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| J Pediatr Gastroenterol Nutr, 1999 Feb, 28(2), 147 - 51 Lymphocyte subset profile of young healthy children residing in a rural area: possible role of recurrent gastrointestinal infections; Granot E et al.; BACKGROUND: Lymphocyte subsets in healthy children are currently characterized by age-related standards . Because antigenic stimuli play a role in maturation of the immune system after birth, there is a question of whether cellular immune development differs in infants whose living conditions entail extensive antigenic exposure and infants growing up in a more protected environment . METHODS: Peripheral blood lymphocyte subsets were studied in two populations of children of similar age and nutritional status; children belonging to a rural population residing in proximity with farm animals and children from an economically privileged urban population . In each population, children studied included a group with an acute diarrheal episode and a healthy control group . RESULTS: Among rural population children, 65% had experienced at least one episode of gastroenteritis within the previous 3-month-period, compared with less than 10% of urban population children . In the rural population group 15% had experienced two or more episodes of gastroenteritis . The proportion of helper T cells was similar in rural population and urban population children . Among helper T cells, the proportion of CD29+ "memory" cells of the total CD4+ helper T cells was more than two times higher than those in rural population children . The proportion of CD8 cells was higher in rural population children than in urban population children, and the proportion of natural killer cells, CD56+ and CD57+, was two to three times higher in rural population children . Within each population, peripheral blood lymphocyte subsets did not differ between the healthy control group and those with acute diarrhea . CONCLUSIONS: In young children exposure to environmental pathogens and specifically to gastrointestinal antigenic stimuli is a major factor affecting development of the cellular immune response . Young children who have experienced enhanced infectious exposure have a peripheral blood lymphocyte profile similar to that of adults. Prog Nucleic Acid Res Mol Biol, 1999, 62, 55 - 108 Degradation of mRNA in Escherichia coli: an old problem with some new twists; Coburn GA et al.; Metabolic instability is a hallmark property of mRNAs in most if not all organisms and plays an essential role in facilitating rapid responses to regulatory cues . This article provides a critical examination of recent progress in the enzymology of mRNA decay in Escherichia coli, focusing on six major enzymes: RNase III, RNase E, polynucleotide phosphorylase, RNase II, poly(A) polymerase(s), and RNA helicase(s) . The first major advance in our thinking about mechanisms of RNA decay has been catalyzed by the possibility that mRNA decay is orchestrated by a multicomponent mRNA-protein complex (the "degradosome") . The ramifications of this discovery are discussed and developed into mRNA decay models that integrate the properties of the ribonucleases and their associated proteins, the role of RNA structure in determining the susceptibility of an RNA to decay, and some of the known kinetic features of mRNA decay . These models propose that mRNA decay is a vectorial process initiated primarily at or near the 5' terminus of susceptible mRNAs and propagated by successive endonucleolytic cleavages catalyzed by RNase E in the degradosome . It seems likely that the degradosome can be tethered to its substrate, either physically or kinetically through a preference for monphosphorylated RNAs, accounting for the usual "all or none" nature of mRNA decay . A second recent advance in our thinking about mRNA decay is the rediscovery of polyadenylated mRNA in bacteria . Models are provided to account for the role of polyadenylation in facilitating the 3' exonucleolytic degradation of structured RNAs . Finally, we have reviewed the documented properties of several well-studied paradigms for mRNA decay in E . coli . We interpret the published data in light of our models and the properties of the degradosome . It seems likely that the study of mRNA decay is about to enter a phase in which research will focus on the structural basis for recognition of cleavage sites, on catalytic mechanisms, and on regulation of mRNA decay. Gene, 1999 Jan 21, 226(2), 365 - 73 Isolation and expression of the genes encoding the acidic ribosomal phosphoproteins P1 and P2 of the medfly Ceratitis capitata; Gagou ME et al.; The genes of the acidic ribosomal proteins P1 and P2 (CcP1 and CcP2) of the medfly Ceratitis capitata were isolated from a genomic library using homologue DNA probes prepared by PCR . Sequencing and characterization of the two genes revealed strong similarities of the encoded amino acid sequence to the homologous proteins of Drosophila melanogaster and other eukaryotic species . The predicted amino acid sequences of the CcP1 and CcP2 proteins shared an almost identical carboxyl terminal sequence of 10 amino acids common to most known acidic ribosomal proteins . The CcP2 gene lacked intervening sequences in contrast to the CcP1 gene, which was interrupted by an intron of 188 nucleotides . Both genes were cloned in expression pT7 vectors and were expressed in Esherichia coli . The 17- and 15-kDa recombinant proteins reacted with a monoclonal antibody specific to the highly conserved carboxyl terminus of eukaryotic acidic ribosomal proteins, confirming their equivalence to these ribosomal components . Both recombinant proteins were electrophoretically identical to acidic proteins extracted from purified ribosomes of C . capitata. Gene, 1999 Jan 21, 226(2), 263 - 71 Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding; Subrahmanyam S et al.; We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein . This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis . By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA . We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E . coli . We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E . coli genome that has a very low binding efficiency compared with the bio operator region. Gene, 1999 Jan 21, 226(2), 233 - 42 Isolation and confirmation of function of the Coccidioides immitis URA5 (orotate phosphoribosyl transferase) gene; Yu JJ et al.; The OPRTase (URA5) gene of the human pathogenic fungus, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped and expressed both by transformation of Escherichia coli and by complementation of wdura5Delta, an auxotrophic strain of Wangiella dermatitidis (Wd) with a disrupted URA5 gene . A functional assay of the recombinant URA5 expressed by E . coli was conducted to ensure that the isolated Ci gene encodes the appropriate enzyme . In the absence of a transformation system for Ci, we also used a reported method of introduction of heterologous DNA into cells of the phylogenetically related fungus, Wangiella dermatitidis, to confirm the function of the Ci URA5 gene . Both the genomic and cDNA sequences of the Ci URA5 gene are presented . The transcription start point and two poly(A) addition sites were confirmed . The gene contains a 714-bp ORF that translates a 238-amino-acid (aa) protein of 25.5kDa and pI of 6.5 . No introns are present . The translated protein contains a single, putative N-glycosylation site . The deduced Ci protein showed 55-63% aa sequence similarity to reported fungal OPRTases . The URA5 gene was mapped to chromosome IV of Ci, and was shown to be a single copy gene by Southern and Northern hybridizations . Transformation of the wdura5Delta mutant to prototrophy was accomplished by electroporation of Wd yeast cells with the Ci URA5 gene . Cellular uptake of the heterologous DNA was confirmed by Southern hybridization . The stable transformants were unable to grow on a medium containing 5-FOA . Expression of the Ci URA5 gene can be used as a selectable marker for a transformation system, and the latter is essential for molecular studies of this pathogenic fungus. Gene, 1998 Dec 11, 224(1-2), 117 - 22 Molecular cloning of Ras cDNA from Penaeus japonicus (Crustacea, decapoda): geranylgeranylation and guanine nucleotide binding; Huang CF et al.; A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning . The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains . Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras . The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30000units/mg of protein, whereas the binding for GDP was 5000units/mg of protein . Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form. Gene, 1998 Dec 28, 225(1-2), 97 - 105 Selection of a cDNA clone for chicken high-mobility-group 1 (HMG1) protein through its unusually conserved 3'-untranslated region, and improved expression of recombinant HMG1 in Escherichia coli; Lee KB et al.; Screening of cDNA libraries for the homologous vertebrate proteins high mobility group (HMG) 1 and 2 using DNA probes based on the coding sequences is likely to result in isolation of both HMG1 and HMG2 clones, as well as pseudogenes, which may be transcribed at low levels . However, the 3'-untranslated regions (UTRs) of HMG1 and 2 are quite distinct, and unusually conserved across species . We have used this property to select the true chicken HMG1 cDNA clone from a chicken lymphocyte cDNA library in lambdagt11, using a probe based on the 3'-UTR of rat HMG1 cDNA . The chicken HMG1 cDNA clone is very similar to all the complete HMG1 cDNA clones isolated so far . We suggest that the sequence designated chicken HMG1 in the GenBank Data Library (Accession number D14314) is, in fact, that of HMG2a {and moreover that the recently reported mouse clone (Accession number AF022465), proposed to encode a new HMG protein, HMG4, is also likely to encode an HMG2a, based on the translated amino-acid sequence and 3'-UTR} . We also report much improved expression of intact recombinant HMG1 in Escherichia coli by the use of chloramphenicol rather than ampicillin selection and conditions that limit cell growth . This should be general for all members of the HMG1 (and 2) family which may be toxic to cells (possibly because of the long acidic tail), and may also prove useful in the production of other such proteins. Gene, 1998 Dec 28, 225(1-2), 31 - 8 Establishment of framework P1 clones for map-based cloning and genome sequencing: direct RFLP mapping of large clones; Shibata D et al.; Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking . The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome . Using a P1-based library of Arabidopsis thaliana with genomic inserts of 70-90kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittier, R.F., 1995 . Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking . Plant J . 7, 351-358), we have now established such a set of framework clones . To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs) . In order to establish framework clones more efficiently, we refined protocols for P1 clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes . This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes . Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap. Biochem J, 1999 Feb 15, 338 ( Pt 1), 41 - 8 Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves; Yoshimura K et al.; We have previously shown that stromal and thylakoid-bound ascorbate peroxidase (APX) isoenzymes of spinach chloroplasts arise from a common pre-mRNA by alternative splicing in the C-terminus of the isoenzymes {Ishikawa, Yoshimura, Tamoi, Takeda and Shigeoka (1997) Biochem . J . 328, 795-800} . To explore the production of mature, functional mRNA encoding chloroplast APX isoenzymes, reverse transcriptase-mediated PCR and S1 nuclease protection analysis were performed with poly(A)+ RNA or polysomal RNA from spinach leaves . As a result, four mRNA variants, one form of thylakoid-bound APX (tAPX-I) and three forms of stromal APX (sAPX-I, sAPX-II and sAPX-III), were identified . The sAPX-I and sAPX-III mRNA species were generated through the excision of intron 11; they encoded the previously identified sAPX protein . Interestingly, the sAPX-II mRNA was generated by the insertion of intron 11 between exons 11 and 12 . The use of this insertional sequence was in frame with the coding sequence and would lead to the production of a novel isoenzyme containing a C-terminus in which a seven-residue sequence replaced the last residue of the previously identified sAPX . The recombinant novel enzyme expressed in Escherichia coli showed the same enzymic properties (except for molecular mass) as the recombinant sAPX from the previously identified sAPX-I mRNA, suggesting that the protein translated from the sAPX-II mRNA is functional as a soluble APX in vivo . The S1 nuclease protection analysis showed that the expression levels of mRNA variants for sAPX and tAPX isoenzymes are in nearly equal quantities throughout the spinach leaves grown under normal conditions . The present results demonstrate that the expression of chloroplast APX isoenzymes is regulated by a differential splicing efficiency that is dependent on the 3'-terminal processing of ApxII, the gene encoding the chloroplast APX isoenzymes. J Mol Biol, 1999 Feb 12, 286(1), 257 - 65 Using loop length variants to dissect the folding pathway of a four-helix-bundle protein; Nagi AD et al.; Rop is a four-helix-bundle protein formed by the association of two helix-loop-helix monomers . The short helix-connecting loop was replaced with a series of polyglycine linkers of increasing length . These mutant proteins all appear to fold via the same general mechanism as that of the wild-type protein, even at the longest loop lengths . Replacement of the wild-type two-residue loop (Asp-Ala) with a (Gly-Gly) linker accelerates both unfolding and refolding rates . These changes in folding and unfolding kinetics likely indicate an alteration in the energy of the transition state . As the length of the glycine linker is further increased, the unfolding rate increases while the refolding rates decrease . The influence of loop length is not limited to these rates, but also impacts upon the stability of the folding intermediate . These dependences underscore the importance of loop closure and help refine the model for Rop's folding, implicating a dimeric intermediate involving hairpin formation . These observations show that loop alteration may be useful as a general technique for dissecting protein folding pathways . J Mol Biol, 1999 Feb 12, 286(1), 219 - 32 Nuclear magnetic resonance and molecular dynamics studies on the interactions of the Ras-binding domain of Raf-1 with wild-type and mutant Ras proteins; Terada T et al.; The Ras protein and its homolog, Rap1A, have an identical "effector region" (residues 32-40) preceded by Asp30-Glu31 and Glu30-Lys31, respectively . In the complex of the "Ras-like" E30D/K31E mutant Rap1A with the Ras-binding domain (RBD), residues 51-131 of Raf-1, Glu31 in Rap1A forms a tight salt bridge with Lys84 in Raf-1 . However, we have recently found that Raf-1 RBD binding of Ras is indeed reduced by the E31K mutation, but is not affected by the E31A mutation . Here, the "Rap1A-like" D30E/E31K mutant of Ras was prepared and shown to bind the Raf-1 RBD less strongly than wild-type Ras, but slightly more tightly than the E31K mutant . The backbone 1H, 13C, and 15N magnetic resonances of the Raf-1 RBD were assigned in complexes with the wild-type and D30E/E31K mutant Ras proteins in the guanosine 5'-O-(beta,gamma-imidotriphosphate)-bound form . The Lys84 residue in the Raf-1 RBD exhibited a large change in chemical shift upon binding wild-type Ras, suggesting that Lys84 interacts with wild-type Ras . The D30E/E31K mutant of Ras caused nearly the same perturbations in Raf-1 chemical shifts, including that of Lys84 . We hypothesized that Glu31 in Ras may not be the major salt bridge partner of Lys84 in Raf-1 . A molecular dynamics simulation of a model structure of the Raf-1 RBD.Ras.GTP complex suggested that Lys84 in Raf-1 might instead form a tight salt bridge with Asp33 in Ras . Consistent with this, the D33A mutation in Ras greatly reduced its Raf-I RBD binding activity . We conclude that the major salt bridge partner of Lys84 in Raf-1 may be Asp33 in Ras . J Mol Biol, 1999 Feb 12, 286(1), 95 - 104 Cross-functional analysis of the Microviridae internal scaffolding protein; Burch AD et al.; The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins . These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle . The genes encoding three Microviridae (phiX174, G4 and alpha3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species . Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles . These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible . There was one condition in which a B protein could not cross-function . The phiX174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21 degreesC . Under these conditions, assembly is arrested early in the morphogenetic pathway, before the first B protein mediated reaction . Two G4 mutants, which can productively utilize the phiX174 B protein at elevated temperatures, were isolated . Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein . The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region . J Mol Biol, 1999 Feb 12, 286(1), 71 - 81 The effect of a hydrophobic N-terminal probe on translational pausing of chloramphenicol acetyl transferase and rhodanese; Tsalkova T et al.; The effect on translational pausing of a hydrophobic probe, coumarin, at the N terminus of nascent peptides was investigated . Two different proteins, bacterial chloramphenicol acetyltransferase and bovine rhodanese, were synthesized by coupled transcription/translation in a cell-free system derived from Escherichia coli . Protein synthesis was initiated with N-formyl-Met-tRNAf or N-acetyl-S-coumarin-Met-tRNAf . Cotranslational incorporation of the coumarin derivative generated nascent polypeptides with a hydrophobic residue at their N termini . The effect of the two N-terminal groups on the size distribution and quantity of the peptides formed by translational pausing was investigated . The N-terminal coumarin caused an accumulation of nascent chloramphenicol acetyltransferase peptides in the mass range of 3.5-4.0 kDa that reflects a delay in translation at this point . No similar effect on rhodanese pause-site peptides was observed . This effect on translational pausing cannot be explained by either mRNA secondary structure or rare codons and tRNA abundance . It is suggested that the effect of N-terminal coumarin on translational pausing is the result of the interaction of the nascent peptide with components of the large ribosomal subunit along the path it follows between the peptidyl transferase center and the exit site on the distal surface . Nature, 1999 Jan 21, 397(6716), 271 - 4 Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase; Harding HP et al.; Protein synthesis and the folding of the newly synthesized proteins into the correct three-dimensional structure are coupled in cellular compartments of the exocytosis pathway by a process that modulates the phosphorylation level of eukaryotic initiation factor-2alpha (eIF2alpha) in response to a stress signal from the endoplasmic reticulum (ER) . Activation of this process leads to reduced rates of initiation of protein translation during ER stress . Here we describe the cloning of perk, a gene encoding a type I transmembrane ER-resident protein . PERK has a lumenal domain that is similar to the ER-stress-sensing lumenal domain of the ER-resident kinase Ire1, and a cytoplasmic portion that contains a protein-kinase domain most similar to that of the known eIF2alpha kinases, PKR and HRI . ER stress increases PERK's protein-kinase activity and PERK phosphorylates eIF2alpha on serine residue 51, inhibiting translation of messenger RNA into protein . These properties implicate PERK in a signalling pathway that attenuates protein translation in response to ER stress. Nature, 1999 Jan 21, 397(6716), 267 - 70 Direct interaction of microtubule- and actin-based transport motors; Huang JD et al.; The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood . For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport . In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination . Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU . As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell . These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules. Biochemistry, 1999 Feb 2, 38(5), 1652 - 8 Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli . Characterization of the beta H91, beta D392, and beta K424 mutants; Hu X et al.; Conserved and semiconserved acidic and basic residues of the beta subunit of the proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli potentially involved in proton pumping were investigated . Out of 16 charged residues studied, 6 have not been previously investigated . The most dramatic effects of mutation were observed with beta H91, beta D392, and beta K424 . beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP+ by NADH (forward reaction) and reduction of 3-acetylpyridine-NAD+ by NADPH (reverse reaction) to lower pH . This mutant catalyzed a cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADP(H) with a pH profile also shifted toward a lower pH . These results are consistent with a mechanism where the normal forward and reverse reactions are indeed limited by protonation/deprotonation of beta H91 . The cyclic reaction was affected by mutations of beta H91, probably through conformational changes involving the active NADP(H) site . The beta D392A mutant was inactive with regard to forward and reverse reactions, but showed a wild-type-like pH dependence for the partly active cyclic reaction . However, Km,app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site . Transhydrogenases contain a conserved beta K424-beta R425-beta S426 sequence that has been proposed to be important for NADP(H) binding . beta K424R was strongly inhibited and showed an 18-fold increased Km,app for NADPH in the reverse reaction as compared to wild type . Consequently, this mutation affected all NADP(H)-linked activities and essentially abolished the unspecific interaction of NAD(H) with this site . The pH dependences of the forward and reverse reactions, as well as the cyclic reaction, were shifted to a lower pH as compared to the wild-type enzyme, and the salt dependence was also altered. Biochemistry, 1999 Feb 2, 38(5), 1643 - 51 Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units; Lau J et al.; Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzyme complexes that are organized into modules, where each module carries the domains needed to catalyze the condensation of an extender unit onto a growing polyketide chain . Each module also dictates the stereochemistry of the chiral centers introduced into the backbone during the chain elongation process . Here we used domain mutagenesis to investigate the role of the acyl transferase (AT) domains of individual modules in the choice and stereochemical fate of extender units . Our results indicate that the AT domains of DEBS do not influence epimerization of the (2S)-methylmalonyl-CoA extender units . Hence, stereochemical control of the methyl-branched centers generated by DEBS most likely resides in the ketosynthase (KS) domains of the individual modules . In contrast, several recent studies have demonstrated that extender unit specificity can be altered by AT domain substitution . In some of these examples, the resulting polyketide was produced at considerably lower titers than the corresponding natural product . We analyzed one such attenuated mutant of DEBS, in which the methylmalonyl transferase domain of module 2 was replaced with a malonyl transferase domain . As reported earlier, the resulting PKS produced only small quantities of the expected desmethyl analogue of 6-deoxyerythronolide B . However, when the same hybrid module was placed as the terminal module in a truncated 2-module PKS, it produced nearly normal quantities of the expected desmethyl triketide lactone . These results illustrate the limits to modularity of these multifunctional enzymes . To dissect the role of specific amino acids in controlling AT substrate specificity, we exchanged several segments of amino acids between selected malonyl and methylmalonyl transferases, and found that a short (23-35 amino acid) C-terminal segment present in all AT domains is the principal determinant of their substrate specificity . Interestingly, its length and amino acid sequence vary considerably among the known AT domains . We therefore suggest that the choice of extender units by the PKS modules is influenced by a "hypervariable region", which could be manipulated via combinatorial mutagenesis to generate novel AT domains possessing relaxed or altered substrate specificity. Biochemistry, 1999 Feb 2, 38(5), 1394 - 401 Allosteric dominance in carbamoyl phosphate synthetase; Braxton BL et al.; A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E . coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration . All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP . Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP . IMP causes only a slight inhibition at 25 degreesC . When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously . The same result was obtained with ornithine and IMP . By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site . When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound . The relationship of this observation to the structure of CPS is discussed. Biochemistry, 1999 Jan 26, 38(4), 1377 - 85 Effects of core mutations on the folding of a beta-sheet protein: implications for backbone organization in the I-state; Lorch M et al.; A series of core mutations were introduced into beta-strand segments of an immunoglobulin fold (the isolated first domain of CD2, CD2.d1) to examine their influence on the rapidly formed intermediate state (I-state) which transiently accumulates in the folding reaction {Parker, M . J., and Clarke, A . R . (1997) Biochemistry 36, 5786-5794} . The residue changes were chemically conservative, each representing the removal of one or two methylene groups from aliphatic side chains . Predictably, the mutations destabilize the folded state with respect to the unfolded state by about 1.1 +/- 0.7 kcal mol-1 per methylene group removed . However, when the folding reaction is dissected by transient kinetic analysis into its component steps, six out of the nine mutations lead to a stabilization of the I-state . The direction and magnitude of these effects on the global stability of the transient intermediate are well correlated with changes in secondary structure propensity occasioned by the substitutions . The results show that, although side chain interactions are extremely weak in this early phase of folding, the beta-strand conformation of the polypeptide chain is established . In the next phase of the reaction, the rate-limiting transition state is attained by the formation of a tightly localized hydrophobic nucleus which includes residues V30, I18, and V78 . Interestingly, in almost all immunoglobulin domains of extracellular proteins, the latter pair are cysteine residues which form a disulfide bridge. Biochemistry, 1999 Jan 26, 38(4), 1371 - 6 Modification of ribonuclease T1 specificity by random mutagenesis of the substrate binding segment; Hubner B et al.; Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far . Therefore, we applied a semirational approach by randomizing the guanine binding site . A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of 6 amino acid positions within the recognition loop was screened on RNase indicator plates . The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S/N43W/N44H/Y45A/E46D (RNaseT1-8/3) exhibits an altered preference toward purine nucleotides . The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type . Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2'3'-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable . This study shows that site-directed random mutagenesis is a powerful additional tool in protein design in order to achieve new enzymatic specificities. Biochemistry, 1999 Jan 26, 38(4), 1346 - 55 Mapping of subunit-subunit contact surfaces on the beta subunit of Escherichia coli RNA polymerase; Nomura T et al.; The RNA polymerase core enzyme of Escherichia coli is composed of 2alpha, 1beta, and 1beta' subunits . Previously we mapped the alpha-alpha, alpha-beta, and alpha-beta' contact sites on the alpha subunit . Here we analyzed the alpha subunit contact sites on the beta subunit by using various experimental approaches: (i) comparison of the proteolytic cleavage map between the unassembled free beta subunit and the alpha2 beta complex; (ii) analysis of the binary complex formation between His6-tagged intact alpha subunit and various truncated beta fragments; and (iii) analysis of the complex formation between the alpha subunit and various His6-tagged beta fragments . The results altogether indicate that two regions of the beta subunit are involved in the full activity of alpha binding, that is, the primary contact site between residues 737 and 904 and the secondary region with assembly control activity downstream from residue 1138 . All of the alpha subunit-beta fragment binary complexes identified in this study were found to bind beta' subunit and form pseudo-core complexes, indicating that the regions of beta involved in alpha subunit contact also participate in interaction with the beta' subunit. Biochemistry, 1999 Jan 26, 38(4), 1316 - 22 Spontaneous propeptide processing of mini-stromelysin-1 mutants blocked by APMA ((4-Aminophenyl)mercuric acetate); Galazka G et al.; Human stromelysin-1 (SL-1) is a member of the stromelysin subfamily of matrix metalloproteinases (MMPs) . The MMPs play a major role in the degradation of the extracellular matrix (ECM) during normal and pathological conditions . SL-1 like the other MMPs can be activated in vitro by the stepwise removal of the propeptide that contains a single unpaired cysteine which coordinates the active site zinc . Other residues in the propeptide also play a role in maintaining the latency of the enzymes . Deletion mutants and single-site amino acid replacements within the propeptide of a carboxyl-terminally truncated stromelysin-1 (mini-SL-1) were constructed and expressed in Escherichia coli to further examine what amino acids within the propeptide of SL-1 are important for maintaining latency . While the natural enzyme displayed some limited tendency to spontaneously (autolytically) convert to lower Mr in a stepwise manner and finally to the fully processed form, all of the truncation mutants of more than 19 amino acids generated in E . coli showed greatly accelerated self-cleavage indicative of diminished stability and/or resistance to proteolysis of the residual propeptide . Mutant Delta63 as well as other mutants in which most of the propeptide had been deleted no longer responded to exposure to the organomercurial APMA by accelerated autolytic processing . Rather, APMA inhibited the autolytic processing in these mutants, further confirming the complexity of the action of this organomercurial in the activation of pro-MMPs. Biochemistry, 1999 Jan 26, 38(4), 1310 - 5 Role of carboxyl-terminal charges on S-modulin membrane affinity and inhibition of rhodopsin phosphorylation; Matsuda S et al.; S-Modulin shows a higher affinity for urea-stripped frog rod outer segment membranes than s26 (a cone homologue of S-modulin) . NaCl at a concentration of several hundred millimolar reduced the membrane affinity of S-modulin to the s26 level . Chimeric S-modulin and s26 whose respective 23 and 29 amino acids at the carboxyl terminus were swapped showed membrane affinites similar to those of s26 and S-modulin, respectively . The membrane affinity of an S-modulin mutant lacking C-terminal positive charges was reduced to the s26 level, while another S-modulin mutant lacking C-terminal negative charges has a higher membrane affinity than wild-type S-modulin . When the molar ratio of recombinant S-modulins to rhodopsin is 0.5, there was no large difference in the inhibition efficiency . However, S-modulin and mutants with high membrane affinities inhibit rhodopsin phosphorylation more efficiently than s26 and mutants with low membrane affinities at the molar ratio of 0.1 . These results indicate that the C-terminal positive charges of these Ca2+-binding proteins enhance the membrane affinity and the inhibitory effect on rhodopsin phosphorylation by increasing the concentration of S-modulin on the membrane. Biochemistry, 1999 Jan 26, 38(4), 1243 - 51 Quaternary structure sensitive tyrosine residues in human hemoglobin: UV resonance raman studies of mutants at alpha140, beta35, and beta145 tyrosine; Nagai M et al.; Recent studies noted the contribution of alpha42Tyr to the T-R-dependent UV resonance Raman (UVRR) spectral changes of HbA {Nagai, M., et al . (1996) J . Mol . Struct . 