P}ATP, and it catalyzed the transfer of the phosphoryl label to RegR . The phosphorylated form of RegS(C) exhibited phosphatase activity on RegR-phosphate . Chemical stability tests and site-specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues . Competition experiments with isolated domains demonstrated that the N-terminal but not the C-terminal domain of RegR interacts with RegS(C) . Band-shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA-binding activity compared with dephosphorylated RegR or the mutant protein RegR-D63N, which cannot be phosphorylated . In conclusion, the RegSR proteins of B . japonicum exhibit functional properties in vitro that are typical of two-component regulatory systems. Eur J Biochem, 1999 Jul, 263(2), 430 - 7 Refolding of recombinant alpha and beta subunits of the Rhodospirillum rubrum F(0)F(1) ATP synthase into functional monomers that reconstitute an active alpha(1)beta(1)-dimer; Du Z et al.; The alpha subunit from the Rhodospirillum rubrum F(0)F(1) ATP synthase (RrF(1)alpha) was over-expressed in unc operon-deleted Escherichia coli strains under various growth conditions only in insoluble inclusion bodies . The functional refolding of urea-solubilized RrF(1)alpha was followed by measuring its ability to stimulate the restoration of ATP synthesis and hydrolysis in beta-less R . rubrum chromatophores reconstituted with pure native or recombinant RrF(1)beta {Nathanson, L . & Gromet-Elhanan, Z . (1998) J . Biol . Chem . 273, 10933-10938} . The refolding efficiency was found to increase with decreasing RrF(1)alpha concentrations and required high concentrations of MgATP, saturating approximately 60% when 50 microgram protein.mL(-1) were refolded in presence of 50 mM MgATP . Size-exclusion HPLC of such refolded RrF(1)alpha revealed a 50-60% decrease in its aggregated form and a parallel appearance of its monomeric peak . RrF(1)beta refolded under identical conditions appeared almost exclusively as a monomer . This procedure enabled the isolation of large amounts of a stable RrF(1)alpha monomer, which stimulated the restoration of ATP synthesis and hydrolysis much more efficiently than the refolded alpha mixture, and bound ATP and ADP in a Mg-dependent manner . Incubation of both RrF(1)alpha and beta monomers, which by themselves had no ATPase activity, resulted in a parallel appearance of activity and assembled alpha(1)beta(1)-dimers, but showed no formation of alpha(3)beta(3)-hexamers . The RrF(1)-alpha(1)beta(1)-ATPase activity was, however, very similar to the activity observed in isolated native chloroplast CF(1)-alpha(3)beta(3), indicating that these dimers contain only the catalytic nucleotide-binding site at their alpha/beta interface . Their inability to associate into an alpha(3)beta(3)-hexamer seems therefore to reflect a much lower stability of the noncatalytic RrF(1) alpha/beta interface. Glycobiology, 1999 Aug, 9(8), 815 - 22 Role of a conserved acidic cluster in bovine beta1,4 galactosyltransferase-1 probed by mutagenesis of a bacterially expressed recombinant enzyme; Zhang Y et al.; The truncated catalytic domain of bovine beta1,4 galactosyltransferase-1 was expressed as inclusion bodies in E.coli and folded to generate 10-15 mg of active enzyme per liter of bacterial culture after extraction and purification under denaturing conditions . Mutations were introduced to investigate the roles of Trp312, Asp318, and Asp320, components of a highly conserved region of sequence in all known beta4GT-1 homologues that includes a cluster of acidic residues . Near and far UV CD spectra of the mutants indicate that the substitutions did not perturb the secondary and tertiary structure of beta4GT-1, and steady state kinetic studies indicate only minor effects on the response to an essential metal cofactor . However substitutions for the two aspartyl residues result in a reduction in catalytic efficiency of a magnitude that suggests they are important for catalysis . It seems possible that this anionic center may act in stabilizing a carbocation formed from the galactose component of the donor substrate in the transition state, reflecting a common reaction mechanism for beta-galactosyltransferase reactions. EMBO J, 1999 Jul 15, 18(14), 4049 - 59 Positioning of sigma(S), the stationary phase sigma factor, in Escherichia coli RNA polymerase-promoter open complexes; Colland F et al.; The sigma(S) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and is required for promoter recognition of many stationary phase genes . We have analysed open complexes of Esigma(S) RNA polymerase, using sigma(S) derivatives carrying single cysteine residues at nine different positions to which the reagent FeBABE has been tethered . All holoenzymes but one formed transcriptionally active open complexes at three different promoters (osmY, galP1 and lacUV5) . The chemical nuclease FeBABE can cleave DNA in proximity to the chelate . The overall cutting pattern of Esigma(S) open complexes does not depend on the nature of the promoter and is similar to that obtained with Esigma(70), but extends towards the downstream part of the promoter . The strongest cleavages are observed with FeBABE positioned on cysteines in regions 2.2 to 3.1 . In contrast to sigma(70), region 2.1 of sigma(S) appears to be far from DNA . Region 4.2 of sigma(S) appears less accessible than its counterpart in sigma(70) and FeBABE positioned in the turn of the helix-turn-helix (HTH) motif in region 4.2 reacts only weakly with the -35 promoter element . This provides a structural basis for the minor role of the -35 sequence in sigma(S)-dependent promoter recognition. J Toxicol Environ Health A, 1999 Jun 25, 57(4), 237 - 45 Sensitivities and gene-expressions of Escherichia coli mutants deficient in DNA repair and reactive oxygen species scavenging capacity exposed to natural sunlight; Yonezawa Y et al.; Sensitivities to sunlight of Escherichia coli mutants deficient in DNA repair capacities such as excision and recombination repair and their wild-type strain were compared . Higher sensitivities to sunlight were clearly observed in the mutant than wild-type strain, indicating that exposure to sunlight induces DNA damage which is repaired by DNA repair mechanisms . In order to assess the role of generation of reactive oxygen species (ROS) in DNA damage induced by exposure to natural sunlight, the kat-sod assay was performed using E . coli mutant strains deficient in ROS scavenging enzymes such as superoxide dismutase and/or catalase . Natural sunlight induced a significant generation of ROS, suggesting a possibility that sunlight induced ROS may be involved in DNA damage in bacterial cells. J Hepatol, 1999 Jun, 30(6), 1146 - 50 Peliosis hepatis with initial presentation as acute hepatic failure and intraperitoneal hemorrhage in children; Jacquemin E et al.; Peliosis hepatis, a condition characterized by the presence of blood-filled lacunar spaces in the liver, usually has a chronic presentation pattern and is mainly reported in adult patients in association with chronic wasting disorders and after administration of various drugs . The present report concerns two previously healthy young children in whom peliosis hepatis initially presented as acute hepatic failure and who had Escherichia coli pyelonephritis . Both patients had active intraperitoneal hemorrhage from the peliotic liver lesions, and liver ultrasonography showed multiple hypoechoic areas of different sizes, which in this context should suggest the diagnosis . One child died from hypovolemic shock and the other recovered . This study indicates that acute peliosis hepatis can be a serious life-threatening disease in children. Plant J, 1999 May, 18(4), 417 - 29 Two MAR DNA-binding proteins of the pea nuclear matrix identify a new class of DNA-binding proteins; Hatton D et al.; Four MAR-binding proteins of 60, 65, 70 and 72 kDa have been detected by South-Western blotting and isolated from pea nuclear matrices . Two cDNAs encoding the 60 and 65 kDa proteins (MARBP-1 and MARBP-2) were isolated from a pea leaf cDNA library by screening with a PCR product obtained using degenerate primers based on an amino acid sequence from the 60 kDa protein . The proteins of 560 and 550 amino acids are 86% identical and contain several KKD/E repeats near the C-terminus . Escherichia coli-expressed MARBP-1 specifically binds A/T-rich MAR DNA . The interaction of MARBP-1/MARBP-2 with MAR DNA involves novel DNA-binding motifs . The MARBP-1 and MARBP-2 genes are expressed in a range of pea tissues and are encoded by genes at different loci . MARBP-1 and MARBP-2 are homologous to yeast nucleolar proteins Nop56p and Nop58p, which are involved in ribosome biogenesis, and to similar highly conserved proteins in other eukaryotes and in archaebacteria . MARBP-1 and MARBP-2 may have multifunctional roles in chromatin organisation and ribosome biogenesis. Plant J, 1999 May, 18(4), 407 - 15 Developmental stage-specific multi-subunit plastid RNA polymerases (PEP) in wheat; Satoh J et al.; Most photosystem I and II plastid genes are transcribed by a plastid encoded Escherichia coli-like RNA polymerase (PEP) . In this study, we show that both promoter selectivity and light-dependency of PEP change dramatically during development in wheat leaves . In the leaf tip, psbA and psbD promoter activities are light induced, whilst psbC, psbE and 16S rRNA promoters do not function efficiently irrespective of light conditions . In contrast to the leaf tip, in the basal portion all PEP promoters studied function in the dark as well as the light, except for psbD . Using in vitro transcription, we found that PEP in the illuminated leaf tip can initiate transcription from the -35 destructed psbA promoter, but the -35 element is essential for transcription in the basal portion . There is an extended -10 element in the psbA promoter, recognized by the PEP in the illuminated leaf tip or purified sigma 70-type Escherichia coli RNA polymerase but not by the PEP in the leaf base . These results suggest that during wheat leaf development, PEP in the leaf base that is functional for most PEP promoters even in the dark is replaced by the light-dependent PEP selectively transcribing the psbA and psbD promoters. Anticancer Drug Des, 1999 Apr, 14(2), 93 - 106 Application of the ADEPT strategy to the MDR resistance in cancer chemotherapy; Desbene S et al.; New prodrugs consisting of a beta-D-glucuronic acid linked to a MDR reversal agent (verapamil, quinine or dipyridamole) through a self-immolative spacer were synthesized . Four of them were selected for their reduced cytoxicity and beta-glucuronidase enzymatic efficient hydrolysis . Combined use of these prodrugs with a beta-D-glucuronyl-spacer-doxorubicin (HMR1826) according to an ADEPT strategy restored in vitro the sensibility of a MDR resistant strain. Anal Chem, 1999 Jul 1, 71(13), 2334 - 9 Detection of double-stranded DNA by IR- and UV-MALDI mass spectrometry; Kirpekar F et al.; Double-stranded DNA ranging from 9 kDa to over 500 kDa were desorbed and analyzed by MALDI TOF mass spectrometry . IR-MALDI with glycerol as matrix yielded excellent results for larger double-stranded DNA by adjustment of the ionic strength through the addition of salts . Very little fragmentation and a routine sensitivity in the subpicomole range were observed in IR-MALDI when double-stranded analytes harboring 70 base pairs or more were probed . In the lower mass range (up to approximately 70 base pairs), UV-MALDI with 6-aza-2-thiothymine as matrix was the ionization method of choice because it allowed specific double-stranded complexes containing relatively few base pairs to be desorbed intactly . In this mode, an essentially quantitative detection of the double-stranded form was observed for a 70-mer . The UV-MALDI was accompanied by a significant fragmentation and a resulting reduced sensitivity and mass resolution . The methods described open MALDI-MS for the analysis of large DNA-DNA and DNA-protein complexes. J Clin Microbiol, 1999 Aug, 37(8), 2719 - 22 Molecular detection and identification of intimin alleles in pathogenic Escherichia coli by multiplex PCR; Reid SD et al.; A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli . The method was tested on 87 strains representing the diarrheagenic E . coli clones . The results show that the PCR assay accurately detects eae and resolves alleles encoding the alpha, beta, and gamma intimin variants. J Clin Microbiol, 1999 Aug, 37(8), 2576 - 80 Serological determination of hepatitis C virus subtypes 1a, 1b, 2a, 2b, 3a, and 4a by a recombinant immunoblot assay; Schroter M et al.; Serological determination of hepatitis C virus (HCV) subtypes has been hampered by the lack of suitable assays . Therefore, a recombinant immunoblot assay has been established for serological differentiation of HCV subtypes 1a, 1b, 2a, 2b, 3a, and 4a . It consists of recombinant HCV proteins from the NS-4 region propagated in Escherichia coli . To confirm the serotyping assay results, the results were compared with those obtained by nucleotide sequencing of the NS-5 region . Sera from 157 patients with chronic HCV infection were examined by this assay, and specific antibodies could be detected in 86% (n = 135) of them . The HCV genotype was determined correctly in all but one sample, and the subtypes determined by the serotyping assay corresponded to the HCV subtypes detected by nucleotide sequencing for 95% (n = 128) of the samples . These data indicate that HCV subtypes can be distinguished serologically . The assay that is described provides an easier means of identification of infection with different HCV subtypes for wider clinical and epidemiological applications. Virology, 1999 Jul 20, 260(1), 182 - 9 Effect of C-terminal mutations of alfalfa mosaic virus coat protein on dimer formation and assembly in vitro; Choi J et al.; The coat protein (CP) of alfalfa mosaic virus (AMV) strain 425 assembles to bacilliform or rod-shaped particles in the presence of nucleic acids or to T = 1 empty icosahedral particles in the absence of nucleic acids . To study the determinants of CP assembly, recombinant CPs (rCPs) that contained a (His)(6) region were expressed in Escherichia coli . Wt rCP and a mutant rCP, which lacked the last nine amino acids of the C terminus (amino acids 213-221), assembled to particles that were identical in electron micrographs . However, a mutant rCP, which lacked the last 18 amino acids of the C terminus (amino acids 204-221), did not assemble . Likewise, a mutant with alanine substitutions at W(191), F(197), and P(198) did not assemble . Furthermore rCP with a single alanine substitution at W(191) did not assemble, whereas the rCP, which had an arginine and an alanine substitution at A(196) and F(197), respectively, formed rod-shaped particles . The mutations that prevented assembly prevented dimer formation, which indicates that dimers are the minimal building blocks of particles . Our results indicate that two separate regions in the C terminus of AMV CP are critical for dimer formation and assembly and that changes in key amino acids in one of the regions affect both assembly and particle morphology . Biochem Biophys Res Commun, 1999 Jul 22, 261(1), 79 - 82 Functional consequences of a carboxyl terminal missense mutation Arg278Cys in human cardiac troponin T; Morimoto S et al.; A carboxyl terminal missense mutant Arg278Cys of human cardiac troponin T that causes familial hypertrophic cardiomyopathy was expressed in Escherichia coli, purified, and exchanged into rabbit cardiac skinned muscle fibers using a troponin exchange technique . Compared to the fibers exchanged with human cardiac wild-type troponin T, the fibers exchanged with the mutant Arg278Cys developed less maximum force with a decreased cooperativity and a slightly increased Ca(2+) sensitivity, resulting in a significant elevation of sub-half-maximal force . Since intact cardiac muscle is thought to never be activated beyond the half-maximum level, the results suggest that an enhanced myofilament response to Ca(2+) may be responsible for the pathogenesis of hypertrophic cardiomyopathy associated with this mutation . The results also provide the first evidence that the carboxyl terminal region of cardiac troponin T plays an important role probably through its interaction with tropomyosin in allowing troponin complex to inhibit the muscle contraction at low Ca(2+), in agreement with the hypothesis deduced from the previous studies on fast skeletal troponin T . Nat Struct Biol, 1999 Jul, 6(7), 648 - 51 Solution structure of a baculoviral inhibitor of apoptosis (IAP) repeat; Hinds MG et al.; Members of the inhibitor of apoptosis (IAP) family of proteins are able to inhibit cell death following viral infection, during development or in cell lines in vitro . All IAP proteins bear one or more baculoviral IAP repeats (BIRs) . Here we describe the solution structure of the third BIR domain from the mammalian IAP homolog B (MIHB/c-IAP-1) . The BIR domain has a novel fold that is stabilized by zinc tetrahedrally coordinated by one histidine and three cysteine residues . The structure consists of a series of short alpha-helices and turns with the zinc packed in an unusually hydrophobic environment created by residues that are highly conserved among all BIRs. Nat Struct Biol, 1999 Jul, 6(7), 643 - 7 EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome; Agrawal RK et al.; Cryo-electron microscopy has been used to visualize elongation factor G (EF-G) on the 70S ribosome in GDP and GTP states . GTP hydrolysis is required for binding of all the domains of EF-G to the pretranslocational complex and for the completion of translocation . In addition, large conformational changes have been identified in the ribosome . The head of the 30S subunit shifts toward the L1 protein side, and the L7/L12 stalk becomes bifurcated upon EF-G binding . Upon GTP hydrolysis, the bifurcation is reversed and an arc-like connection is formed between the base of the stalk and EF-G. Nat Struct Biol, 1999 Jul, 6(7), 628 - 33 Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone; Lee YH et al.; The crystal structure of recombinant TroA, a zinc-binding protein component of an ATP-binding cassette transport system in Treponema pallidum, was determined at a resolution of 1.8 A . The organization of the protein is largely similar to other periplasmic ligand-binding proteins (PLBP), in that two independent globular domains interact with each other to create a zinc-binding cleft between them . The structure has one bound zinc pentavalently coordinated to residues from both domains . Unlike previous PLBP structures that have an interdomain hinge composed of beta-strands, the N- and C-domains of TroA are linked by a single long backbone helix . This unique backbone helical conformation was possibly adopted to limit the hinge motion associated with ligand exchange. Nat Biotechnol, 1999 Jul, 17(7), 696 - 701 Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase; Matsumura I et al.; The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde . We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme . A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening . Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated . Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function . We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters. Nat Biotechnol, 1999 Jul, 17(7), 691 - 5 Rapid protein-folding assay using green fluorescent protein; Waldo GS et al.; Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein . We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP . Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone . We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E . coli into closely related proteins that fold robustly and are fully soluble and functional . This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution. Nat Biotechnol, 1999 Jul, 17(7), 683 - 90 An in vivo library-versus-library selection of optimized protein-protein interactions; Pelletier JN et al.; We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides . Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli . Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth . Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs . Using more weakly associating mDHFR fragments, we increased the stringency of selection . We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation . This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates . We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies. Comput Chem, 1999 Jun 15, 23(3-4), 387 - 400 A bayesian statistical algorithm for RNA secondary structure prediction; Ding Y et al.; A Bayesian approach for predicting RNA secondary structure that addresses the following three open issues is described: (1) the need for a representation of the full ensemble of probable structures; (2) the need to specify a fixed set of energy parameters; (3) the desire to make statistical inferences on all variables in the problem . It has recently been shown that Bayesian inference can be employed to relax or eliminate the need to specify the parameters of bioinformatics recursive algorithms and to give a statistical representation of the full ensemble of probable solutions with the incorporation of uncertainty in parameter values . In this paper, we make an initial exploration of these potential advantages of the Bayesian approach . We present a Bayesian algorithm that is based on stacking energy rules but relaxes the need to specify the parameters . The algorithm returns the exact posterior distribution of the number of destabilizing loops, stacking energy matrices, and secondary structures . The algorithm generates statistically representative structures from the full ensemble of probable secondary structures in exact proportion to the posterior probabilities . Once the forward recursions for the algorithm are completed, the backward recursive sampling executes in O(n) time, providing a very efficient approach for generating representative structures . We demonstrate the utility of the Bayesian approach with several tRNA sequences . The potential of the approach for predicting RNA secondary structures and presenting alternative structures is illustrated with applications to the Escherichia coli tRNA(Ala) sequence and the Xenopus laevis oocyte 5S rRNA sequence. Structure Fold Des, 1999 Jun 15, 7(6), 691 - 8 Crystal structure of the N-terminal domain of the DnaB hexameric helicase; Fass D et al.; BACKGROUND: The hexameric helicase DnaB unwinds the DNA duplex at the Escherichia coli chromosome replication fork . Although the mechanism by which DnaB both couples ATP hydrolysis to translocation along DNA and denatures the duplex is unknown, a change in the quaternary structure of the protein involving dimerization of the N-terminal domain has been observed and may occur during the enzymatic cycle . This N-terminal domain is required both for interaction with other proteins in the primosome and for DnaB helicase activity . Knowledge of the structure of this domain may contribute to an understanding of its role in DnaB function . RESULTS: We have determined the structure of the N-terminal domain of DnaB crystallographically . The structure is globular, highly helical and lacks a close structural relative in the database of known protein folds . Conserved residues and sites of dominant-negative mutations have structurally significant roles . Each asymmetric unit in the crystal contains two independent and identical copies of a dimer of the DnaB N-terminal domain . CONCLUSIONS: The large-scale domain or subunit reorientation that is seen in DnaB by electron microscopy might result from the formation of a true twofold symmetric dimer of N-terminal domains, while maintaining a head-to-tail arrangement of C-terminal domains . The N-terminal domain of DnaB is the first region of a hexameric DNA replicative helicase to be visualized at high resolution . Comparison of this structure to the analogous region of the Rho RNA/DNA helicase indicates that the N-terminal domains of these hexameric helicases are structurally dissimilar. Int J Parasitol, 1999 May, 29(5), 723 - 30 Expression and characterisation of a Plasmodium falciparum protein containing domains homologous to sarcalumenin and a tyrosine kinase substrate, eps15; McDaniel JP et al.; We have identified in Plasmodium falciparum a novel gene encoding a putative bi-functional protein, termed PfPast-1, from genomic and cDNA libraries . Analysis indicated that the sequence encodes a 62 kDa protein of 529 amino acid residues with two distinctive domains: a sarcalumenin-like domain of approximately 320 amino acids at the amino half of the molecule, which shares homology to a major sarcoplasmic reticulum lumenal protein, sarcalumenin, and an eps15 homology domain of about 90 amino acids located at the carboxyl terminus . The eps15 homology domain, first identified in a tyrosine kinase substrate, eps15, and found in increasing numbers of mammalian proteins, has recently been suggested as a protein-protein interaction domain involved in intracellular sorting . Genomic sequences encoding similar proteins containing both the sarcalumenin-like and eps15 homology domains have been identified in humans and Drosophila . RNA blot analysis revealed the presence of a single messenger RNA transcript approximately 3.7 kb in size, which is expressed in all the developmental stages examined with the highest level in extracellular gametes followed by erythrocytic asexual stages, and the lowest in the gametocytes . In the attempt to define its biological function, we have expressed a full-length recombinant PfPast-1 protein in Escherichia coli . Specific immune serum directed against the recombinant protein recognised a approximately 55 kDa protein in the parasite lysate . Further characterisation of PfPast-1 may help in elucidation of its functions in P . falciparum. Biotechnol Bioeng, 1999 Sep 5, 64(5), 590 - 8 On-line detection of acetate formation in Escherichia coli cultures using dissolved oxygen responses to feed transients; Akesson M et al.; Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation . It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate . Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived . The model predicts a clear change in the character of the transient response when acetate formation starts . The predicted effect was verified in fed-batch cultivations of E . coli TOPP1 and E . coli BL21(DE3), both before and after induction of recombinant protein production . It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction . On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding . Biochem Biophys Res Commun, 1999 Jul 14, 260(3), 676 - 81 Human prostate CYP3A5: identification of a unique 5'-untranslated sequence and characterization of purified recombinant protein; Yamakoshi Y et al.; We have isolated a cDNA clone coding for CYP3A5 from a human prostate cDNA library . The human prostate CYP3A5 cDNA had a unique 5'-untranslated sequence, suggesting that the prostate specific regulation of CYP3A5 is different from liver . Hybridization screening using a human genomic BAC library yielded four positive clones, two of which were shown to contain the unique 5'-untranslated sequence by Southern blot analysis . The CYP3A5 recombinant protein expressed in Escherichia coli using the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 4.4 nmol of P450/mg of protein . This P450 exhibited 6beta-hydroxylation activity toward both testosterone and progesterone . No polar metabolite of 5alpha-dihydrotestosterone (DHT) was detected . The apparent K(m) values for testosterone and progesterone 6beta-hydroxylation were 143 and 114 microM, respectively, with V(max) values of 0.48 and 0 . 21 nmol/min/nmol of P450, respectively . This is the first report that a particular form of P450, CYP3A5, has been isolated from human prostate and that the purified recombinant protein of CYP3A5 has been shown to be active in the metabolism of sex hormones . Biochem Biophys Res Commun, 1999 Jul 5, 260(2), 527 - 33 Real time analysis of interaction between inositol 1,4, 5-trisphosphate receptor type I and its ligand; Natsume T et al.; Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger that releases intracellular Ca(2+) by binding to its specific receptor, inositol 1,4,5-trisphosphate receptor (IP(3)R), in a wide range of cellular processes . We report here large-scale expression and purification of N-terminal 604 amino acids of IP(3)R type 1 (T604) expressed in E . coli, which contains the ligand binding domain . Surface plasmon resonance biosensor studies showed that purified T604 could bind to its ligands with binding specificity identical to that of full-length native IP(3)R type 1 . Kinetic parameters of T604 for IP(3) consisted of a fast association rate constant (K(ass) = 1.2 x 10(6) M(-1) s(-1)) and a rapid dissociation rate constant (k(diss) = 1 s(-1)), and the equilibrium dissociation constant was determined to be 336 nM, at 150 mM NaCl and pH 7.4 . However, association and dissociation patterns depended on the pH level and ionic strength . These results pave the way toward detail analysis of structure-function analysis of the ligand binding domain of IP(3)R type 1 for its ligands . Dev Genet, 1999, 25(1), 49 - 63 Gene trap screening using negative selection: identification of two tandem, differentially expressed loci with potential hematopoietic function; Cannon JP et al.; A fusion gene between Escherichia coli lacZ and herpes simplex virus thymidine kinase (HSV-tk) was constructed and used in a gene trap screen for hematopoietic loci in mouse embryonic stem (ES) cells . This gene, galtek, allowed both convenient histochemical detection of expression as well as ablation of expressing cells under 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) selection . Individual ES cell clones bearing gene trap insertions were differentiated in the presence of FIAU and scored for erythropoietic activity at day 9 of differentiation . Screening of a total of 235 independent gene trap lines identified one clone, F3, which consistently demonstrated FIAU-sensitive erythropoiesis during in vitro differentiation . Cloning of endogenous transcribed sequences from the F3 insertion site identified two distinct transcription units, F3-1 and F3-2, encoding mRNAs of approximately 1.3 kb and 3.35 kb, respectively . The transcripts were unrelated and did not exhibit similarity to known sequences . Both loci demonstrated similar relative levels of expression in the heart, testis, kidney, and lung as assessed by Northern blot hybridization . Whole-mount in situ hybridization detected F3-2 expression at multiple sites in embryonic day (E) 10.5 embryos, including the genital ridges, the aortic endothelium, and endothelium-associated cell clusters within the aortic lumen . Expression of F3-2 in the aortic endothelium and endothelium-associated clusters overlapped that of gata-2, a gene required for hematopoietic development . The FIAU sensitivity of hematopoiesis in F3 embryoid bodies may result from expression of galtek during the formation of early hematopoietic cells, directed by regulatory signals from one or both of these endogenous loci. Vaccine, 1999 May 4, 17(18), 2237 - 44 Modified M2 proteins produce heterotypic immunity against influenza A virus; Frace AM et al.; Vaccination with the influenza A transmembrane protein M2 provides enhanced viral clearance and recovery from influenza A virus infection in mice . However, the high degree of hydrophobicity of the protein limits its purification for vaccine purposes . We have attempted to alter the structure of the M2 protein to allow high level recombinant expression in Escherichia coli, to reduce its hydrophobicity and improve protein solubility, thus improving its properties as a vaccine subunit candidate . Constructs investigated include deletion of the transmembrane domain of M2 (residues 26-43) and an extended deletion (residues 26-55) . A full-length M2 protein was not pursued because of poor expression, even in the presence of amantadine . Expressed as glutathione S-transferase fusion proteins and used to vaccinate mice, either deletion construct was found to raise M2-specific serum antibodies and enhance viral clearance in mice challenged with homologous and heterologous influenza A viruses . Enzymatic cleavage from the GST fusion domain produces soluble protein giving similar results . The results demonstrate that large alterations of M2 protein structure can improve its isolation and purification characteristics without detracting from its immunogenic properties. Lab Anim Sci, 1999 Jun, 49(3), 254 - 9 Characterization of the interaction of Escherichia coli heat-stable enterotoxixn (STa) with its intestinal putative receptor in various age groups of mice, using flow cytometry and binding assays; Al-Majali AM et al.; BACKGROUND AND PURPOSE: Enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) is a major cause of diarrhea in young animals . Age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa was investigated . METHODS: Four age groups (2-day-, 1- and 2-week-, and 2-month-old) of Swiss Webster mice were studied (8 to 10 mice/group) . Flow cytometry and radiolabeled STa (125I-STa) assays were used as reliable quantitative measures for characterization of STa-enterocyte receptor interaction . RESULTS AND CONCLUSIONS: Interaction of STa with its putative receptor was stronger for enterocytes of 2-day-old mice . Scatchard analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at higher numbers on enterocytes from 2-day-old (7.2 nmol/mg) than older (0.30, 0.36, and 0.40 nmol/mg for 1-week-, 2-week-, and 2-month-old mice, respectively) . Additionally, receptors from 2-day-old mice had greater affinity for STa (Kd = 75 nM) than did receptors from older mice (Kd = 125, 1,430, and 1,111 nM for 1-week-, 2-week-, and 2-month-old mice, respectively) . Density of STa receptors on enterocytes and their affinity to STa may determine extent of binding and severity of the secretory response, and may explain the high susceptibility of newborn animals and human infants to STa-mediated diarrhea. FEBS Lett, 1999 Jun 18, 453(1-2), 215 - 8 Functional roles of the two cyclic AMP-dependent forms of cyclic AMP receptor protein from Escherichia coli; Mukhopadhyay J et al.; The cyclic AMP receptor protein activates transcription in Escherichia coli, only when complexed with cyclic AMP . The cyclic AMP receptor protein-cyclic AMP complex formed at low concentrations of cyclic AMP has a different conformation from either cyclic AMP receptor protein alone or its complex with cyclic AMP formed at high cyclic AMP concentrations . Various biophysical data suggest that the latter complex resembles free cyclic AMP receptor protein . We have examined the conformational and biological properties of cyclic AMP receptor protein as a function of cyclic AMP concentrations, using the gal operon of E . coli . A biphasic behavior is observed . It is shown that only the complex formed at lower concentrations of cyclic AMP is the transcriptionally active form . This difference between the complexes at different levels of cyclic AMP arises from a decreased ability of the cyclic AMP receptor protein-cyclic AMP complex at high cyclic AMP concentrations to bind to DNA at specific sites. FEBS Lett, 1999 Jun 18, 453(1-2), 179 - 82 Mutation of the mitochrondrially encoded ATPase 6 gene modeled in the ATP synthase of Escherichia coli; Ogilvie I et al.; Defects of respiratory chain protein complexes and the ATP synthase are becoming increasingly implicated in human disease . Recently, mutations in the ATPase 6 gene have been shown to cause several different neurological disorders . The product of this gene is homologous to the a subunit of the ATP synthase of Escherichia coli . Here, mutations equivalent to those described in humans have been introduced into the a subunit of E . coli by site-directed mutagenesis, and the effects of these mutations on the ATPase activity, ATP synthesis and ability of the enzyme to pump protons studied in detail . The effects of the mutations varied considerably . The mutation L262P (9185 T-C equivalent) caused a 70% loss of ATP synthesis activity, reduced DCCD sensitivity, and lowered proton pumping activity . The L207P (8993 T-C equivalent) reduced ATP synthesis by 50%, affected DCCD sensitivity, while proton pumping was only marginally affected when measured by the standard AMCA quenching assay . The other mutations studied affected the functioning of the ATP synthase much less . The results confirm that modeling of these point mutations in the E . coli enzyme is a useful approach to determining how alterations in the ATPase 6 gene affect enzyme function and, therefore, how a pathogenic effect can be exerted. FEBS Lett, 1999 Jun 18, 453(1-2), 15 - 9 Formation of adenosine 5'-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli; Bouhss A et al.; The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate . The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols . Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5'-tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes. Jpn J Thorac Cardiovasc Surg, 1999 May, 47(5), 204 - 9 Study of gene delivery in a rabbit vein graft model . Improvement of the efficiency of gene transfer into vein grafts; Chikada M et al.; Gene therapy is a therapeutic strategy in treating cardiovascular disease . Vein graft failure, the major limitation on coronary artery bypass surgery, may be amenable to gene approaches . Some studies describe gene therapies using functioning genes to prevent vein graft stenosis . Gene transfer efficiency remains a major issue . In this rabbit vein graft model, we studied gene delivery using a replication-deficit recombinant adenovirus to improve gene transfer efficiency into vein grafts . The adenovirus vector that contains the E.coli lacZ gene encoding beta gal was used because this vector is widely used and thought to be effective . Gene transfer was detected by X-gal staining . We hypothesized that dimethylsulfoxide and hyaluronidase, both drug delivery enhancers, would improve efficiency and that, in transfer to adventitia, direct injection would be more effective than dwelling . We studied 3 gene delivery methods to intima and media (controls, using dimethylsulfoxide and using hyaluronidase before transfection) and 2 delivery methods to adventitia (direct injection and dwelling) . We used 6 rabbits per delivery method . X-gal stained positive cell rates were counted using light microscopy . Our findings indicate that (1) dimethylsulfoxide increased the efficiency of transfection to media and intima, (2) hyaluronidase increased intimal transfection efficiency, (3) direct injection to adventitia was more effective than dwelling . These findings suggest that in vein grafting, our methods are feasible for improving gene transfer efficiency. Br J Pharmacol, 1999 Jun, 127(3), 685 - 92 Protective effects of a superoxide dismutase mimetic and peroxynitrite decomposition catalysts in endotoxin-induced intestinal damage; Salvemini D et al.; 1 . The relative contributions of superoxide anion (O2-) and peroxynitrite (PN) were evaluated in the pathogenesis of intestinal microvascular damage caused by the intravenous injection of E . coli lipopolysaccharide (LPS) in rats . The superoxide dismutase mimetic (SODm) SC-55858 and the active peroxynitrite decomposition catalysts 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulphonatophenyl)-por phyrinato iron (III) and 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)-porphyrinato iron (III) (FeTMPS, FeTMPyP respectively) were used to assess the roles of O2- and PN respectively . 2 . The intravenous injection of LPS elicited an inflammatory response that was characterized by a time-dependent infiltration of neutrophils, lipid peroxidation, microvascular leakage (indicative of microvascular damage), and epithelial cell injury in both the duodenum and jejunum . 3 . Administration of the SODm SC-55858, FeTMPS or FeTMPyP at 3 h post LPS reduced the subsequent increase in microvascular leakage, lipid peroxidation and epithelial cell injury . Inactive peroxynitrite decomposition catalysts exhibited no protective effects . Only, SC-55858 inhibited neutrophil infiltration . 4 . Our results suggest that O2 and peroxynitrite play a significant role in the pathogenesis of duodenal and intestinal injury during endotoxaemia and that their remoyal by SODm and peroxynitrite decomposition catalysts offers a novel approach to the treatment of septic shock or clinical conditions of gastrointestinal inflammation . Furthermore, the remarkable protection of the intestinal epithelium by these agents suggests their use during chemo- and radiation therapy, cancer treatments characterized by gastrointestinal damage . Potential mechanisms through which these radicals evoke damage are discussed. Hum Mol Genet, 1999 Aug, 8(8), 1523 - 8 Identification and characterization of three novel missense mutations in mevalonate kinase cDNA causing mevalonic aciduria, a disorder of isoprene biosynthesis; Houten SM et al.; Mevalonic aciduria is a rare autosomal recessive metabolic disorder, characterized by psychomotor retardation, failure to thrive, hepatosplenomegaly, anemia and recurrent febrile crises . The disorder is caused by a deficient activity of mevalonate kinase due to mutations in the encoding gene . Thus far, only two disease-causing mutations have been identified . We now report four different missense mutations including three novel ones, which were identified by sequence analysis of mevalonate kinase cDNA from three mevalonic aciduria patients . All mutations affect conserved amino acids . Heterologous expression of the corresponding mutant mevalonate kinases as fusion proteins with glutathione S -transferase in Escherichia coli showed a profound effect of each of the mutations on enzyme activity . In addition, immunoblot analysis of fibroblast lysates from patients using specific antibodies against mevalonate kinase identified virtually no protein . These results demonstrate that the mutations affect not only the activity but also the stability of the mutant proteins. J Virol, 1999 Aug, 73(8), 6729 - 42 Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs; Harvey BG et al.; Administration of adenovirus (Ad) vectors to immunologically naive experimental animals almost invariably results in the induction of systemic anti-Ad neutralizing antibodies . To determine if the human systemic humoral host responses to Ad vectors follow a similar pattern, we evaluated the systemic (serum) anti-Ad serotype 5 (Ad5) neutralizing antibodies in humans after administration of first generation (E1(-) E3(-)) Ad5-based gene transfer vectors to different hosts . AdGVCFTR.10 (carrying the normal human cystic fibrosis {CF} transmembrane regulator cDNA) was sprayed (8 x 10(7) to 2 x 10(10) particle units {PU}) repetitively (every 3 months or every 2 weeks) to the airway epithelium of 15 individuals with CF . AdGVCD.10 (carrying the Escherichia coli cytosine deaminase gene) was administered (8 x 10(8) to 8 x 10(9) PU; once a week, twice) directly to liver metastasis of five individuals with colon cancer and by the intradermal route (8 x 10(7) to 8 x 10(9) PU, single administration) to six healthy individuals . AdGVVEGF121.10 (carrying the human vascular endothelial growth factor 121 cDNA) was administered (4 x 10(8) to 4 x 10(9.5) PU, single administration) directly to the myocardium of 11 individuals with ischemic heart disease . Ad vector administration to the airways of individuals with CF evoked no or minimal serum neutralizing antibodies, even with repetitive administration . In contrast, intratumor administration of an Ad vector to individuals with metastatic colon cancer resulted in a robust antibody response, with anti-Ad neutralizing antibody titers of 10(2) to >10(4) . Healthy individuals responded to single intradermal Ad vector variably, from induction of no neutralizing anti-Ad antibodies to titers of 5 x 10(3) . Likewise, individuals with ischemic heart disease had a variable response to single intramyocardial vector administration, ranging from minimal neutralizing antibody levels to titers of 10(4) . Evaluation of the data from all trials showed no correlation between the peak serum neutralizing anti-Ad response and the dose of Ad vector administered (P > 0.1, all comparisons) . In contrast, there was a striking correlation between the peak anti-Ad5 neutralizing antibody levels evoked by vector administration and the level of preexisting anti-Ad5 antibodies (P = 0.0001) . Thus, unlike the case for experimental animals, administration of Ad vectors to humans does not invariably evoke a systemic anti-Ad neutralizing antibody response . In humans, the extent of the response is dictated by preexisting antibody titers and modified by route of administration but is not dose dependent . Since the extent of anti-Ad neutralizing antibodies will likely modify the efficacy of administration of Ad vectors, these observations are of fundamental importance in designing human gene therapy trials and in interpreting the efficacy of Ad vector-mediated gene transfer. J Virol, 1999 Aug, 73(8), 6405 - 14 Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus; Smith GA et al.; A full-length clone of the 142-kb pseudorabies virus (PRV) genome was constructed as a stable F plasmid in Escherichia coli . The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, deletions, or inversions . The transfection of pBecker1 into susceptible eukaryotic cells resulted in productive viral infection . Virus isolated following transfection was indistinguishable from wild-type virus in a rodent model of infection and spread to retinorecipient regions of the brain following inoculation in the vitreous body of the eye . Mutagenesis of pBecker1 in E . coli with a mini-Tn5-derived transposon enabled the rapid isolation of insertion mutants, identification of essential viral genes, and simplified construction of viral revertants . The serial passage of a viral insertion mutant demonstrated the transposon insertion to be stable . However, the F-plasmid insertion present in the viral gG locus was found to undergo a spontaneous deletion following transfection into eukaryotic cells . The implications of F-plasmid insertion into the viral genome with regard to phenotype and genomic stability are discussed. J Virol, 1999 Aug, 73(8), 6257 - 64 Membrane permeabilization by small hydrophobic nonstructural proteins of Japanese encephalitis virus; Chang YS et al.; Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells . We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or alpha-sarcine, to which mock-infected cells were insensitive . However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability . Using an inducible Escherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability . We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so . When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability . Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition . Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control . Furthermore, when cotransfected with a reporter gene luciferase or beta-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells . Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells. J Biol Chem, 1999 Jul 16, 274(29), 20479 - 88 Randomization of the receptor alpha chain recruitment epitope reveals a functional interleukin-5 with charge depletion in the CD loop; Wu SJ et al.; We report the functional phage display of single chain human interleukin-5 (scIL-5) and its use for receptor-binding epitope randomization . Enzyme-linked immunosorbent assays and optical biosensor analyses verified expression of scIL-5 on the phage surface and binding of scIL-5 phage to interleukin-5 receptor alpha chain . Furthermore, an asymmetrically disabled but functional scIL-5 mutant, (wt/A5)scIL-5, was displayed on phage . (wt/A5)scIL-5 was constructed from an N-terminal half containing the original five charged residues (88EERRR92) in the CD loop, including the Glu89 and Arg91 believed key in the alpha chain recognition site, combined with a C-terminal half containing a disabled CD loop sequence (88AAAAA92) missing the key recognition residues . This asymmetric variant was used as a starting point to generate an scIL-5 library in which the intact 88-92 N-terminal CD loop was randomized . From this epitope library, a receptor-binding variant of IL-5 was detected, (SLRGG/A5)scIL-5, in which the only charged residue in the CD loop is an Arg at position 90 . Characterization of this variant expressed as a soluble protein in E . coli shows that the IL-5 pharmacophore for receptor alpha chain binding can function with a single positive charge in the CD loop . Charge-depleted CD loop mimetics of IL-5 suggest the importance of charge distribution in functional IL-5 receptor recruitment. J Biol Chem, 1999 Jul 16, 274(29), 20351 - 7 Chaperone activity of a chimeric GroEL protein that can exist in a single or double ring form; Erbse A et al.; The molecular chaperone GroEL is a protein complex consisting of two rings each of seven identical subunits . It is thought to act by providing a cavity in which a protein substrate can fold into a form that has no propensity to aggregate . Substrate proteins are sequestered in the cavity while they fold, and prevented from diffusion out of the cavity by the action of the GroES complex, that caps the open end of the cavity . A key step in the mechanism of action of GroEL is the transmission of a conformational change between the two rings, induced by the binding of nucleotides to the GroEL ring opposite to the one containing the polypeptide substrate . This conformational change then leads to the discharge of GroES from GroEL, enabling polypeptide release . Single ring forms of GroEL are thus predicted to be unable to chaperone the folding of GroES-dependent substrates efficiently, since they are unable to discharge the bound GroES and unable to release folded protein . We describe here a detailed functional analysis of a chimeric GroEL protein, which we show to exist in solution in equilibrium between single and double ring forms . We demonstrate that whereas the double ring form of the GroEL chimera functions effectively in refolding of a GroES-dependent substrate, the single ring form does not . The single ring form of the chimera, however, is able to chaperone the folding of a substrate that does not require GroES for its efficient folding . We further demonstrate that the double ring structure of GroEL is likely to be required for its activity in vivo. J Biol Chem, 1999 Jul 16, 274(29), 20259 - 64 Molecular and biochemical analysis of MalK, the ATP-hydrolyzing subunit of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis; Greller G et al.; We report the cloning, sequencing, and expression of malK encoding the ATP-hydrolyzing subunit of the maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis . According to the deduced amino acid sequence, MalK consists of 372 amino acids with a calculated molecular weight of 41,787 . It shows 47% identity with the MalK protein of Escherichia coli and high sequence conservation in important regions . C-terminal His-tagged MalK was purified . The soluble protein appeared monomeric by molecular sieve chromatography and showed ATPase activity . Enzymatic activity was highest at 80 degrees C with a Km of 150 microM and a Vmax of 0.55 micromol of ATP hydrolyzed/min/mg of protein . ADP was not a substrate but a competitive inhibitor (Ki 230 microM) . GTP and CTP were also hydrolyzed . ATPase activity was inhibited by N-ethylmaleimide but not by vanadate . The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the ABC transporters in these two phylogenetic branches. J Biol Chem, 1999 Jul 16, 274(29), 20075 - 8 Sec-dependent pathway and DeltapH-dependent pathway do not share a common translocation pore in thylakoidal protein transport; Asai T et al.; Thylakoidal proteins of plant chloroplasts are transported to thylakoids via several different pathways, including the DeltapH-dependent and the Sec-dependent pathways . In this study, we asked if these two pathways utilize a common translocation pore . A fusion protein consisting of a 23-kDa subunit of the oxygen evolving complex and Escherichia coli biotin carboxyl carrier protein was biotinylated in E . coli cells and purified . When incubated with isolated pea thylakoids in the absence of avidin, the purified fusion protein was imported into the thylakoids via the DeltapH-dependent pathway . However in the presence of avidin, the fusion protein became lodged in the thylakoid membranes, with its N terminus reaching the thylakoidal lumen, while its C-terminal segment complexed with avidin exposed on the thylakoidal surface . The translocation intermediate of the fusion protein inhibited the import of authentic 23-kDa subunit, suggesting that it occupies a putative translocation pore for the DeltapH-dependent pathway . However the intermediate did not block import of the 33-kDa subunit of the oxygen evolving complex, which is a substrate for the Sec-dependent pathway . These results provide evidence against the possibility of a common translocation pore shared by the Sec-dependent pathway and the DeltapH-dependent pathway. J Biol Chem, 1999 Jul 16, 274(29), 20068 - 70 The signal recognition particle-targeting pathway does not necessarily deliver proteins to the sec-translocase in Escherichia coli; Cristobal S et al.; ProW is an Escherichia coli inner membrane protein that consists of a 100-residue-long periplasmic N-terminal tail (N-tail) followed by seven