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 spontaneou