379, 65-75; Huang, S., et al . (1997) Biochemistry 36, 6197-6206}, but the observed UVRR changes of the Tyr residue cannot be fully interpreted with alpha42Tyr alone . To identify the remaining contributions, the 235 nm-excited UVRR spectra of Tyr mutant Hbs at alpha140, beta35, and beta145 were investigated here . The Fe-His stretching mode demonstrated that all of these mutant Hbs take the T structure in the deoxy form under these experimental conditions . The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond . The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible . In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift . In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected . This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes . It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145) . These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments. Biochemistry, 1999 Jan 26, 38(4), 1193 - 202 Functions of His107 in the catalytic mechanism of human glutathione S-transferase hGSTM1a-1a; Patskovsky YV et al.; Domain interchange analyses and site-directed mutagenesis indicate that the His107 residue of the human subunit hGSTM1 has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions, and a H107S substitution accounts for the marked differences in the properties of the homologous hGSTM1-1 (His107) and hGSTM4-4 (Ser107) glutathione S-transferases . Reciprocal replacement of His107 and Ser107 in chimeric enzymes results in reciprocal conversion of catalytic properties . With 1-chloro-2, 4-dinitrobenzene as a substrate, the His107 residue primarily influences the pH dependence of catalysis by lowering the apparent pKa of kcat/Km from 7.8 for the Ser107-containing enzymes to 6.3 for the His107-containing enzymes . There is a parallel shift in the pKa for thiolate anion formation of enzyme-bound GSH . Y6F mutations have no effect on the pKa for these enzymes . Crystal structures of hGSTM1a-1a indicate that the imidazole ring of His107 is oriented toward the substrate binding cleft approximately 6 A from the GSH thiol group . Thus, His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation . All wild-type enzymes and H107S chimera have nearly identical equilibrium constants for formation of enzyme-GSH complexes (Kd values of 1-2 x 10(-)6 M); however, KmGSH and Ki values for S-methylglutathione inhibition determined by steady-state kinetics are nearly 100-fold higher . The functions of His107 of hGSTM1a-1a are unexpected in view of a substantial body of previous evidence that excluded participation of histidine residues in the catalytic mechanisms of other glutathione S-transferases . Consequences of His107 involvement in catalysis are also substrate-dependent; in contrast to 1-chloro-2,4-dinitrobenzene, for the nucleophilic addition reaction of GSH to ethacrynic acid, the H107S substitution has no effect on catalysis presumably because product release is rate-limiting. J Neurochem, 1999 Feb, 72(2), 549 - 56 A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein; Watanabe T et al.; Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids . It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease . To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol . Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa) . UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates . APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody . These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain . In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP. Protein Eng, 1998 Dec, 11(12), 1285 - 92 Human pancreatic RNase1-human epidermal growth factor fusion: an entirely human 'immunotoxin analog' with cytotoxic properties against squamous cell carcinomas; Psarras K et al.; The gene encoding human pancreatic ribonuclease 1 (hpRNasel) was fused with a gene encoding human epidermal growth factor (hEGF) . The hybrid human protein was isolated from Escherichia coli inclusion bodies, refolded and purified to homogeneity . The fusion protein competed with 125I-hEGF for binding to hEGF receptors (EGFR) and had ribonucleolytic activities approaching those of hpRNase1 . Several conformations having different enzymatic activities could be detected after reversed-phase high-performance liquid chromatographic analysis, the less hydrophobic molecules being the most active . The hybrid protein was specifically cytotoxic to A431, an EGFR overexpressing squamous carcinoma cell line, with an IC50 of approximately 10(-7) M . In contrast, recombinant hpRNase1 had an IC50 higher than 10(-4) M . A mixture of free hEGF and free hpRNasel was not more cytotoxic than hpRNasel alone and no cytotoxicity was detected in EGFR-deficient control cells . Taken together, these data suggest that this construct might be useful for targeted therapy of esophageal, lung and other squamous cell carcinomas and also breast cancers overexpressing EGFR, which correlate with a poor prognosis and cannot be cured by surgery alone . Engineering hybrid molecules with endogenous human proteins for targeted therapy may alleviate the dose-limiting immunogenicity and toxicity of conventional immunotoxins. Protein Eng, 1998 Dec, 11(12), 1257 - 65 Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein; Dimasi N et al.; The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases . The HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent complex, which participates in processing the viral polyprotein . This proteolytic activity is believed to be essential for virus proliferation and thus the NS3 protease is a prime target for developing anti-HCV pharmacological agents . Recent X-ray crystallography structural studies have revealed the nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chain polypeptide . To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of making a beta-turn, to the N-terminus of the NS3 protease domain . This engineered single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/ NS4A non-covalent complex . Moreover, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor . The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineering for further structural studies and in vitro selection of potential drug-resistant mutants . These are important steps towards developing effective anti-protease compounds. Protein Eng, 1998 Dec, 11(12), 1249 - 56 Expression of apolipoprotein(a) kringle IV type 9 in Escherichia coli: demonstration of a specific interaction between kringle IV type 9 and apolipoproteinB-100; Rahman M et al.; A number of studies have provided evidence that lipoprotein(a) {Lp(a)} assembly is a two-step process in which initial non-covalent interactions between apolipoprotein(a) {apo(a)} and apolipoproteinB-100 (apoB-100) precede specific disulfide bond formation . We have designed a construct encoding apo(a) kringle IV type 9 (KIV9) in which the unpaired cysteine at position 67 in this kringle is replaced with a tyrosine . The single kringle was expressed in bacteria and purified to homogeneity from cell homogenates . The purified derivative (designated KIV9deltaCys) was assessed for its ability to bind to purified human LDL . This interaction was detected either by ELISA using immobilized LDL or by column chromatography in which LDL binding to KIV9deltaCys immobilized on Ni2+-Sepharose was determined . In both cases, the interaction of KIV9deltaCys and LDL was observed . Further, we demonstrated that the binding interaction was sensitive to the addition of amino acids including lysine, the lysine analogue epsilon-aminocaproic acid, arginine, phenylalanine and proline, with arginine and lysine having the greatest inhibitory effect . Binding of KIV9deltaCys to an immobilized apoB peptide spanning residues 3732-3745 of apoB was also demonstrated by ELISA . As was the case for LDL, this binding interaction was sensitive to the addition of arginine and lysine . Computer modeling of KIV9 demonstrated an excellent fit with residues 3732-3738 (PSCKLDF) of the apoB peptide . The modeling predicts the presence of overlapping lysine and phenylalanine-binding pockets in KIV9 which explains the inhibitory effects of lysine, arginine and phenylalanine which were observed in the binding assays . In summary, this study represents the first demonstration that KIV9 can interact directly with LDL through non-covalent interactions which may contribute to the first step of Lp(a) formation. Protein Eng, 1998 Dec, 11(12), 1243 - 7 Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length; Bottomley SP et al.; The exposed Serpin reactive centre loop controls the specificity of the serpin proteinase interaction . Mutations within this region have been used to generate novel potentially therapeutic inhibitors . In this study we examine the effect of the serpin scaffold and reactive centre loop length upon the generation of such inhibitors . The reactive centre loop regions, P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and ACT/Serp1, respectively . AT/Serp1 formed SDS stable complexes with a range of proteinases with association rate constants for plasmin, tissue plasminogen activator, urokinase, thrombin and factor Xa of approximately 10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for all of them . ACT/Serp1, however, formed SDS-stable complexes with only plasmin and thrombin with association rate constant 100-fold slower than AT/Serp1 and an increased stoichiometry of inhibition . The reactive centre loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1 . These four additional residues (VETR) were inserted into AT/Serp1 to form AT/Serp1(VETR) . AT/Serp1(VETR) formed SDS stable complexes with plasmin, thrombin and tissue plasminogen activator similar to AT/Serp1, however, the association rate constants were 10-fold slower than those observed