closely spaced transmembrane segments . N-tail translocation presumably proceeds in a C-to-N-terminal direction and represents a poorly understood aspect of membrane protein biogenesis . Here, using an in vivo depletion approach, we show that N-tail translocation in a ProW derivative comprising the N-tail and the first transmembrane segment fused to the globular P2 domain of leader peptidase depends both on the bacterial signal recognition particle (SRP) and the Sec-translocase . Surprisingly, however, a deletion construct with only one transmembrane segment downstream of the N-tail can assemble properly even under severe depletion of SecE, a central component of the Sec-translocase, but not under SRP-depletion conditions . To our knowledge, this is the first demonstration that the SRP-targeting pathway does not necessarily deliver SRP-dependent inner membrane proteins to the Sec-translocase . The data further suggest that N-tail translocation can proceed in the absence of a functional Sec-translocase. J Bacteriol, 1999 Jul, 181(14), 4435 - 6 Growth phase-dependent regulation of Vsr endonuclease may contribute to 5-methylcytosine mutational hot spots in Escherichia coli; Macintyre G et al.; Using rabbit polyclonal antibodies, we have shown that the Dcm cytosine methylase of Escherichia coli is maintained at a constant level during cell growth, while Vsr endonuclease levels are growth phase dependent . Decreased production of Vsr relative to Dcm during the log phase may contribute substantially to the mutability of 5-methylcytosine. J Bacteriol, 1999 Jul, 181(14), 4430 - 4 Biochemical and genetic evidence for participation of DevR in a phosphorelay signal transduction pathway essential for heterocyst maturation in Nostoc punctiforme ATCC 29133; Hagen KD et al.; In a test of the hypothesis that DevR is a response regulator protein that functions in a phosphorelay signal transduction system involved in heterocyst development in Nostoc punctiforme ATCC 29133, purified affinity-tagged DevR was shown to be phosphorylated in vitro by the noncognate sensor kinase EnvZ . Site-directed mutagenesis was used to generate N . punctiforme mutants with single amino acid substitutions at the putative phosphorylation site of DevR . These mutants exhibited a Fox- phenotype like the original devR insertion mutant UCD 311, consistent with a phosphotransferase role for DevR. J Bacteriol, 1999 Jul, 181(14), 4374 - 80 Mutational analysis of the cbb operon (CO2 assimilation) promoter of Ralstonia eutropha; Jeffke T et al.; PL promoters direct the transcription of the duplicated cbb operons from the facultative chemoautotroph Ralstonia eutropha H16 . The operons encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle required for CO2 assimilation . Their transcription depends on the activator protein CbbR . Structure-function relationships in the cloned chromosomal promoter region were analyzed by site-directed mutagenesis . PL was altered in its presumed hexameric -35 and/or -10 box or in the spacer region between the boxes to achieve a greater or lesser resemblance to the structure of the sigma70 consensus promoter of Escherichia coli . PL::lacZ transcriptional fusions of various promoter variants were assayed in transconjugant strains of R . eutropha as well as in corresponding cbbR deletion mutants . Mutations increasing the similarity of the -35 and/or -10 box to the consensus sequence stimulated PL activity to various extents, whereas mutations deviating from the consensus decreased the activity . The length of the spacer region also proved to be critical . The conversion of the boxes, either individually or simultaneously, into the consensus sequences resulted in a highly active PL . All improved PL mutants, however, retained the activation under inducing or derepressing growth conditions, although the full-consensus promoter was nearly constitutive . They were also activated in the cbbR mutants . The activity of the overlapping, divergently oriented cbbR promoter was less affected by the mutations . The half- and full-consensus PL mutants were comparably active in E . coli . Two major conclusions were drawn from the results: (i) the location and function of PL were verified, and (ii) indirect evidence was obtained for the involvement of another regulator(s), besides CbbR, in the transcriptional control of the R . eutropha cbb operons. J Bacteriol, 1999 Jul, 181(14), 4318 - 25 Identification and characterization of a new lipoprotein, NlpI, in Escherichia coli K-12; Ohara M et al.; We report here the identification of a new lipoprotein, NlpI, in Escherichia coli K-12 . The NlpI structural gene (nlpI) is located between the genes pnp (polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E . coli chromosome . The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein . An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments . The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone . Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions . NlpI may be important for an as-yet-undefined step in the overall process of cell division. J Bacteriol, 1999 Jul, 181(14), 4170 - 5 Increased rrn gene dosage causes intermittent transcription of rRNA in Escherichia coli; Voulgaris J et al.; When the number of rRNA (rrn) operons in an Escherichia coli cells is increased by adding an rrn operon on a multicopy plasmid, the rate of rRNA expression per operon is reduced to maintain a constant concentration of rRNA in the cell . We have used electron microscopy to examine rRNA transcription in cells containing a multicopy plasmid carrying rrnB . We found that there were fewer RNA polymerase molecules transcribing the rrn genes, as predicted from previous gene dosage studies . Furthermore, RNA polymerase molecules were arranged in irregularly spaced groups along the operon . No apparent pause or transcription termination sites that would account for the irregular spacing of the groups of polymerase molecules were observed . We also found that the overall transcription elongation rate was unchanged when the rrn gene dosage was increased . Our data suggest that when rrn gene dosage is increased, initiation events, or promoter-proximal elongation events, are interrupted at irregular time intervals. Bioorg Med Chem, 1999 May, 7(5), 789 - 94 Chemoenzymatic synthesis of an unnatural tetramethyl cobalt corphinoid; Santander PJ et al.; The chemoenzymatic synthesis and structural characterization by 13C NMR of a tetramethyl cobalt-corphinoid produced by methylation of cobalt-precorrin-3 using CbiF are described. Immunol Rev, 1999 Apr, 168, 23 - 31 Exploitation of major histocompatibility complex class I molecules and caveolae by simian virus 40; Parton RG et al.; Simian virus 40 (SV40), a non-enveloped DNA virus, is transported from the cell surface to the nucleus where virus replication occurs . This pathway of virus uptake involves binding to surface MHC class I molecules, entry via non-coated pits, and subsequent transport to the endoplasmic reticulum (ER) . At some stage in this pathway the virus must cross a membrane to reach the cytosol . In the present review, the cellular machinery which the virus has utilized to enter the cell will be examined . In particular, we will consider recent evidence for the involvement of caveolae in the infectious entry step and propose a model involving recruitment of caveolar proteins around the membrane-bound virus . We also speculate that a similar mechanism may have been exploited by bacterial pathogens . The subsequent steps by which SV40 reaches the ER remain unclear but recent evidence suggests that this pathway may be shared with several other proteins that are transported from surface caveolae to the ER. Pflugers Arch, 1999 Aug, 438(3), 361 - 4 Glutathione (GSH) reduces the open probability of mechanosensitive channels in Escherichia coli protoplasts; Koprowski P et al.; The effects of glutathione (reduced GSH, and oxidized GSSG) on mechanosensitive (MS) ion channels from Escherichia coli protoplasts were investigated using excised, inside-out membrane patches . Our studies demonstrate here that 5 mM GSH irreversibly reduces the activities of the 560-pS MS channel by approximately 70-75% whereas 5 mM GSSG did not alter the MS channel currents . In addition, millimolar concentrations of dithiothreitol had similar although weaker effects to GSH . The physiological concentration of GSH in E . coli cytoplasm ranges from 3.5 mM to 6.6 mM, which may indicate that under normal conditions these MS channels open less due to membrane stretch. Curr Microbiol, 1999 Aug, 39(2), 68 - 72 Bile salt activation of stress response promoters in Escherichia coli; Bernstein C et al.; Bile salts are prevalent in the mammalian intestine, a natural habitat of Escherichia coli . The bile salts deoxycholate, chenodeoxycholate, ursodeoxycholate, and glycocholate were tested for their effect on induction of 13 specific stress response genes . The most consistently activated E . coli promoters were those for genes micF, osmY, and dinD . MicF and osmY gene products are associated with membrane functions and are responsive to oxidative stress . DinD is induced by DNA damage as part of the SOS response . These results indicate that bile acids, to which E . coli are naturally exposed, induce expression of specific stress response genes, possibly in response to membrane perturbation, oxidative stress, and DNA damage . Altered expression of stress-response genes may also promote interaction of E . coli with cells of the colonic epithelium. J Mol Biol, 1999 Jul 23, 290(4), 825 - 37 Discrimination by Escherichia coli initiation factor IF3 against initiation on non-canonical codons relies on complementarity rules; Meinnel T et al.; Translation initiation factor IF3, one of three factors specifically required for translation initiation in Escherichia coli, inhibits initiation on any codon other than the three canonical initiation codons, AUG, GUG, or UUG . This discrimination against initiation on non-canonical codons could be due to either direct recognition of the two last bases of the codon and their cognate bases on the anticodon or to some ability to "feel" codon-anticodon complementarity . To investigate the importance of codon-anticodon complementarity in the discriminatory role of IF3, we constructed a derivative of tRNALeuthat has all the known characteristics of an initiator tRNA except the CAU anticodon . This tRNA is efficiently formylated by methionyl-tRNAfMettransformylase and charged by leucyl-tRNA synthetase irrespective of the sequence of its anticodon . These initiator tRNALeuderivatives (called tRNALI) allow initiation at all the non-canonical codons tested, provided that the complementarity between the codon and the anticodon of the initiator tRNALeuis respected . More remarkably, the discrimination by IF3, normally observed with non-canonical codons, is neutralised if a tRNALIcarrying a complementary anticodon is used for initiation . This suggests that IF3 somehow recognises codon-anticodon complementarity, at least at the second and third position of the codon, rather than some specific bases in either the codon or the anticodon . J Mol Recognit, 1999 Mar-Apr, 12(2), 141 - 52 Targetting of the N-terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric ScFvs blocks the E6-mediated degradation of cellular p53; Giovane C et al.; The E6 protein of cancer-associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway . In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6-mediated p53 in vitro degradation . The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N-terminal end of 16E6 . The variable regions of these antibodies were cloned and expressed in E . coli as single chain Fvs (scFvs) . Purified scFvs were present in monomeric form and totally abolished 16E6-mediated p53 degradation by preventing the formation of E6/p53 protein complexes . Our results demonstrate that monovalent binding of scFvs to the N-terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein . Biopolymers, 1999 Sep, 50(3), 303 - 18 High-resolution calorimetric and optical melting profiles of DNA plasmids: resolving contributions from intrinsic melting domains and specifically designed inserts; Volker J et al.; We demonstrate that differential scanning calorimetry (DSC) can be used to yield high-resolution melting profiles for DNA plasmids that agree in all major features with the corresponding plasmid melting profiles derived using more traditional optical techniques . We further demonstrate that by combining information derived from both calorimetric and optical melting profiles one can glean insights that are unavailable from either melting curve alone . By using both optical and calorimetric observables, we show how one can resolve, identify, and measure the thermodynamic properties of particular sequences/domains of interest within a plasmid . We also show that complementary DSC and optical melting studies on plasmids with and without specifically designed inserts can provide fundamental advantages over the corresponding melting studies on other model system constructs for thermodynamically characterizing nucleic acid sequences/structures . J Infect Dis, 1999 Aug, 180(2), 419 - 25 Participation of ABH glycoconjugates in the secretory response to Escherichia coli heat-labile toxin in rabbit intestine; Galvan EM et al.; The ability of membrane ABH blood group-active glycoconjugates to act as receptors of the heat-labile enterotoxin of Escherichia coli (LTh) was studied in vitro and in vivo when GM1 was blocked by the cholera toxin B subunit . Rabbits were classified as AB or H based on intestinal ABH-antigenic activities . Brush border membranes from AB rabbits contained 4 times more LTh binding sites than the H ones . LTh interaction could be inhibited by lectins that recognize ABH determinants . LTh induced a similar dose-dependent secretory response in ligated ileal loops of both types of animals . Anti-AB antibodies and Ulex europaeus I lectin could significantly reduce the fluid accumulation in AB and H rabbits, respectively . LTh caused adenylate cyclase activation even when GM1 was blocked, and this effect was abolished by the addition of specific ABH ligands . These results suggest that ABH glycoconjugates are involved in the host secretory response to LTh in rabbit intestine. Biotechnol Bioeng, 1999 Aug 5, 64(3), 333 - 41 Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations; Schneider S et al.; Utilising whole cells of recombinant Escherichia coli K27 (pCYP102, pGEc47) containing active cytochrome P450BM-3 monooxygenase {E.C . 1 . 14.14.1}, multiple oxidations of saturated and unsaturated fatty acids were performed by the enzyme under conditions of excess oxygen . The amount of oxygen dissolved in the culture medium strongly influenced the regioselectivity of the reaction, as reflected in the distribution and amount of oxidised products . We have verified by gas chromatography/mass spectrometry that the products of in vivo biotransformation of pentadecanoic acid by cytochrome P450BM-3 are identical to those formed in cell-free extracts containing the enzyme . The formation of keto- and dihydroxy acids, side products which are characteristic for in vitro conversions with purified cytochrome P450BM-3 in the presence of excess oxygen, has been observed as well . Thus, by varying the oxygen concentration, we could control the regioselectivity of oxidation and the number of products made . Under oxygen limiting conditions, only monooxidised 12-, 13-, and 14-hydroxy-pentadecanoic acids were obtained . Consequently, unwanted side products could be excluded by modulating the amount of oxygen used in the bioconversion . Furthermore, whole cell oxidation of two unsaturated long-chain fatty acids, cis-pentadec-10-enoic and cis-hexadec-9-enoic acid, resulted in the production of epoxides, various subterminal hydroxyalkenoic acids and keto- and hydroxyalkanoic acids . Although we obtained higher activities of C15:0 conversion in vitro, the whole cell biocatalyst proved to be useful for specific oxidations of long-chain fatty acids since there is no need to add the costly cofactor NADPH . This biooxidation by E . coli K27 (pCYP102, pGEc47) under oxygen limitation has been demonstrated at the 2-L scale, showing that 12-, 13-, and 14-hydroxypentadecanoic acids can be produced in the g L-1 range . Biotechnol Bioeng, 1999 Aug 5, 64(3), 272 - 83 Modeling, simulation, and kinetic analysis of a heterogeneous reaction system for the enzymatic conversion of poorly soluble substrate; Lee DC et al.; We developed a kinetic model that describes a heterogeneous reaction system consisting of a solid substrate suspension for the production of D-amino acid using D-hydantoinase . As a biocatalyst, mass-produced free and whole cell enzymes were used . The heterogeneous reaction system involves dissolution of a solid substrate, enzymatic conversion of the dissolved D-form substrate, spontaneous racemization of an L-form substrate to D-form, and deactivation of the enzyme . In the case of using whole cell enzymes, transfer of the dissolved substrate and product through the cell membrane was considered . The kinetic parameters were determined from experiments, literature data, and by using Marquardt's method of nonlinear regression analysis . The model was simulated using the kinetic parameters and compared with experimental data, and a good agreement was observed between the experimental results and the simulation ones . Factors affecting the kinetics of the heterogeneous reaction system were analyzed on the basis of the kinetic model, and the efficiency of the reaction systems using free and whole cell enzymes was also compared . Biotechnol Bioeng, 1999 Jun 20, 63(6), 654 - 62 Kinetics of retrovirus production and decay; Le Doux JM et al.; There has been only limited success in using recombinant retroviruses to transfer genes for the purposes of human gene therapy, in part because the average number of genes delivered to the target cells (transduction efficiency) is often too low to achieve the desired therapeutic effect {Miller, AD . 1990 . Blood 76:271-278; Mulligan RC . 1993 . Science 260:926-932; Orkin SH, Motulsky AG . 1995 . Report and recommendations of the panel to assess the NIH investment in research on gene therapy . Bethesda, MD: National Institutes of Health.} . One strategy to improve transduction efficiency is to focus on understanding and improving the processes used to produce recombinant retroviruses . In this report, we characterized the dynamics of retrovirus production and decay in batch cultures of virus producer cells using a simple mathematical model, a recombinant retrovirus encoding the Escherichia coli lacZ gene, and quantitative assays for virus activity and number . We found that the rate at which recombinant retroviruses spontaneously lose their activity (decay) is a strong function of temperature, decreasing roughly 2-fold for every 5 degrees C reduction in temperature, whereas the rate at which retroviruses are produced is only weakly affected by temperature, decreasing about 10% for every 5 degrees C reduction in temperature . In addition, we developed a simple mathematical model of virus production and decay that predicted that the virus titer in batch cultures of virus producer cells would reach a maximum steady-state at a rate that is inversely proportional to the virus decay rate and to a level that is proportional to the ratio of the virus production rate to the virus decay rate . Consistent with the model, we observed that the steady-state levels of virus titer increased more than 3-fold when the cell culture temperature was reduced from 37 to 28 degrees C . Despite their higher titers, virus stocks produced at 28 degrees C, when used in undiluted form so as to mimic human gene transfer protocols, did not transduce substantially more cells than virus stocks produced at 37 degrees C . The implications of our findings on the production of retroviruses for use in human gene therapy protocols are discussed . Biopolymers, 1999 Sep, 50(3), 273 - 86 Addition of positively charged tripeptide to N-terminus of the Fos basic region leucine zipper domain: implications on DNA bending, affinity, and specificity; Mahmoudi T et al.; GKH-Fos(139-211)/Jun(248-334) (GKH: glycine-lysine-histidine) is a modified Fos/Jun heterodimer designed to contain a metal binding motif in the form of a GKH tripeptide at the amino terminus of Fos bZIP domain dimerized with the Jun basic region leucine zipper (bZIP) domain . We examined the effect of the addition of positively charged GKH motif to the N-terminus of Fos(139-211) on the DNA binding characteristics of the Fos(139-211)/Jun(248-334) heterodimer . Binding studies indicate that while the nonspecific DNA binding affinity of the GKH modified heterodimer increases 4-fold, it specifically binds the activating protein-1 (AP-1) site 6-fold less tightly than the control unmodified counterpart . Furthermore, helical phasing analysis indicates that GKH-Fos(139-211)/Jun(248-334) and control Fos(139-211)/Jun(248-334) both bend the DNA at the AP-1 site toward the minor groove . However, due to the presence of the positively charged GKH motif on Fos, the degree of the induced bend by GKH- Fos(139-211)/Jun(248-334) is greater than that induced by the unmodified Fos/Jun heterodimer . Our results suggest that the unfavorable energetic cost of the increased DNA bending by GKH-Fos(139-211)/Jun(248-334) results in a decrease in both specificity and affinity of binding of the heterodimer to the AP-1 site . These findings may have important implications in protein design as well in our understanding of DNA bending and factors responsible for the functional specificity of different members of the bZIP family of transcription factors . Mol Biol Cell, 1999 Jul, 10(7), 2441 - 59 Rho-dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22; Brancolini C et al.; Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies . We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells . In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed . Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase . Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities . RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response . Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes . Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes . Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid-induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells . We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization. Mol Biol Cell, 1999 Jul, 10(7), 2163 - 73 In vitro studies with purified components reveal signal recognition particle (SRP) and SecA/SecB as constituents of two independent protein-targeting pathways of Escherichia coli; Koch HG et al.; The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared . This was achieved in a novel cell-free system from E . coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4 . 5S RNA, and FtsY . The integration of two membrane proteins into inside-out plasma membrane vesicles of E . coli required all three SRP components and could not be driven by SecA, SecB, and DeltamicroH+ . In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive . Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E . coli . Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E . coli . Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel. Pharm Res, 1999 Jun, 16(6), 808 - 12 Investigation of protein-surfactant interactions by analytical ultracentrifugation and electron paramagnetic resonance: the use of recombinant human tissue factor as an example; Jones LS et al.; PURPOSE: The purpose of this work is to utilize electron paramagnetic resonance (EPR) spectroscopy in conjunction with analytical ultracentrifugation (AUC) to investigate the binding of surfactants to proteins with a transmembrance domain . As an example these methods have been used to study the interaction of a nonionic surfactant, C12E8, to recombinant human tissue factor (rhTF) in liquid formulations . The complementary nature of the two techniques aids in data interpretation when there is ambiguity using a single technique . In addition to binding stoichiometries, the possibility of identifying the interacting domains by using two forms of rhTF is explored . METHODS: Two recombinant, truncated forms of human tissue factor were formulated in the absence of phospholipids . Neither of the recombinant proteins, produced in E . coli, contains the cytoplasmic domain . Recombinant human tissue factor 243 (rhTF 243) consists of 243 amino acids and includes the transmembrane sequences . Recombinant human tissue factor 220 (rhTF 220), however, contains only the first 221 amino acids of the human tissue factor, lacking those of the transmembrane region . EPR and AUC were used to investigate the interactions between these two forms of rhTF and polyoxyethylene 8 lauryl ether, C12E8 . RESULTS: Binding of C12E8 to rhTF 243 is detected by both EPR spectroscopy and AUC . Although a unique binding stoichiometry was not determined, EPR spectroscopy greatly narrowed the range of possible solutions suggested by the AUC data . Neither technique revealed an interaction between rhTF 220 and C12E8 . CONCLUSIONS: The complementary nature of EPR spectroscopy and AUC make the combination of the two techniques useful in data interpretation when studying the interactions between rhTF and C12E8 . By utilizing these techniques in this study, the binding stoichiometry of rhTF 243 to C12E8 ranges from 1.2:1 to 1.3:0.6 based on an aggregation number of 120 . This binding is consistent with previously reported activity data that showed an increase in clotting rate when rhTF 243 is in the presence of C12E8 micelles . From the rhTF 220 data, it can further be concluded that the transmembrane domain of rhTF is necessary for interactions with C12E8. Acta Biochim Pol, 1998, 45(4), 883 - 94 Possible evolution of factors involved in protein biosynthesis; Nyborg J; The elongation factors of protein biosynthesis are well preserved through out evolution . They catalyze the elongation phase of protein biosynthesis, where on the ribosome amino acids are added one at a time to a growing peptide according to the genetic information transcribed into mRNA . Elongation factor Tu (EF-Tu) provides the binding of aminoacylated tRNA to the ribosome and protects the aminoester bond against hydrolysis until a correct match between the codon on mRNA and the anticodon on tRNA can be achieved . Elongation factor G (EF-G) supports the translocation of tRNAs and of mRNA on the ribosome so that a new codon can be exposed for decoding . Both these factors are GTP binding proteins, and as such exist in an active form with GTP and an inactive form with GDP bound to the nucleotide binding domain . Elongation factor Ts (EF-Ts) will catalyze the exchange of nucleotide on EF-Tu . This review describes structural work on EF-Tu performed in our laboratory over the last eight years . The structural results provide a rather complete picture of the major structural forms of EF-Tu, including the so called ternary complex of aa-tRNA:EF-Tu:GTP . The structural comparison of this ternary complex with the structure of EF-G:GDP displays an unexpected macromolecular mimicry, where three domains of EF-G mimick the shape of the tRNA in the ternary complex . This observation has initiated much speculation on the evolution of all factors involved in protein synthesis, as well as on the details of the ribosomal function in one part of elongation. Crit Care Med, 1999 Jun, 27(6), 1164 - 7 Inhibition of tumor necrosis factor-alpha release in rat experimental endotoxemia by treatment with the 21-aminosteroid U-74389G; Lehmann C et al.; OBJECTIVE: To determine the effect of the 21-aminosteroid U-74389G on tumor necrosis factor (TNF)-alpha release in experimental endotoxemia . DESIGN: Prospective, randomized, controlled animal study . SETTING: Experimental laboratory . SUBJECTS: Twenty-one male Wistar rats weighing 190+/-40 g . INTERVENTIONS: The rats were divided equally into 3 groups: a) control; b) endotoxemia (5 mg/kg lipopolysaccharide {LPS} from Escherichia coli 055:B5); and c) endotoxemia and U-74389G administration 30 mins before (3 mg/kg) and 60 mins after (1.5 mg/kg) endotoxin challenge . MEASUREMENTS AND MAIN RESULTS: At 0, 120, and 240 mins, serum levels of TNF-alpha were measured using a specific rat TNF-alpha ELISA kit . U-74389G-treated endotoxemic animals showed significantly reduced TNF-alpha release 120 mins after endotoxin challenge (control, 2.5+/-2.1 pg/mL; LPS, 4041+/-871 pg/mL; U-74389G, 1627+/-474 pg/mL {p < .05}) . Two hundred forty minutes after LPS administration, TNF-alpha levels decreased, whereas values in the untreated LPS group remained twice as high as those in the U-74389G group (LPS, 863+/-182 pg/mL; U-74389G, 369+/-54 pg/mL {p < .05}) . CONCLUSIONS: The study demonstrated that administration of U-74389G, which has radical-scavenging and membrane-stabilizing properties, decreased TNF-alpha release during endotoxemia . Thus, 21-aminosteroids may lend themselves to evaluation in the treatment of septic states. Avian Dis, 1999 Apr-Jun, 43(2), 320 - 5 Evaluation of scratches as an essential element in the development of avian cellulitis in broiler chickens; Norton RA et al.; Currently, the published cellulitis models do not adequately address the actual pathogenesis as seen in the commercial broiler industry . In this model, small dermal scratches were made on the skin of broiler chickens, which were then placed on litter seeded with avian cellulitis-associated Escherichia coli . The research confirms scratches are required for the induction of avian cellulitis . The research also confirms that "type I" cellulitis lesions or those previously thought to be due to hatchery-borne infections can be induced with scratches . The described methods provide a realistic model for cellulitis development that will improve the reliability of prophylactic and therapeutic-regimen efficacy testing data, thereby providing information more directly useful to the commercial broiler industry. Injury, 1999 Jan, 30(1), 9 - 14 Subcellular membrane impairment and application of phospholipase A2 inhibitors in endotoxic shock; Song SM et al.; The study aims at elucidating the mechanism involved in the cell dysfunction or impairment and the protective effects of phospholipase A2 (PLA2) inhibitors in endotoxin shock . Thirty-four rabbits were divided randomly into four groups: (1) normal control group (NC, n = 6), receiving saline intravenously; (2) endotoxin shock group (ES, n = 12), receiving 3 mg/kg of E . coli endotoxin; (3) chloroquine pretreated group (CQ, n = 8), receiving 3 mg/kg of chloroquine 3 min before endotoxin injection and (4) chlorpromazine pretreated group (CPZ, n = 8), receiving 0.3 mg/kg of chlorpromazine 30 min before endotoxin injection . Hepatic mitochondria were extracted either 8 h after commencement of the experiment or when the animals died for detecting PLA2 activity, membrane fluidity, membrane bound succinate dehydrogenate (SDH) and malondialdehyde (MDA) . Mitochondria of the lung, heart and kidney were also used for detection of the membrane fluidity . It was revealed that the survival rate of 8 h was 100% (NC), 58% (ES), 87.5% (CQ) and 75% (CPZ), respectively . Mean arterial pressure (MAP) dropped soon after endotoxin injection and descended continuously afterwards in the ES group (P < 0.01) . Fluorescence polarization, microviscosity and anisotrophy with a DPH probe were elevated above control levels (P < 0.01) . SDH was decreased obviously following endotoxin infusion (P < 0.01) . Chloroquine and chlorpromazine, serving as PLA2 inhibitors, could abate cellular dysfunction and increase survival rate . It is proposed that PLA2 plays a pivotal role in cellular injury in endotoxin shock . PLA2 inhibitor might serve as a useful adjunct in combating sepsis and shock. J Mol Biol, 1999 Jul 16, 290(3), 667 - 84 In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites; Harrison L et al.; When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS) . In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed . The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand . Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand . When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg . Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site.To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively . In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand . However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG . Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced . Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away . Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break . J Mol Biol, 1999 Jul 16, 290(3), 653 - 66 Dimerisation mutants of Lac repressor . I . A monomeric mutant, L251A, that binds Lac operator DNA as a dimer; Dong F et al.; Dimer formation between monomers of the Escherichia coli Lac repressor is substantially specificed by the interactions between three alpha-helices in each monomer which form a hydrophobic interface . As a first step in analysing the specificity of this interaction, we examined the mutant L251A . LacR bearing this mutation in a background lacking the C-terminal heptad repeats is completely incapable of forming dimers in solution, with a dimer-monomer equilibrium dissociation constant, or Kd, higher than 10(-5)M . This correlates with a 200-fold decrease in its ability to repress the lac operon in vivo compared to dimeric LacR . Surprisingly, the mutant is still capable of forming dimers upon binding to short operator DNA in vitro . Analysis of the kinetic parameters of binding of the mutant to operator DNA reveals a 2000 to 3000-fold increase in the equilibrium dissociation constant (Kd) of the mutant-DNA complex in comparison to dimeric LacR-operator complexes, with the change almost entirely due to a greater than 1000-fold decrease in association rate . The dissociation rate varies only by a factor of about two, in comparison to dimeric LacR . This change reflects a kinetic pathway in which dimer formation, in solution or on DNA, is the rate-limiting step . These findings have implications for the specificity and stability of the protein-protein interface in question . Genomics, 1999 Jul 1, 59(1), 90 - 6 Cloning and chromosomal mapping of the human DNA polymerase theta (POLQ), the eighth human DNA polymerase; Sharief FS et al.; We have cloned the cDNA for the eighth human DNA polymerase, DNA polymerase θ . The human cDNA encodes a putative DNA polymerase of 1762 amino acids with a calculated molecular mass of 198 kDa . The derived protein sequence is homologous to the Drosophila melanogaster mus308 protein product, a putative DNA polymerase-helicase involved in repair of interstrand crosslinks . The C-terminal region contains the canonical DNA polymerase motifs A, B, and C found in the family A type of DNA polymerases, which includes Escherichia coli polymerase I . The N-terminal region contains a putative ATP binding domain but not motifs for a helicase . The gene was mapped by radiation hybrid analysis to chromosome 3q within an interval flanked by proximal marker D3S1303 and distal marker D3S3576 and, based on proximity to a gene that has been mapped cytogenetically, within band 3q13.31 . Arch Biochem Biophys, 1999 Jul 15, 367(2), 348 - 53 Pea chloroplast glyceraldehyde-3-phosphate dehydrogenase has uracil glycosylase activity; Wang X et al.; Pea (Pisum sativum) chloroplastic glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) was tested for uracil DNA glycosylase activity . It was found that both the chloroplast and the recombinant subunit B dehydrogenases remove uracil from poly(dA{3H}dU) . The glycosylase activity of the recombinant subunit B enzyme and that of a truncated form corresponding in length to subunit A were associated with the dehydrogenase activity in gel-filtration experiments . Both activities of the chloroplast enzyme were inhibited by antisera raised against recombinant subunit B, and both activities of the recombinant subunit B enzyme were inhibited by antisera raised against pea chloroplast glyceraldehyde-3-P dehydrogenase . Antisera raised against Escherichia coli uracil glycosylase did not affect the glycosylase activity of the recombinant subunit B enzyme . The glycosylase pH activity profile of the chloroplast dehydrogenase was unique . It is distinct from the dehydrogenase pH activity profile and from the pH activity profiles of other plant glycosylases . The glycosylase activity, but not the dehydrogenase activity, of the recombinant subunit B enzyme was inhibited by uracil . Pyridine nucleotides stimulated the glycosylase activity . To our knowledge this is the first example of a nonhuman glyceraldehyde-3-P dehydrogenase, and of an NADP-dependent glyceraldehyde-3-P dehydrogenase, that exhibits uracil glycosylase activity . Arch Biochem Biophys, 1999 Jul 15, 367(2), 216 - 24 The role of human glutathione S-transferases hGSTA1-1 and hGSTA2-2 in protection against oxidative stress; Zhao T et al.; In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human alpha-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver . We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli . Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide . In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates . For example, the catalytic efficiency (kcat/Km) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s-1 mM-1, respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s-1 mM-1, respectively . Immunotitration studies with human liver extracts showed that the antibodies against human alpha-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively . GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates . These results show that the alpha-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver . Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetraenoic acid, an important intermediate in the 5-lipoxygenase pathway . Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7905 - 9 Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene; Rocklin AM et al.; The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) . In addition to ACC, Fe(II), O2, CO2, and ascorbate are required for in vitro enzyme activity . Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme.(substrate or cofactor).NO complexes . These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O2 complexes . It is shown here that the ternary Fe(II)ACCO.ACC.NO complex is readily formed, but no Fe(II)ACCO.ascorbate.NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site . The binding modes of ACC and the structural analog alanine specifically labeled with 15N or 17O were examined by using Q-band electron nuclear double resonance (ENDOR) . The data indicate that these molecules bind directly to the iron through both the alpha-amino and alpha-carboxylate groups . These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O2 bind to the iron as a step in O2 activation . We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O2, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7871 - 6 Single-nucleotide polymorphisms can cause different structural folds of mRNA; Shen LX et al.; Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man . Genes containing one or more SNPs can give rise to two or more allelic forms of mRNAs . These mRNA variants may possess different biological functions as a result of differences in primary or higher order structures that interact with other cellular components . Here we report the observation of marked differences in mRNA secondary structure associated with SNPs in the coding regions of two human mRNAs: alanyl tRNA synthetase and replication protein A, 70-kDa subunit (RPA70) . Enzymatic probing of SNP-containing allelic fragments of the mRNAs revealed pronounced allelic differences in cleavage pattern at sites 14 or 18 nt away from the SNP, suggesting that a single-nucleotide variation can give rise to different mRNA folds . By using phosphorothioate oligodeoxyribonucleotides complementary to the region of different allelic structures in the RPA70 mRNA, but not extending to the SNP itself, we find that the SNP exerts an allele-specific effect on the accessibility of its flanking site in the endogenous human RPA70 mRNA . This further supports the allele-specific structural features identified by enzymatic probing . These results demonstrate the contribution of common genetic variation to structural diversity of mRNA and suggest a broader role than previously thought for the effects of SNPs on mRNA structure and, ultimately, biological function. Curr Biol, 1999 Jul 1, 9(13), R475 - 7 DNA repair: Polymerases for passing lesions; Bridges BA; Replicative DNA polymerases generally cannot pass lesions in the template strand . Now there is accumulating evidence for the widespread existence of a separate class of DNA polymerases that can carry out translesion synthesis in both mutagenic and error-free ways. J Med Chem, 1999 Jul 1, 42(13), 2394 - 402 Apstatin analogue inhibitors of aminopeptidase P, a bradykinin-degrading enzyme; Maggiora LL et al.; Membrane-bound aminopeptidase P (AP-P) participates in the degradation of bradykinin in several vascular beds . We have developed an inhibitor of AP-P called apstatin (1) (N-{(2S, 3R)-3-amino-2-hydroxy-4-phenyl-butanoyl}-L-prolyl-L-prolyl-L-al aninam ide); IC50,human = 2.9 microM . In the rat, apstatin can potentiate the vasodilatory effect of bradykinin, reduce blood pressure in an aortic-coarctation model of hypertension, and reduce cardiac damage and arrhythmias induced by ischemia/reperfusion . In this study, we have determined structure-activity relationships for apstatin analogues as well as for other chemical classes of inhibitors using AP-P isozymes from different sources . The most potent inhibitor was one in which the N-terminal residue of apstatin was replaced with a (2S,3R)-3-amino-2-hydroxy-5-methyl-hexanoyl residue (6, IC50,human = 0.23 microM) . The (2R,3S)-analogue of 6 was equipotent with 6 while the (2S,3S)- and (2R,3R)-analogues were considerably less potent . Apstatin analogues lacking the L-alanine or having hydroxyproline in place of the proline in the second position had reduced affinity . Certain thiol-, carboxylalkyl-, and hydroxamate-containing compounds were inhibitory in the low micromolar range . Human cytosolic AP-P isozymes and Escherichia coli AP-P exhibited different inhibitor profiles than mammalian membrane-bound AP-P isozymes . The effects of the compounds on X-Pro dipeptidase (prolidase) and leucyl aminopeptidase are also presented. J Microbiol Methods, 1999 Jul, 37(1), 93 - 6 Identification of phosphate-regulated genes by differential expression in the UV-irradiated host system; Chuang SE et al.; The UV-irradiated host system has been used for identifying protein products of genes cloned in a phage vector . By starving the host cells for phosphate immediately before UV-irradiation, we demonstrate that phosphate-regulated genes can be easily identified . By employing this new technique, we also provide evidence showing that the gpsA gene might be a new member of the phosphate starvation-inducible (psi) genes of E . coli. J Protein Chem, 1999 Apr, 18(3), 387 - 96 Nucleotide and Mg2+ induced conformational changes in GroEL can be detected by sulfhydryl labeling; Jai EA et al.; The accessibility of fluorescein-5-maleimide to sulfhydryl groups in the molecular chaperone GroEL was used to follow structural rearrangements in the protein triggered by binding Mg2+ and/or adenine nucleotides . Three peptides, each containing one of the cysteines of GroEL (C138, C458 and C519) were identified . GroEL labeled in 50 mM TrisHCl, pH 7.8, incorporated approximately 0.3 labels each on C138 and C458 . With 10 mM MgCl2, the labeling increased to approximately 0.8 labels each on C138 and C458 . The increase was partially due to a conformational change which occurred upon Mg2+ binding as well as to an increase in ionic strength . When ADP, ATP, or AMP-PNP were added to a solution of GroEL and Mg2+, C138 incorporated approximately 0.8 labels, while C458 incorporated approximately 0.1 labels . These results suggest that the binding of adenine nucleotides changed the conformation of GroEL and made a previously highly exposed sulfhydryl group inaccessible . GroEL slowly dissociated into monomers when it was extensively labeled at C458 . GroEL labeled with fluorescein-5-maleimide, under any of the conditions examined, was able to bind but not release active rhodanese . The observed variations in sulfhydryl accessibility are consistent with mechanisms that suggest binding of GroES to GroEL differs from the binding of substrate protein to GroEL, and that the binding of Mg2+ or Mg-adenine nucleotides results in conformational changes in GroEL. J Protein Chem, 1999 Apr, 18(3), 379 - 86 Site-directed mutagenesis evidence for arginine-384 residue at the active site of maize branching enzyme II; Cao H et al.; Essential arginine residues are suggested to be located at the active sites of maize branching enzymes (BE) based on the evidence that two arginine residues are conserved in all BE from various species and that as little as one arginine residue is located at the active site of maize BE by phenylglyoxal (PGO) modification . To determine the exact location of the active-site arginine residue in BE, we employed peptide mapping and site-directed mutagenesis approaches . A single trypsin-digested, {14C}PGO-labeled peptide was purified from maize BEII by two rounds of HPLC separation, but we failed to obtain amino acid sequencing information . Site-directed mutagenesis was then used to create one mutant (arginine-384 to alanine-384), R384A . Immunoblotting result showed that BEII protein was expressed at a similar level in the wild type and the R384A mutant . However, BE activity in the R384A mutant was only 1.4% of the wild type . These results support the conclusion that the conserved arginine-384 residue is important in BEII catalysis. Plant Mol Biol, 1999 May, 40(1), 99 - 110 Characterization of the pea rDNA replication fork barrier: putative cis-acting and trans-acting factors; Lopez-Estrano C et al.; It was previously shown that in pea (Pisum sativum), rDNA repeats contain a polar replication fork barrier that blocks progression of the replication machinery moving in the direction opposite to transcription . This barrier maps in the untranscribed spacer close to the 3' end of the 25S gene . Very similar barriers are also found in the rDNA of yeast, Xenopus and mammalian cultured cells . This high conservation indicates that the rDNA barrier plays a relevant biological role . Progression of replication forks through the DNA sequence where the barrier maps in pea was investigated in plasmids replicating in Escherichia coli and Saccharomyces cerevisiae . No barrier was detected in these heterologous systems, indicating that the DNA sequence by itself was insufficient to block the replication machinery . Therefore, trans-acting factors were likely to be required . Taking advantage of the natural sequence heterogeneity in pea rDNA, we obtained evidence that a 27 bp imperfect tandem repeat is involved in the arrest of replication . Moreover, nuclear protein(s) specifically bound to this repeat suggesting that this DNA/protein complex is responsible for the polar arrest of replication forks. Plant Mol Biol, 1999 May, 40(1), 45 - 54 Disruption of specific flavonoid genes enhances the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings; Pelletier MK et al.; Polyclonal antibodies were developed against the flavonoid biosynthetic enzymes, CHS, CHI, F3H, FLS, and LDOX from Arabidopsis thaliana . These antibodies were used to perform the first detailed analysis of coordinate expression of flavonoid metabolism at the protein level . The pattern of flavonoid enzyme expression over the course of seedling development was consistent with previous studies indicating that chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS) are encoded by 'early' genes while leucoanthocyanidin dioxygenase (LDOX) is encoded by a 'late' gene . This sequential expression may underlie the variations in flavonoid end-products produced during this developmental stage, as determined by HPLC analysis, which includes a shift in the ratio of the flavonols, quercetin and kaempferol . Moreover, immunoblot and HPLC analyses revealed that several transparent testa lines blocked at intermediate steps of the flavonoid pathway actually accumulated higher levels of specific flavonoid enzymes and end-products . These results suggest that specific intermediates may act as inducers of flavonoid metabolism. Plant Mol Biol, 1999 May, 40(1), 13 - 21 Distribution of two isoforms of NADP-dependent isocitrate dehydrogenase in soybean (Glycine max); Park KS et al.; Two different cDNAs that encode NADP-specific isocitrate dehydrogenase (NADP-IDH) isozymes of soybean (Glycine max) were characterized . The nucleotide sequences of the coding regions of these cDNAs have 74% identity to each other and give predicted amino acid sequences that have 83% identity to each other . Using PCR techniques, their coding regions were subcloned into a protein overexpression vector, pQE32, to yield pIDH4 and pIDH1, respectively . Both IDH4 and IDH1 enzymes were expressed in Escherichia coli as catalytically active His6 tagged proteins, purified to homogeneity by affinity chromatography on nickel chelate resin and rabbit polyclonal antibodies to each were generated . Surprisingly, antiserum to IDH4 did not react with IDH1 protein and IDH1 antiserum reacted only very weakly with IDH4 protein . IDH4 antibody reacts with a protein of expected molecular weight in cotyledon, young leaf, young root, mature root and nodules but the reaction with mature leaf tissue was low compared to other tissues . Western blot results show that IDH1 was not expressed in young roots but a protein that reacts with the IDH1 antibody was highly expressed in leaves, showing that there was tissue-specific accumulation of NADP-IDH isozymes in soybean. Appl Biochem Biotechnol, 1999 Apr, 80(1), 23 - 37 Heat-shock and stringent responses have overlapping protease activity in Escherichia coli . Implications for heterologous protein yield; Harcum SW et al.; The cellular response of a heat-shocked controlled chemostat of Escherichia coli JM105 {pSH101} was characterized and compared to that of a similar culture induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) . The proteases elicited by the IPTG pulse were previously shown to be upregulated by the stringent stress response and were shown here to be upregulated by heat shock, although to a lesser extent . Owing to the apparent overlap between these responses, a relaxed mutant (rel-, devoid of the stringent response; JM109) was examined for its response to both a chemically imposed stringent response and to IPTG induction in controlled chemostats . There was no significant upregulation of protease activity under either imposed stress . More important, a nine-fold increase of chloramphenicol acetyl-transferase (CAT) activity was found for the IPTG-induced relaxed mutant culture . Additionally, the responses from heat shock and IPTG induction were examined in batch cultures . The culture that was simultaneously IPTG-induced and heat-shocked was observed to have the highest CAT activity as well as the most rapid loss in activity after a maximum . Control experiments indicated that the heat shock did not affect loss of CAT activity; instead, the loss of activity correlated with the amount of CAT synthesized . Furthermore, an increase in CAT expression was found during heat shock . Results indicated that heat shock and, alternatively, the use of stringent response-mutant hosts could both be used to facilitate increased recombinant protein yields in the E . coli expression system. Appl Biochem Biotechnol, 1999 Apr, 80(1), 13 - 22 Production and characterization of an antibody specific for a novel protein serine/threonine kinase, MPK38, highly expressed in hematopoietic cells; Yang Y et al.; We report an antibody that selectively recognizes MPK38, a new protein serine/threonine kinase closely related to the SNF1 serine/threonine kinase family . This antibody recognized a region of the N-terminal kinase catalytic domain and part of the remaining C-terminal portion and was sensitive enough to detect a 72-kDa recombinant MPK38 in insect cells by Western blotting . Immunoblot analysis showed that the recombinant MPK38 was expressed in a time-dependent manner and reached a maximum after 48 h postinfection . In addition, the immune complex kinase assay revealed that the recombinant and endogenous MPK38 protein autophosphorylated in vitro . Phosphoamino acid analysis of autophosphorylated MPK38 protein showed that the phosphorylation was exclusively on serine and threonine residues, suggesting that MPK38 is a protein serine/threonine kinase . Thus, this antibody could be helpful for elucidating the biological functions of MPK38 in the MPK38-expressing cells. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 8178 - 83 Structural basis of chaperone self-capping in P pilus biogenesis; Hung DL et al.; PapD is an immunoglobulin-like chaperone that mediates the assembly of P pili in uropathogenic strains of Escherichia coli . It binds and caps interactive surfaces on pilus subunits to prevent their premature associations in the periplasm . We elucidated the structural basis of a mechanism whereby PapD also interacts with itself, capping its own subunit binding surface . Crystal structures of dimeric forms of PapD revealed that this self-capping mechanism involves a rearrangement and ordering of the C2-D2 and F1-G1 loops upon dimerization which might ensure that a stable dimer is not formed in solution in spite of a relatively large dimer interface . An analysis of site directed mutations revealed that chaperone dimerization requires the same surface that is otherwise used to bind subunits. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7809 - 14 Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation; Tien M et al.; The ability of peroxynitrite to modify amino acid residues in glutamine synthetase (GS) and BSA is greatly influenced by pH and CO2 . At physiological concentrations of CO2 (1.3 mM), the generation of carbonyl groups (0.2-0.4 equivalents/subunit) is little affected by pH over the range of 7.2-9.0, but, in the absence of CO2, carbonyl formation increases (from 0.1- 1.2 equivalents/subunit) as the pH is raised from 7.2 to 10.5 . This increase is attributable, in part but not entirely, to the increase in peroxynitrite (PN) stability with increasing pH . Of several amino acid polymers tested, only those containing lysine residues yielded carbonyl derivatives . In contrast, the nitration of tyrosine residues of both GS and BSA at pH 7.5 almost completely depends on the presence of CO2 . However, the pH profiles of tyrosine nitration in GS and BSA are not the same . With both proteins, nitration decreases approximately 65% with increasing pH over the range of 7.2-8.4, but, then in the case of GS only, there is a 3.4-fold increase in the level of nitration over the range pH 8.4-8.8 . The oxidation of methionine residues in both proteins and in the tripeptide Ala-Met-Ala was inhibited by CO2 at both high and low pH values . These results emphasize the importance of controlling the pH and CO2 concentrations in studies involving PN and indicate that PN is not likely to contribute appreciably to carbonyl formation or oxidation of methionine residues of proteins at physiological pH and CO2 concentrations. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7785 - 90 Structure of the subunit c oligomer in the F1Fo ATP synthase: model derived from solution structure of the monomer and cross-linking in the native enzyme; Dmitriev OY et al.; The structure of the subunit c oligomer of the H+-transporting ATP synthase of Escherichia coli has been modeled by molecular dynamics and energy minimization calculations from the solution structure of monomeric subunit c and 21 intersubunit distance constraints derived from cross-linking of subunits . Subunit c folds in a hairpin-like structure with two transmembrane helices . In the c12 oligomer model, the subunits pack to form a compact hollow cylinder with an outer diameter of 55-60 A and an inner space with a minimal diameter of 11-12 A . Phospholipids are presumed to pack in the inner space in the native membrane . The transmembrane helices pack in two concentric rings with helix 1 inside and helix 2 outside . The calculations strongly favor this structure versus a model with helix 2 inside and helix 1 outside . Asp-61, the H+-transporting residue, packs toward the center of the four transmembrane helices of two interacting subunits . From this position at the front face of one subunit, the Asp-61 carboxylate lies proximal to side chains of Ala-24, Ile-28, and Ala-62, projecting from the back face of a second subunit . These interactions were predicted from previous mutational analyses . The packing supports the suggestion that a c-c dimer is the functional unit . The positioning of the Asp-61 carboxyl in the center of the interacting transmembrane helices, rather than at the periphery of the cylinder, has important implications regarding possible mechanisms of H+-transport-driven rotation of the c oligomer during ATP synthesis. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7780 - 4 The gamma-subunit rotation and torque generation in F1-ATPase from wild-type or uncoupled mutant Escherichia coli; Omote H et al.; The rotation of the gamma-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase (FOF1) . The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type . A fluorescently labeled actin filament connected to the gamma-subunit of the F1 sector rotated on addition of ATP . This progress enabled us to analyze the gammaM23K (the gamma-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation . We found that the F1 sector produced essentially the same frictional torque, regardless of the mutation . These results suggest that the gammaM23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa. Biochemistry, 1999 Jul 6, 38(27), 8808 - 19 Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids; Short GF 3rd et al.; An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs . The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E . coli release factor 1 (RF1) . Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E . coli dihydrofolate reductase . The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest . The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E . coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG) . When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation . It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds. Acta Crystallogr D Biol Crystallogr, 1999 Jul, 55 ( Pt 7), 1368 - 9 Rhombohedral crystals of 2-dehydro-3-deoxygalactarate aldolase from Escherichia coli; Blackwell NC et al.; 2-Dehydro-3-deoxygalactarate (DDG) aldolase (E.C . 4.1.2.20) catalyzes the reversible aldol cleavage of DDG and 2-dehydro-3-deoxyglucarate to pyruvate and tartronic semialdehyde . Rhombohedral crystals of recombinant DDG aldolase from Escherichia coli K-12 were obtained . The crystals belong to space group R32 with unit-cell parameters a = 93 A, alpha = 85 degrees . The crystals diffract to beyond 1.8 A resolution on a Cu Kalpha rotating-anode generator . The asymmetric unit is likely to contain two molecules, corresponding to a packing density of 1.34 A3 Da-1. Acta Crystallogr D Biol Crystallogr, 1999 Jul, 55 ( Pt 7), 1350 - 2 N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms; Gil F et al.; The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E . coli, allowing enzyme purification in three steps . NAGK exhibits high specific activity (1.1 micromol s-1 mg-1), lacks Met1 and forms dimers (shown by cross-linking) . Crystals of unliganded NAGK diffract to 2 A and belong to space group P6122 or its enantiomorph P6522 (unit-cell parameters a = b = 78.6, c = 278.0 A) with two monomers in the asymmetric unit . Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 A and belong to space group C2221 (unit-cell parameters a = 60.0, b = 71.9, c = 107.4 A), with one monomer in the asymmetric unit . NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase. Acta Crystallogr D Biol Crystallogr, 1999 Jul, 55 ( Pt 7), 1309 - 19 Errors and reproducibility in electron-density map interpretation; Mowbray SL et al.; Three investigators, with varying levels of experience, independently built and refined the structure of Escherichia coli ribokinase at 2.6 A resolution . At the end of the refinement/rebuilding processes the models had essentially converged, although each had its own particular pattern of remaining errors . The subsequent refinement of the same structure at 1.8 A resolution allowed an overall quality check of each of the lower resolution models, and an analysis of which graphics-based tools were generally most efficient in locating these errors . Criteria which are useful in the application of Ramachandran, main-chain and side-chain database and real-space fit analyses are presented. Acta Crystallogr D Biol Crystallogr, 1999 Jul, 55 ( Pt 7), 1257 - 63 Structure of the histidine-containing phosphotransfer (HPt) domain of the anaerobic sensor protein ArcB complexed with the chemotaxis response regulator CheY; Kato M et al.; The three-dimensional structure of the HPt domain of ArcB complexed with CheY has been determined using the molecular-replacement method . The structure was refined to a crystallographic R factor of 18.3% at 2.68 A resolution . The final model included 1899 protein atoms (117 residues from the HPt domain and 128 residues from CheY), one sulfate ion and 44 solvent molecules . In the crystal, CheY molecules stacked along the a axis of the cell with no interactions between neighbouring rows and the HPt domain bridged the CheY molecules . The phosphodonor residue His715 was fully exposed to the solvent region, even though the HPt domain was in contact with four molecules of CheY . CheY showed significant conformational change . This indicates that the HPt domain has a rigid structure when complexed with CheY. Biochemistry, 1999 Jul 6, 38(27), 8831 - 8 HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis; Trent MS et al.; Internal fatty acylation of proteins is a recognized means of modifying biological behavior . Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor . Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis . Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis . TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH . NAI had no effect . Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity . Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH . Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity. Biochemistry, 1999 Jul 6, 38(27), 8751 - 61 Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process; Tsai CH et al.; Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D) . These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp . We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs . The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions . The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect . r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka . Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect . Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature . r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure . The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity . Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect . Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process . There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process . Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule. Biochemistry, 1999 Jul 6, 38(27), 8657 - 70 The solution structure of oxidized Escherichia coli cytochrome b562; Arnesano F et al.; The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts . The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state . The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues . The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller . This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2 . The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained . Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility . The pKa values affecting the hyperfine-shifted signals are also discussed . Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity. Intervirology, 1999, 42(1), 17 - 21 A novel protein tag from herpes simplex virus type 1 DNA polymerase; Schreiner U et al.; An epitope (HPOL) derived from the so-called thumb region of the herpes simplex virus type 1 DNA polymerase in combination with a monoclonal antibody (MAb 1051c) was tested for protein tagging . Using a conventional expression vector, a DNA cassette encoding the HPOL epitope was fused to the C-terminus of the dihydrofolate reductase (DHFR) gene such that the recombinant DHFR contained both a N-terminal HIS-tag and a C-terminal HPOL tag . Expression of recombinant DHFR in Escherichia coli cells was compared by Western blot analysis using either mouse RGS.HIS antibody or MAb 1051c . Immunostaining revealed that both antibodies reacted specifically with DHFR, but the detection sensitivity achieved with MAb 1051c was about 15-fold greater using a standard staining protocol . An HPOL antibody column was successfully applied for affinity purification of DHFR, demonstrating the usefulness of the HPOL epitope/MAb 1051c system for protein tagging, expression monitoring and purification of HPOL-tagged recombinant proteins. J Biochem (Tokyo), 1999 Jul, 126(1), 218 - 25 A cambialistic SOD in a strictly aerobic hyperthermophilic archaeon, Aeropyrum pernix; Yamano S et al.; The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized . The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6 kDa . The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs . The metal binding residues conserved in Mn- and Fe-SODs were also found in A . pernix SOD . When the SOD gene was expressed in E . coli cells, the product formed a homodimer, and contained both Mn and Fe . Metal reconstitution experiments showed that A . pernix SOD is cambialistic, i.e . active with either Fe or Mn . The specific activities were 906 U/mg with Mn and 175 U/mg with Fe . No loss of activity of Mn-reconstituted SOD was observed at 105 degrees C even after 5 h incubation . Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A . pernix even at concentrations up to 400 mM . This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe . With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe. J Biochem (Tokyo), 1999 Jul, 126(1), 194 - 9 Effects of subunit I mutations on redox-linked conformational changes of the Escherichia coli bo-type ubiquinol oxidase revealed by Fourier-transform infrared spectroscopy; Yamazaki Y et al.; Cytochrome bo is the heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli, and functions as a redox-coupled proton pump . As an extension to our mutagenesis and Fourier-transform infrared studies on ion pumps, we examined the effects of subunit I mutations on redox-linked protein structural changes in cytochrome bo . Upon photo-reduction in the presence of riboflavin, Y288F and H333A showed profound effects in their peptide backbone vibrations (amide-I and amide-II), probably due to the loss of CuB or replacement of high-spin heme o with heme B . In the frequency region of protonated carboxylic C=O stretching vibrations, negative 1,743 cm-1 and positive 1,720 cm-1 bands were observed in the wild-type; the former shifted to 1,741 cm-1 in E286D but not in other mutants including D135N . This suggests that Glu286 in the D-channel is protonated in the air-oxidized state and undergoes hydrogen bonding changes upon reduction of the redox metal centers . Two pairs of band shifts at 2,566 (+)/2,574 (-) and 2,546 (+)/2,556 (-) cm-1 in all mutants indicate that two cysteine residues not in the vicinity of the metal centers undergo redox-linked hydrogen bonding changes . Cyanide had no effect on the protein structural changes because of the rigid local protein structure around the binuclear center or the presence of a ligand(s) at the binuclear center, and was released from the binuclear center upon reduction . This study establishes that cytochrome bo undergoes unique redox-linked protein structural changes . Localization and time-resolved analysis of the structural changes during dioxygen reduction will facilitate understanding of the molecular mechanism of redox-coupled proton pumping at the atomic level. J Biochem (Tokyo), 1999 Jul, 126(1), 98 - 103 Fluoride-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption and EPR spectroscopies; Tsubaki M et al.; Cytochrome bd-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli contains two hemes b (b558 and b595) and one heme d as redox metal centers . To clarify the structure of the reaction center, we analyzed the fully oxidized enzyme by visible and EPR spectroscopies using fluoride ion as a monitoring probe . The visible spectral changes upon fluoride-binding were typical of ferric iron-chlorine species, indicating heme d as a primary binding site . The negative peak at 645 nm in the difference spectrum indicates that heme b595 also provides the low-affinity fluoride-binding site . Fluoride-binding caused a complete disappearance from the EPR spectra of the low-spin signals ascribable to heme d and spectral changes in both rhombic and axial high-spin signals . After fluoride-binding, each component of the rhombic high-spin signal showed superhyperfine splitting arising from the interaction of the unpaired spin of the heme d iron with the nuclear magnetic moment of 19F . The axial high-spin species was converted to a new rhombic high-spin species assignable to heme b595-fluoride . The g = 2 component of this new species also gave 19F-superhyperfine splitting . These results indicate that both heme d and heme b595 can coordinate with a fluoride ion with different affinities in the fully oxidized state. J Biochem (Tokyo), 1999 Jul, 126(1), 78 - 83 Function of the propeptide region in recombinant expression of active procathepsin L in Escherichia coli; Ogino T et al.; In order to determine the functional role of the procathepsin L propeptide region for the preparation of active recombinant rat cathepsin L (CL), cDNAs encoding two short-length propeptides (C-terminal 2 and 27 residues) and the full-length (96 residues) one plus the entire CL were expressed as two soluble fusion proteins with a fragment of maltose-binding protein and an insoluble fusion protein with glutathione-S-transferase in Escherichia coli, respectively . After refolding of the insoluble fusion protein, each gene product was purified to homogeneity by amylose or glutathione-Sepharose-4B affinity column, and digestion with factor Xa and alpha-thrombin under alkaline conditions (pH approximately 8.0) led to the elution of two pure short-length procathepsin Ls (PCLs) and a full-length one, respectively . The enzymatic activity, estimated by hydrolytic assaying of benzoxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide under acidic conditions (pH 5.5), indicated that the two short-length PCLs exhibited in a great loss of the activity, as compared with the full-length PCL . The CD spectra of the short-length PCLs were different from that of the full-length one . The present results clearly show that the full-length propeptide is essential for construction of the active tertiary structure of CL at the stage of recombinant protein expression, although the expression of CL itself in E . coli does not require the propeptide . Based on the tertiary structure of PCL, the propeptide region necessary for the construction of the CL active structure has been discussed. Mutat Res, 1999 Jun 25, 442(2), 79 - 87 Evaluation of Escherichia coli DJ4309 expressing human P450 1A2 in mutagenicity testing of complex food mixtures; Constable A et al.; Heterocyclic aromatic amines (HAAs) are potent bacterial mutagens and potential human carcinogens formed in heat processed proteins . The Ames test (strain TA98) is a useful mutagenicity test system to screen food products for these compounds . HAAs require activation to their genotoxic forms, and in the Ames test, a rat liver S-9 preparation is normally used . In order to better understand the mechanisms of mutagen activation with respect to human metabolism, new bacterial strains containing human cytochrome P450s and other metabolic enzymes have recently been developed . We have investigated the capacity of one of these strains, DJ4309 {Josephy et al., Chem . Res . Toxicol . 