with AT/Serp1, while the stoichiometry of inhibition remained around 1 . These results suggest that the additional reactive centre loop residues effect the rate of initial complex formation by placing the reactive centre loop in a non-ideal conformation . This study demonstrates that both reactive centre loop length and serpin scaffold are important in defining the inhibitory characteristics of a serpin. Protein Eng, 1998 Dec, 11(12), 1229 - 34 Engineered human carboxypeptidase B enzymes that hydrolyse hippuryl-L-glutamic acid: reversed-polarity mutants; Edge M et al.; Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli . By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C-terminal acidic amino acid residues instead of the normal C-terminal basic residues . This was achieved by mutating Asp253 at the base of the S1' specificity pocket, which normally interacts with the basic side-chain of the substrate, to either Lys or Arg . The resulting enzymes had the desired reversed polarity and enzyme activity was improved significantly with further mutations at residue 251 . The {G251T,D253K}HCPB double mutant was 100 times more active against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the single mutant, {D253K}HCPB . Triple mutants, containing additional changes at Ala248, had improved activity against hipp-Glu substrate when position 251 was Asn . These reversed-polarity mutants of a human enzyme have the potential to be used in antibody-directed enzyme prodrug therapy of cancer. Protein Eng, 1998 Dec, 11(12), 1219 - 27 Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and characterization of the recombinant enzyme; Koga Y et al.; The Pk-glpK gene, which encodes glycerol kinase (GK) from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, was cloned and expressed in Escherichia coli . The amino acid sequence of this enzyme (Pk-GK) deduced from the nucleotide sequence showed 57% identity with that of E . coli GK and 47% identity with that of human GK . Pk-GK, which has a molecular weight of 55902 (497 amino acid residues), was purified from E . coli and characterized . Despite the high sequence similarity, Pk-GK and E . coli GK are greatly divergent in structure and function from each other . Unlike E . coli GK, which exists as a tetramer, Pk-GK exists as a dimer . The preferred divalent cation for Pk-GK is Co2+, instead of Mg2+ . The optimum pH and temperature for Pk-GK activity are 8.0 and 80 degrees C, respectively . Pk-GK can utilize other nucleoside triphosphates than ATP as a phosphoryl donor . It is fairly resistant to an allosteric inhibitor of E . coli GK, fructose-1,6-bisphosphate . Determination of the kinetic parameters indicates that the Km value of the enzyme is 15.4 microM for ATP and 111 microM for glycerol and its kcat value is 940 s(-1) . The enzyme was shown to be fairly resistant to irreversible heat inactivation and still retained 50% of its enzymatic activity even after heating at 100 degrees C for 30 min . Construction of a model for the three-dimensional structure of the enzyme suggests that the formation of extensive ion-pair networks is responsible for the high stability of this enzyme. Protein Eng, 1998 Dec, 11(12), 1211 - 7 Misfolding of chloramphenicol acetyltransferase due to carboxy-terminal truncation can be corrected by second-site mutations; Van der Schueren J et al.; Folding of chloramphenicol acetyltransferase (CAT) in Escherichia coli is hampered by deletion of the carboxy-terminal tail including the last residue of the carboxy-terminal alpha-helix . Such truncated CAT polypeptides quantitatively aggregate into cytoplasmic inclusion bodies, which results in absence of a chloramphenicol-resistant phenotype for the producing host . In this paper, a genetic approach is presented to examine this aggregation process in more detail . Random mutagenesis of inactive CAT followed by direct phenotypic selection for revertants with restored chloramphenicol resistance was used to isolate second-site suppressors of inactive truncation mutants of CAT . Two random mutagenesis procedures, independently of each other, yielded a unique substitution of Phe for Leu at amino acid position 145 . This second-site mutation does not drastically affect the proteins' stability under normal growth conditions of E . coli . Hence, the introduction of Phe at amino acid position 145 improves the ability of the protein to fold into a soluble, enzymatically active conformation . The conservative character of the Leu145Phe replacement indicates that limited changes at crucial positions can have important effects on protein folding in vivo. Scand J Gastroenterol, 1998 Dec, 33(12), 1256 - 61 Extracts of Helicobacter pylori reduce gastric mucosal blood flow through a VacA- and CagA-independent pathway in rats; Atuma C et al.; BACKGROUND: Helicobacter pylori may interfere with gastroduodenal protective mechanisms . Such effects could be due to a direct interaction with gastric epithelial cells but also to the action of a wide range of secreted and membrane-bound virulence factors . Our aim was to study the acute effects of water extracts produced from H . pylori on gastric mucosal blood flow and acid secretion and to relate them to VacA and CagA activity . METHOD: Extracts were produced from strains 88-23 and A5, both wild type; A5VacA, an isogenic mutant lacking expression of the vacuolating cytotoxin (VacA) and the immunodominant antigen (CagA); and Escherichia coli strain ATCC-25922 . Bacterial extracts were applied on the exteriorized gastric corporal mucosa in inactin-anaesthetized rats after removal of as much as possible of the mucus layer, during intravital microscopy . Blood flow was measured by means of laser-Doppler flowmetry . RESULTS: All H . pylori extracts, including the extract from 88-23 heated to 100 degrees C for 30 min, significantly reduced blood flow by 15%-19%, whereas E . coli had no significant effect on blood flow . CONCLUSION: A factor or a combination of factors, other than VacA and CagA released from H . pylori, might compromise the natural defence of the gastric corporal mucosa by reducing mucosal blood flow . The factor is heat-stable and lacking or less potent in E . coli. Gene Ther, 1998 Nov, 5(11), 1571 - 4 Provision of positive and negative selections in retroviral vectors containing the cytosine deaminase gene; Shiau AL et al.; The E . coli cytosine deaminase (CD) provides a negative selection system for suicide gene therapy as CD transfectants are eliminated following 5-fluorocytosine (5FC) treatment . Here we report a positive selection system for the CD gene using 5-fluorouracil (5FU) and cytosine in selection medium to screen for CD-positive transfectants . It is based on the relief of 5FU toxicity by uracil which is converted from cytosine via CD catalysis, as uracil competes with the toxic 5FU in subsequent pyrimidine metabolism . Hence, a retroviral vector containing the CD gene may provide both positive and negative selections after gene transfer . The CD transfectants selected with the positive selection system showed susceptibility to 5FC in subsequent negative selection in vitro and in vivo . Therefore, this dual selection system is useful not only for combination therapy with transgene and CD gene, but can also act to eliminate selectively transduced cells after the transgene has furnished its effects or upon undesired conditions if 5FC is applied for negative selection in vivo. Gene Ther, 1998 Nov, 5(11), 1481 - 7 Gene transfer vectors derived from equine infectious anemia virus; Olsen JC; Equine infectious anemia virus (EIAV) is a lentivirus in the retrovirus family of viruses . Replication-defective EIAV vectors have been constructed that encode bacterial puromycin-N-acetyl transferase and E . coli beta-galactosidase . These vectors could be prepared with titers greater than 10(5) infectious units/ml and were able to act as vehicles to carry genes into cultured human cells . In addition, stable helper cell lines were created by modifying human 293 cells to express EIAV proteins . Unlike retroviral vectors based on murine leukemia virus, EIAV lentiviral vectors transduce nondividing (aphidicolin-arrested) cells . These properties make EIAV vectors promising gene transfer vehicles. Arch Virol, 1998, 143(12), 2461 - 9 The nucleotide sequence of the 3'-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production; Li RH et al.; A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA . The sequence of the 3'-terminal 3158 nucleotides, which consisted of the 3'-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57-68% similar at the nucleotide level and 72-82% similar at the amino acid level when compared with other potyviruses . Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus . A recombinant DsMV-Ch CP (approximately 39 kDa) expressed in E . coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA. Arch Virol, 1998, 143(12), 2443 - 51 Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants; Yang SJ et al.; A full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator . This construct was found infectious when inoculated to lettuce plants . The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity . The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E . coli . Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out . The symptoms induced in lettuce by either the intron-less or the intro-containing constructs were identical to those caused by the wild-type virus . However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA. Rocz Panstw Zakl Hig, 1998, 49(3), 293 - 8 The level of endotoxin contamination in biopreparations; Aleksandrowicz J et al.; Bacterial endotoxins as contamination of biopreparations have been estimated by chromogenic LAL test . Study on some compounds (aluminium hydroxide, formaldehyde and merthiolate) being components of vaccines showed no effect on the result of LAL test . The level of endotoxins in virus vaccines with the limits defined in producers certificate was adequate, the level of endotoxin was also low in virus vaccines of undefined requirements . The concentration of endotoxin in bacterial vaccines was differentiated . Considering the results of our experiments, as well as the fact, that the requirements for endotoxin contamination of bacterial vaccines are not available it seems necessary to establish the limits for these group of biopreparations. Genetika, 1998 Oct, 34(10), 1338 - 44 {Further genetic study of tandem duplication formation in the region of the deo operon in the process of Escherichia coli K-12 conjugational recombination}; Sukhodolets VV; The formation of heterozygous duplications in the region of the deo operon was studied in conjugational matings (male) HfrH deoC deoD thr::Tn9 thyA x HfrH deoA deoB::Tn5 thyA . When recombinants that inherited the donor marker thr::Tn9 (Cml r) were selected on a medium containing thymine and chloramphenicol, but not threonine, more than 80% of the offspring were heterozygous tandem duplications extending to the region of the deo operon . In matings with a thymidine-dependent HfrH deoA deoB::Tn5 thyA strain as a recipient, when recombinants were selected on a medium containing thymine, i.e., under conditions of thymine starvation of merozygotes, the recombinogenic effect was observed . However, this effect did not change the frequency of duplication formation . The integration of genetic markers via homologous recombination into the chromosomal regions adjacent to duplications occurred at a lower frequency . An analysis of the formation of haploid segregants by duplications showed that, in most cases of duplication formation, the proximal segment of the donor chromosome is integrated into the distal position, i.e., after the homologous segment of the recipient chromosome. Bioorg Khim, 1998 Oct, 24(10), 756 - 9 {Expression of mutant horse cytochrome c genes in Escherichia coli}; Dolgikh DA et al.; Here we describe genetically engineered constructs for the expression in Escherichia coli of genes for horse cytochrome c mutants . These constructs allow the expression of the cytochrome c genes together with hemeligase, an enzyme which covalently links heme to cytochrome . Careful selection of producer strains and the adjustment of the conditions of expression provided for expression levels of 10-15 mg of protein per liter of culture . This is by an order of magnitude greater than the expression previously achieved in yeast . A series of horse cytochrome c mutants were obtained in this way. Biophys J, 1999 Feb, 76(2), 709 - 15 Single-molecule imaging of RNA polymerase-DNA interactions in real time; Harada Y et al.; Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of lambda-phage DNA suspended in solution by optical traps . The interactions of RNA polymerase molecules were not homogeneous along DNA . They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of approximately 0.66 s-1, which was more than severalfold smaller than the rate at other positions . The association rate constant for the slow dissociation sites was 9.2 x 10(2) bp-1 M-1 s-1 . The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions . RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA . The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA . The present method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the process of transcription. FEBS Lett, 1999 Jan 15, 442(2-3), 241 - 5 Conformational stabilities of the rat alpha- and beta-parvalbumins; Henzl MT et al.; It is widely believed that beta-parvalbumin (PV) isoforms are intrinsically less stable than alpha-parvalbumins, due to greater electrostatic repulsion and an abbreviated C-terminal helix . However, when examined by differential scanning calorimetry, the apo-form of the rat beta-PV (i.e . oncomodulin) actually displays greater thermal stability than the alpha-PV . Whereas the melting temperature of the a isoform is 45.8 degrees C at physiological pH and ionic strength, the Tm for the beta isoform is more than 7 degrees higher (53.6 degrees C) . This result suggests that factors besides net charge and C-terminal helix length strongly influence parvalbumin conformational stability . Extension of the F helix in the beta-PV, by insertion of Ser-109, has a modest stabilizing effect, raising the Tm, by 1.1 degrees . Truncation of the alpha-PV F helix, by removal of Glu-108, has a more profound impact, lowering the Tm by 4.0 degrees. FEBS Lett, 1999 Jan 15, 442(2-3), 231 - 4 Induction of cytokines in a human colon epithelial cell line by Shiga toxin 1 (Stx1) and Stx2 but not by non-toxic mutant Stx1 which lacks N-glycosidase activity; Yamasaki C et al.; Stx1 and Stx2 produced by Shiga toxin-producing Escherichia coli are cytotoxic due to their N-glycosidase activity on 28S rRNA . In this study, we have shown that proinflammatory cytokine mRNAs, especially IL-8, were induced by Stx1 and Stx2 in Caco-2 cells . A non-toxic mutant of Stxl which lacks N-glycosidase activity did not induce cytokine mRNAs . IL-8 production at the protein level was enhanced by Stx1 and Stx2, but not by the mutant Stx1 . These results demonstrate that Shiga toxins induce expression and synthesis of cytokines in Caco-2 cells and their N-glycosidase activity is essential for the induction. FEBS Lett, 1999 Jan 15, 442(2-3), 198 - 202 Purification of histidine tagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli; Hohenfeld IP et al.; Bacteriorhodopsin (BR) from Halobacterium salinarum as well as halorhodopsin (pHR) and sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were functionally expressed in E . coli using the method of Shimono et al . IFEBS Lett . (1997) 420, 54-56} . The histidine tagged proteins were purified with yields up to 1.0 mg/l cell culture and characterized by ESI mass spectrometry and their photocycle . The pSRII and pHR photocycles were indistinguishable from the wild type proteins . The BR photocycle was considerably prolonged . pSOII is located in the cytoplasmic membrane and the C-terminus is oriented towards the cytoplasm as determined by immunogold labelling. FEBS Lett, 1999 Jan 15, 442(2-3), 183 - 8 A recombinant single-chain antibody fragment that neutralizes toxin II from the venom of the scorpion Androctonus australis hector; Mousli M et al.; Monoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis . The cDNAs encoding the variable regions of this antibody were isolated by PCR-mediated cloning . A single-chain Fv gene was engineered and expressed in Escherichia coli . The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo . We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single-chain Fv, which opens new perspectives for the treatment of envenomizations. FEBS Lett, 1999 Jan 15, 442(2-3), 143 - 6 Calcium-dependent interaction of annexin I with annexin II and mapping of the interaction sites; Lee KH et al.; Annexins are multifunctional intracellular proteins with Ca2+- and phospholipid-binding properties . Their structures consist of four conserved repeat domains that form the core and a diverse N-terminal tail, from which their functional differences may arise . We searched for cellular proteins that interact with the N-terminal tail plus domain I of annexin I (ANX1) by using the yeast two-hybrid method . Screening of a HeLa cell cDNA library yielded annexin II (ANX2) cDNA . The interaction between ANX1 and ANX2 also occurred in vitro in a Ca2+-dependent manner . Mapping of the interaction sites revealed that interaction between domain I of ANX1 and domain IV of ANX2 was stronger than the other combinations. Annu Rev Genet, 1998, 32, 437 - 59 Evolution and mechanism of translation in chloroplasts; Sugiura M et al.; The entire sequence (120-190 kb) of chloroplast genomes has been determined from a dozen plant species . The genome contains from 87 to 183 known genes, of which half encode components involved in translation . These include a complete set of rRNAs and about 30 tRNAs, which are likely to be sufficient to support translation in chloroplasts . RNA editing (mostly C to U base changes) occurs in some chloroplast transcripts, creating start and stop codons and changing codons to retain conserved amino acids . Many components that constitute the chloroplast translational machinery are similar to those of Escherichia coli, whereas only one third of the chloroplast mRNAs contain Shine-Dalgarno-like sequences at the correct positions . Analyses conducted in vivo and in vitro have revealed the existence of multiple mechanisms for translational initiation in chloroplasts. Annu Rev Genet, 1998, 32, 163 - 84 The genetics of disulfide bond metabolism; Rietsch A et al.; Disulfide bonds are required for the stability and function of a large number of proteins . Genetic analysis in combination with biochemical studies have elucidated the main catalysts involved in facilitating these processes in the cell . All enzymes involved in thiol-disulfide metabolism have a conserved active site that consists of two cysteine residues, separated by two intervening amino acids, the Cys-Xaa-Xaa-Cys motif . While these enzymes are capable of catalyzing both disulfide bond formation and reduction, they have evolved to perform one or the other reaction more efficiently . In the cytoplasm, multiple pathways are involved in the reduction of disulfide bonds that occur as part of the catalytic cycle of a variety of metabolic enzymes . In the bacterial periplasm, a system for the efficient introduction as well as isomerization of disulfide bonds is in place . In eukaryotes, disulfide bonds are introduced into proteins in the endoplasmic reticulum . Genetic studies have recently begun to reveal new features of this process . While the enzyme mechanisms of thiol-disulfide oxidoreductases have been the subject of much scrutiny, questions remain regarding where and when they act in vivo, their specificities, and the maintenance of the redox environment that determines their function. Annu Rev Genet, 1998, 32, 59 - 94 Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli; Danese PN et al.; Escherichia coli must actively transport many of its proteins to extracytoplasmic compartments such as the periplasm and outer membrane . To perform this duty, E . coli employs a collection of Sec (secretion) proteins that catalyze the translocation of various polypeptides through the inner membrane . After translocation across the inner membrane, periplasmic and outer-membrane proteins are folded and targeted to their appropriate destinations . Here we review our knowledge of protein translocation across the inner membrane . We also discuss the various signal transduction systems that monitor extracytoplasmic protein folding and targeting, and we consider how these signal transduction systems may ultimately control these processes. Ann N Y Acad Sci, 1998 Dec 13, 864, 131 - 5 Properties of artificial proteins with random sequences; Yomo T et al.; A library of artificial proteins of 141 amino acid residues, of which 95 are random and which include 20 kinds of amino acids, was prepared . As the properties of the artificial random proteins are free from the evolutionary constraint, they can be used as a standard to discriminate the specialized properties of natural proteins . Out of the 25 identified random proteins, 5 are soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble . Therefore, as natural soluble or insoluble proteins can arise from the line of soluble or insoluble ancestry, respectively, solubility does not seem a specialized