11 (1998) 70-74} as a screening tool for mutagens in food products . DJ4309 expresses the human P450 1A2, human NADPH cytochrome reductase and the bacterial acetyl CoA:arylamine N-acetyltransferase . This strain is as sensitive as the Ames system to the mutagenic effects of the heterocyclic aromatic amines 2-amino-3-methylimidazo{4,5-f}quinoline, 2-amino-3, 4-dimethylimidazo{4,5-f}quinoline and 2-amino-3,8-dimethylimidazo{4, 5-f}quinoxaline, but less sensitive to 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine . However, the mutagenicity of the arylamine 2-aminofluorene is considerably higher in DJ4309 than in the Ames test system . Meat extracts with a total HAA content ranging from less than 2 ng/g to 20 ng/g are efficiently detected by the Ames TA98 strain with rat liver S-9 activation . DJ4309 is less sensitive, with fewer revertants induced over the same dose range . Unknown compounds present in the meat extracts appear to inhibit the activity of the P450 1A2 enzyme in the DJ4309 strain . We have therefore demonstrated that although DJ4309 is a useful tool for mechanistic studies in chemical carcinogenesis, the screening of complex food matrices for HAAs by this bacterial strain must be conducted with caution . Mutat Res, 1999 Jun 25, 442(2), 61 - 8 Distinct responses of a recA::luxCDABE Escherichia coli strain to direct and indirect DNA damaging agents; Min J et al.; The recombinant Escherichia coli strain DPD2794 containing a recA::luxCDABE fusion is used to detect genotoxicity of various chemicals . Genotoxic agents were previously categorized into two groups, Direct DNA Damaging (DDD) agents and Indirect DNA Damaging (IDD) agents; these two groups have been distinguished with this strain . Minimum detectable concentrations of the DDD agents were about one to five orders of magnitude lower than those of the IDD agents . The response patterns of this strain to DDD agents differed from those to IDD agents in terms of kinetics and the forms of the dose-dependent response . Mutat Res, 1999 Jun 30, 427(2), 89 - 97 Comparison of selectable and plaque assay systems to detect menadione- and UV-induced lacI mutations in mammalian cells; Andrew SE et al.; We have compared the spontaneous mutation frequency and spectrum of lacI genes recovered from a rat embryonic fibroblast line transfected with a lambda-phage shuttle vector (Rat2lambdalacI) using both the traditional plaque assay as well as a positive selection assay . In addition, mutation frequencies and spectrum were determined after treatment of the cells with either the intracellular superoxide-generating compound, menadione, or UVC light . The differences in mutation frequency between the two systems suggested that the selectable assay was better at discerning relatively small mutation frequency increases, more rapidly and at lower cost, than the plaque assay method . Some novel lacI mutations were observed in mutants derived from the selectable assay . This indicates that the selectable assay system may be a useful tool for assessing the mutagenic potential of different agents . EMBO J, 1999 Jul 1, 18(13), 3868 - 75 A human DNA editing enzyme homologous to the Escherichia coli DnaQ/MutD protein; Hoss M et al.; Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis . However, editing exonucleases can be functions of separate polypeptides . We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame . The protein expressed from the cloned human sequence exhibits 3' exonuclease activity . The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon . The gene maps to human chromosome 3p21.2-21.3 . In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease. EMBO J, 1999 Jul 1, 18(13), 3856 - 67 Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli; Uga H et al.; In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences . To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region . The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis . Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity . The mutated incC mini-F plasmids lost the function to control the copy number . The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE . These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein . Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important . The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number. EMBO J, 1999 Jul 1, 18(13), 3800 - 7 Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome; Pape T et al.; The fidelity of aminoacyl-tRNA (aa-tRNA) selection by the bacterial ribosome is determined by initial selection before and proofreading after GTP hydrolysis by elongation factor Tu . Here we report the rate constants of A-site binding of a near-cognate aa-tRNA . The comparison with the data for cognate aa-tRNA reveals an additional, important contribution to aa-tRNA discrimination of conformational coupling by induced fit . It is found that two rearrangement steps that limit the chemical reactions of A-site binding, i.e . GTPase activation (preceding GTP hydrolysis) and A-site accommodation (preceding peptide bond formation), are substantially faster for cognate than for near-cognate aa-tRNA . This suggests an induced-fit mechanism of aa-tRNA discrimination on the ribosome that operates in both initial selection and proofreading . It is proposed that the cognate codon-anticodon interaction, more efficiently than the near-cognate one, induces a particular conformation of the decoding center of 16S rRNA, which in turn promotes GTPase activation and A-site accommodation of aa-tRNA, thereby accelerating the chemical steps . As kinetically favored incorporation of the correct substrate has also been suggested for DNA and RNA polymerases, the present findings indicate that induced fit may contribute to the fidelity of template-programed systems in general. EMBO J, 1999 Jul 1, 18(13), 3793 - 9 SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA); Karzai AW et al.; In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of the partially synthesized nascent polypeptide chain . The SsrA-tagged protein is then degraded by C-terminal-specific proteases . SmpB, a unique RNA-binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality-control system . Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA-defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs . Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo . Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain . SsrA RNA is present at wild-type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors. EMBO J, 1999 Jul 1, 18(13), 3776 - 82 Assaying RNA chaperone activity in vivo using a novel RNA folding trap; Clodi E et al.; In the absence of proteins, RNAs often misfold in vitro due to alternative base pairings which result from the molecule being trapped in inactive conformations . We identify an in vivo folding trap in the T4 phage td gene, caused by nine base pairs between a sequence element in the upstream exon of the td gene and another at the 3' end of the intron . During translation, the ribosome resolves this interaction; consequently the intron folds correctly and splicing occurs . The introduction of a stop codon upstream of this base pairing prevents resolution of the inactive structure so that splicing cannot proceed . We have used this folding trap to probe for RNA binding proteins which, when overexpressed, either resolve the misfolded structure or impede its formation in vivo . We distinguish between proteins which recognize the intron structure and those which bind non-specifically and apparently ignore the intron . The first class, e.g . Neurospora crassa CYT-18, can rescue the exonic trap and intron mutants which cause a structural defect . However, known RNA chaperones such as Escherichia coli StpA and S12 and the HIV protein NCp7, only resolve the exonic trap without suppressing intron mutations . Thus, this structural trap enables detection of RNA chaperone activity in vivo. EMBO J, 1999 Jul 1, 18(13), 3558 - 63 Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0 A resolution; Williams KA et al.; Electron cryomicroscopy of frozen-hydrated two-dimensional crystals of NhaA, a Na+/H+ antiporter from Escherichia coli predicted to have 12 transmembrane alpha-helices, has facilitated the calculation of a projection map of NhaA at 4.0 A resolution . NhaA was homologously expressed in E.coli with a His6 tag, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography . Two-dimensional crystals were obtained after reconstitution of purified protein with E.coli lipids . The projection map reveals that this secondary transporter has a highly asymmetric structure in projection . NhaA exhibits overall dimensions of approximately 38x48 A with a ring-shaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density containing several peaks indicative of transmembrane helices . Two crystal forms with p22121 symmetry show tightly packed dimers of NhaA which differ in the interactions between adjacent dimers . This work provides the first direct glimpse into the structure of a secondary transporter. Development, 1999 Aug, 126(15), 3415 - 24 Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality; Hamada Y et al.; Notch family genes encode transmembrane proteins involved in cell-fate determination . Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E . coli (beta)-galactosidase gene . The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity . Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable . Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart . Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice . Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice . Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se . In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1 . In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice . The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function. Vet Microbiol, 1999 Jun 1, 67(1), 61 - 74 Monoclonal antibodies recognising fimbriae F107 (F18) of an oedema disease causing strain of Escherichia coli; Rosocha J et al.; Escherichia coli isolated from experimentally induced oedema disease in pigs was used for the isolation and purification of F107 fimbriae . The reference strain was probed using membrane DNA hybridisation for the presence of fed A gene . F107 fimbriae were purified on FPLC and purity was checked on HPLC and SDS PAGE . A protein with major subunit of 18.9 kDa was used for Mabs preparation . Mabs reacted with 18.9 kDa protein previously classified as a major fimbrial subunit and were able to detect F107 fimbriae in immunoelectron microscopy on the surface of the strains 107/86 and 8872 . Other strains used in this study did not express any fimbriae . Western blot analysis and F107 ELISA confirmed, that Mabs react with 18.9 kDa subunit whereas strains passaged many times in laboratory did not express F107 fimbriae. Vet Parasitol, 1999 Jun 1, 83(1), 55 - 64 Lymphocyte responses to mitogens and rickettsial antigens in sheep experimentally infected with Ehrlichia (Cytoecetes) phagocytophila; Gokce HI et al.; Infection of sheep with Ehrlichia (Cytoecetes) phagocytophila, the causative agent of tick-borne fever (TBF), was characterised by a significant reduction in lymphocyte reactivity to the mitogens phytohaemagglutinin, concanavalin A, pokeweed mitogen and Escherichia coli lipopolysaccharide during the period of rickettsiaemia . The addition of the prostaglandin inhibitor, indomethacin, or the nitric oxide inhibitor, N(G)-monomethyl-L-arginine, had no significant effect on the suppressive effects of E . phagocytophila on lymphocyte reactivity to the mitogens . However, peripheral blood lymphocytes obtained from primed sheep proliferated in the presence of live or heat-inactivated E . phagocytophila . Antigen-specific proliferation was detected in lymphocytes samples obtained 11 to 21 days post-inoculation with E . phagocytophila. Virus Res, 1999 Apr, 60(2), 137 - 45 Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus; Tibbles KW et al.; Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans . In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets . The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro . A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification . Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive . From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1 . This tentatively completes the processing map for the ORF1 region with respect to 3CLpro. J Biochem Biophys Methods, 1999 May 13, 39(3), 143 - 51 Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract; Sun H et al.; A two-dimensional (2-D) gel electrophoresis system in which native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) are performed subsequently to analyze protein mixtures is described . Reasonably good resolution and excellent reproducibility was obtained when the proteins in the soluble protein extract from E . coli cells were separated using this procedure . Perhaps more importantly, the relevance of this native/SDS-2-D PAGE for the detection of protein interactions in a complicated protein mixture was examined using the interaction between interleukin-2 (IL-2) and its receptor alpha chain (IL-2Ralpha) in the E . coli protein extract as a model system . Native gel was used to preserve the interactions between the two molecules and SDS gel was used to maximize the separation of the denatured proteins . Mobility changes of these two proteins on 2-D maps resulted from the formation of IL-2/IL-2-2Ralpha complex were clearly observed despite of the presence of a large number of other protein spots . Thus, this approach is a useful complement to the standard 2-D gel electrophoresis system for analyzing complicated protein mixture, especially for the study of protein interactions. J Biol Chem, 1999 Jul 9, 274(28), 19913 - 8 Delta psi stimulates membrane translocation of the C-terminal part of a signal sequence; van Dalen A et al.; For several proteins in Escherichia coli it has been shown that the protonmotive force (pmf) dependence of translocation can be varied with the signal sequence composition, suggesting an effect of the pmf on the signal sequence . To test this possibility, we analyzed the effect of the membrane potential on translocation of the signal sequence . For this purpose, a precursor peptide was used (SP+7), corresponding to the signal sequence of PhoE with the first seven amino acids of the mature part that can be processed by purified leader peptidase . Translocation was studied in pure lipid vesicles containing leader peptidase, with its active site inside the vesicles . In the presence of a positive inside Delta psi, the amount of processing of SP+7 was significantly higher than without a Delta psi, indicating that the translocation of the cleavage region is stimulated by Delta psi . Replacement of the helix-breaking glycine residue at position -10 in the signal sequence for a leucine abolished the effect of Delta psi on the translocation of the cleavage region . It is concluded that Delta psi directly acts on the wild type signal sequence by stimulating the translocation of its C terminus . We propose that Delta psi acts on the signal sequence by stretching it into a transmembrane orientation. J Biol Chem, 1999 Jul 9, 274(28), 19723 - 30 Mutational analysis of the PapB transcriptional regulator in Escherichia coli . Regions important for DNA binding and oligomerization; Xia Y et al.; PapB is a transcriptional regulator in the control of pap operon expression in Escherichia coli . There are PapB homologous proteins encoded by many fimbrial gene systems that are involved in the regulation of fimbriae-adhesin production, and previous studies suggested that PapB binds DNA through minor groove contact . Both deletion and alanine-scanning mutagenesis were used to identify functionally important regions of the PapB protein . Mutations altering Arg61 or Cys65 caused deficiency in DNA binding, indicating that these residues are critical for PapB binding to DNA . Alanine substitutions at positions 35-36, 53-56, and 74-76 resulted in mutants that were impaired in oligomerization . All these amino acid residues are conserved among the PapB homologous proteins, suggesting their importance in the whole family of regulatory proteins . The transcriptional efficiency of all the mutants was clearly reduced as compared with that of wild-type PapB . Taken together, we have localized regions in the PapB protein that are involved in DNA binding and oligomerization, and our results show that both functions are required for its activity as a transcriptional regulator.
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