property of natural proteins . The soluble random proteins RP3-42 and RP3-45 were purified and their properties were investigated. Nucleic Acids Res, 1999 Feb 15, 27(4), 1198 - 204 Double strand break rejoining by mammalian mitochondrial extracts; Lakshmipathy U et al.; DNA end-joining was measured by incubating linearized plasmid DNA with mitochondrial protein extracts . A spectrum of end-joined molecules ranging from re-circularized monomer to dimer and higher molecular weight forms was observed . The DNA end-joining reaction required ATP and Mg2+, and was inhibited by sodium chloride . Both cohesive- and blunt-ended DNA molecules were end-joined, although the former were more efficient substrates . Molecular analysis of rejoined molecules revealed that >95% of the linearized DNA were precisely end-joined . The few imprecisely end-joined molecules recovered, sustained deletions that spanned direct repeat sequences . The deletions observed are strikingly similar to those present in mitochondrial genomes of patients with Kearns-Sayre or Pearson syndromes, certain ophthalmic myopathies and the aged . These results suggest that mammalian mitochondria possess a DNA double strand break repair activity similar to that seen in the nucleus, and that this repair pathway may play a role in the generation of mitochondrial DNA deletions associated with a number of human pathologies. Nucleic Acids Res, 1999 Feb 15, 27(4), 1118 - 25 Thermodynamics of RNA hairpins containing single internal mismatches; Meroueh M et al.; Thermodynamic parameters and circular dichroism spectra are presented for RNA hairpins containing single internal mismatches in the stem regions . Three different sequence contexts for the G*U mismatch and two contexts for C*A, G*A, U*U, A*C and U*G mismatches were examined and compared with Watson-Crick base-pair stabilities . The RNA hairpins employed were a microhelix and tetraloop representing the Escherichia coli tRNAAlaacceptor stem and sequence variants that have been altered at the naturally occurring G*U mismatch site . UV melting studies were carried out under different conditions to evaluate the effects of sodium ion concentration and pH on the stability of mismatch-containing hairpins . Our main findings are that single internal mismatches exhibit a range of effects on hairpin stability . In these studies, the size and sequence of the loop and stem are shown to influence the overall stability of the RNA, and have a minor effect on the relative mismatch stabilities . The relationship of these results to RNA-ligand interactions involving mismatch base-pairs is discussed. Nucleic Acids Res, 1999 Feb 15, 27(4), 1094 - 103 Vaccine candidate MSP-1 from Plasmodium falciparum: a redesigned 4917 bp polynucleotide enables synthesis and isolation of full-length protein from Escherichia coli and mammalian cells; Pan W et al.; The Plasmodium falciparum malaria parasite is the causative agent of malaria tropica . Merozoites, one of the extracellular developmental stages of this parasite, expose at their surface the merozoite surface protein-1 complex (MSP-1), which results from the proteolytic processing of a 190-200 kDa precursor . MSP-1 is highly immunogenic in humans and numerous studies suggest that this protein is an effective target for a protective immune response . Although its function is unknown, there are indications that it may play a role during invasion of erythrocytes by merozoites . The parasite-derived msp-1 gene, which is approximately 5000 bp long, contains 74% AT . This high AT content has prevented stable cloning of the full-size gene in Escherichia coli and consequently its expression in heterologous systems . Here, we describe the synthesis of a 4917 bp gene encoding MSP-1 from the FCB-1 strain of P . falciparum adjusted for human codon preferences . The synthetic msp-1 gene (55% AT) was cloned, maintained and expressed in its entirety in E.coli as well as in CHO and HeLa cells . The purified protein is soluble and appears to possess native conformation because it reacts with a panel of mAbs specific for conformational epitopes . The strategy we used for synthesizing the full-length msp-1 gene was toassemble it from DNA fragments encoding all of the major proteolytic fragments normally generated at the parasite's surface . Thus, after subcloning we also obtained each of these MSP-1 processing products as hexahistidine fusion proteins in E.coli and isolated them by affinity chromatography on Ni2+agarose . The availability of defined preparations of MSP-1 and its major processing products open up new possibilities for in-depth studies at the structural and functional level of this important protein, including the exploration of MSP-1-based experimental vaccines. Nucleic Acids Res, 1999 Feb 15, 27(4), 1015 - 24 Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage; Bourdat AG et al.; Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage . One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) . In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method . For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed . The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry . The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA . In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates . The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported. Nucleic Acids Res, 1999 Feb 15, 27(4), 979 - 83 Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase; Bessho T; An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans . Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol . Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol . The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins . These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein. Bioinformatics, 1998, 14(10), 839 - 45 SALSA: improved protein database searching by a new algorithm for assembly of sequence fragments into gapped alignments; Rognes T et al.; MOTIVATION: Optimal sequence alignment based on the Smith-Waterman algorithm is usually too computationally demanding to be practical for searching large sequence databases . Heuristic programs like FASTA and BLAST have been developed which run much faster, but at the expense of sensitivity . RESULTS: In an effort to approximate the sensitivity of an optimal alignment algorithm, a new algorithm has been devised for the computation of a gapped alignment of two sequences . After scanning for high-scoring words and extensions of these to form fragments of similarity, the algorithm uses dynamic programming to build an accurate alignment based on the fragments initially identified . The algorithm has been implemented in a program called SALSA and the performance has been evaluated on a set of test sequences . The sensitivity was found to be close to the Smith-Waterman algorithm, while the speed was similar to FASTA (ktup = 2) . AVAILABILITY: Searches can be performed from the SALSA homepage at using a wide range of databases . Source code and precompiled executables are also available . CONTACT: torbjorn.rognes@labmed.uio.no Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 939 - 44 Model of maltose-binding protein/chemoreceptor complex supports intrasubunit signaling mechanism; Zhang Y et al.; The Tar protein of Escherichia coli is unique among known bacterial chemoreceptors in that it generates additive responses to two very disparate ligands, aspartate and maltose . Aspartate binds directly to the periplasmic (extracytoplasmic) domain of Tar . Maltose first binds to maltose-binding protein (MBP) . MBP then assumes a closed conformation in which it can interact with the periplasmic domain of Tar . MBP residues critical for binding Tar were identified in a screen of mutations that cause specific defects in maltose chemotaxis . Mutations were introduced into a plasmid-borne malE gene that encodes a mutant form of MBP in which two engineered Cys residues spontaneously generate a disulfide bond in the oxidizing environment of the periplasmic space . This disulfide covalently crosslinks the NH3-terminal and COOH-terminal domains of MBP and locks the protein into a closed conformation . Double-Cys MBP confers a dominant-negative phenotype for maltose taxis, and we reasoned that third mutations that relieve this negative dominance probably alter residues that are important for the initial interaction of MBP with Tar . The published three-dimensional structures of MBP and the periplasmic domain of E . coli Tar were docked in a computer simulation that juxtaposed the residues in MBP identified in this way with residues in Tar that have been implicated in maltose taxis . The resulting model of the MBP-Tar complex exhibits good complementarity between the surfaces of the two proteins and supports the idea that aspartate and MBP may each initiate an attractant signal through Tar by inducing similar conformational changes in the chemoreceptor. Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 863 - 8 TOXCAT: a measure of transmembrane helix association in a biological membrane; Russ WP et al.; The noncovalent association of transmembrane alpha-helices is a fundamental event in the folding of helical membrane proteins . In this work, a system (TOXCAT) is developed for the study of transmembrane helix-helix oligomerization in a natural membrane environment . This assay uses a chimeric construct composed of the N-terminal DNA binding domain of ToxR (a dimerization-dependent transcriptional activator) fused to a transmembrane domain (tm) of interest and a monomeric periplasmic anchor (the maltose binding protein) . Association of the tms results in the ToxR-mediated activation of a reporter gene encoding chloramphenicol acetyltransferase (CAT) . The level of CAT expression indicates the strength of tm association . The assay distinguishes between a known dimerizing tm and a mutant in which dimerization is disrupted . In addition, modulation of the chimera concentration shows that the dimerization exhibits concentration dependence in membranes . TOXCAT also is used to select oligomeric tms from a library of randomized sequences, demonstrating the potential of this system to reveal novel oligomerization motifs . The TOXCAT system has been used to investigate glycophorin A tm-mediated dimerization . Although the overall sensitivity of glycophorin A tm dimerization to mutagenesis is found to be similar in membranes and in detergent micelles, several significant differences exist . Mutations to polar residues, which are generally disruptive in SDS, exhibit sequence specificity in membranes, demonstrating both the limitations of detergent micelles and the wider range of application of the TOXCAT system. Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 823 - 8 Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition; Kai Y et al.; The crystal structure of phosphoenolpyruvate carboxylase (PEPC; EC 4 . 1.1.31) has been determined by x-ray diffraction methods at 2.8-A resolution by using Escherichia coli PEPC complexed with L-aspartate, an allosteric inhibitor of all known PEPCs . The four subunits are arranged in a "dimer-of-dimers" form with respect to subunit contact, resulting in an overall square arrangement . The contents of alpha-helices and beta-strands are 65% and 5%, respectively . All of the eight beta-strands, which are widely dispersed in the primary structure, participate in the formation of a single beta-barrel . Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers . The location of the catalytic site is likely to be near the C-terminal side of the beta-barrel . The binding site for L-aspartate is located about 20 A away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector binding . The participation of Arg-587 is unexpected, because it is known to be catalytically essential . Because this residue is in a highly conserved glycine-rich loop, which is characteristic of PEPC, L-aspartate seemingly causes inhibition by removing this glycine-rich loop from the catalytic site . There is another mobile loop from Lys-702 to Gly-708 that is missing in the crystal structure . The importance of this loop in catalytic activity was also shown . Thus, the crystal-structure determination of PEPC revealed two mobile loops bearing the enzymatic functions and accompanying allosteric inhibition by L-aspartate. Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 811 - 7 Origins of DNA-binding specificity: role of protein contacts with the DNA backbone; Schildbach JF et al.; A central question in protein-DNA recognition is the origin of the specificity that permits binding to the correct site in the presence of excess, nonspecific DNA . In the P22 Arc repressor, the Phe-10 side chain is part of the hydrophobic core of the free protein but rotates out to pack against the sugar-phosphate backbone of the DNA in the repressor-operator complex . Characterization of a library of position 10 variants reveals that Phe is the only residue that results in fully active Arc . One class of mutants folds stably but binds operator with reduced affinity; another class is unstable . FV10, one member of the first class, binds operator DNA and nonoperator DNA almost equally well . The affinity differences between FV10 and wild type indicate that each Phe-10 side chain contributes 1.5-2.0 kcal to operator binding but less than 0.5 kcal/mol to nonoperator binding, demonstrating that contacts between Phe-10 and the operator DNA backbone contribute to binding specificity . This appears to be a direct contribution as the crystal structure of the FV10 dimer is similar to wild type and the Phe-10-DNA backbone interactions are the only contacts perturbed in the cocrystal structure of the FV10-operator complex. J Surg Res, 1999 Feb, 81(2), 156 - 63 Concomitant increase in neutrophil adhesion to inflammatory peritoneum and remote organs during peritonitis; Fukatsu K et al.; BACKGROUND: Neutrophils contribute to the host defense mechanism, but they can cause remote organ injury in peritonitis . The purpose of this study was to examine neutrophil adhesion to the peritoneum and remote organs simultaneously in peritonitis using a fluorescence microscopic method . STUDY DESIGN: Experiment 1: Sprague-Dawley rats (n = 16) were injected intraperitoneally (ip) with saline solution or 10(5), 10(7), or 10(9) Escherichia coli . Five hours after challenge, 1 x 10(6) fluorescein-labeled neutrophils were infused . Two minutes after neutrophil injection, five peritoneal samples (the greater omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy . Lung myeloperoxidase (MPO) activity was also determined . Experiment 2: Rats (n = 23) were given 10(9) E . coli ip . Before challenge (0 h) or at 1, 5, or 10 h after challenge, labeled neutrophils were infused . Then, the labeled neutrophil numbers in organs and lung MPO activities were assessed as described for Experiment 1 . Hemodynamic and arterial blood gas data were also obtained in another set of rats before and at 1, 5, 8 and 10 h after 10(9) E . coli ip challenge . RESULTS: Experiment 1: The labeled neutrophil numbers in the peritoneum, lungs, and kidney showed significant positive correlations with the injected bacterial numbers . Lung MPO also positively correlated with E . coli number and labeled neutrophil number in the lungs . Experiment 2: Labeled neutrophil numbers in the peritoneum and kidney peaked at 5 h . The pulmonary labeled neutrophil number rose, reaching a plateau at 5 h . No remarkable change was observed in the hepatic labeled neutrophil number . There was a positive correlation between lung MPO activity and pulmonary labeled neutrophil number . Hemodynamic and blood gas data reflected a hyperdynamic state . CONCLUSIONS: Concomitant dose-dependent increases in neutrophil adhesion in the peritoneum, lungs, and kidney were observed in this peritonitis model . Increased neutrophil adhesion was transient in the peritoneum and kidney but persistent in the lungs . Strategies modulating neutrophil adhesion in organs are anticipated to be useful for the treatment of peritonitis . J Surg Res, 1999 Feb, 81(2), 129 - 38 Hepato-splanchnic blood flow and oxygen extraction capabilities during experimental tamponade: effects of endotoxin; Zhang H et al.; We studied the hepato-splanchnic vascular response and changes in O2 extraction capabilities to a reduction in blood flow following endotoxemia . Fourteen anesthetized and mechanically ventilated dogs were divided into two groups of seven each . Group 1 received 2 mg/kg of E . coli endotoxin, and group 2 served as a control . After initial fluid resuscitation following endotoxic shock, regional blood flow estimated by an ultrasonic technique increased similarly in the hepatic artery, portal vein, and mesenteric artery, but microvascular blood flow estimated by a laser Doppler technique was lower in the liver than in the intestinal mucosa . When blood flow was reduced by cardiac tamponade, endotoxin-treated animals had greater whole body and regional critical O2 delivery (DO2crit) and lower whole body, liver, and intestinal critical O2 extraction ratios (O2ERcrit) . DO2crit was higher in the liver than in intestine but O2ERcrit was similar in the two organs . Whole body DO2crit at the onset of organ O2 supply dependency was similar under control (9.4 +/- 1.9 mL/kg . min for whole body, 10.3 +/- 4.7 mL/kg . min for liver, and 10.0 +/- 2.6 mL/kg . min for intestine) and endotoxic conditions (13.6 +/- 3.2 mL/kg . min for whole body, 15.6 +/- 2.7 mL/kg . min for liver, and 15.4 +/- 8.7 mL/kg . min for intestine) . We conclude that fluid-resuscitated endotoxic shock in dogs is characterized by blood flow redistribution within the liver and intestine . Microvascular depression may be more severe in the liver than in the intestinal mucosa, although the whole body, the liver, and the intestine became O2 supply-dependent simultaneously . Genetics, 1999 Feb, 151(2), 439 - 46 Escherichia coli mutM suppresses illegitimate recombination induced by oxidative stress; Onda M et al.; DNA damage by oxidative stress is one of the causes of mutagenesis . However, whether or not DNA damage induces illegitimate recombination has not been determined . To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of lambdabio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination . To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process . The frequency of lambdabio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type . Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination . Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total lambdabio transducing phages in the wild type or in the mutM mutant, respectively . The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA. EMBO J, 1999 Feb 1, 18(3), 771 - 83 The internal workings of a DNA polymerase clamp-loading machine; Turner J et al.; Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins . The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader . We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme . The gamma complex uses ATP to open the beta clamp and assemble it onto DNA . Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp . The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it . The delta' subunit acts as a modulator of the interaction between delta and beta . On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface . The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA . Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex. EMBO J, 1999 Feb 1, 18(3), 595 - 604 Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding; Munck Petersen C et al.; We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP) . The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain . We now show that the truncation results from cellular processing . Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide . We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin . The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity . Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. EMBO J, 1999 Feb 1, 18(3), 555 - 64 Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function; Poon PP et al.; ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity . They rely on the actions of GTPase-activating proteins (GAPs) for their function . The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport . However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist . We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity . Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)-Golgi stage of vesicular transport . Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi . The glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER-Golgi alleviates the effects of a glo3Delta mutation . An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins . These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER. Am J Respir Crit Care Med, 1999 Feb, 159(2), 610 - 2 Contaminated aerosol recovery from pulmonary function testing equipment; Hiebert T et al.; Clinically, the spread of infectious agents between subjects undergoing spirometry is quite uncommon . There is almost no documentation in the medical literature on this subject . We studied the retrieval of nonpathogenic Escherichia coli after aerosolizing organisms into standard pulmonary function tubing of a type that is frequently used by volume-sensing spirometers . The arrival of the aerosol at the distal end of the tubing was documented by culture . After delays of 0, 1, 5, and 10 min, respectively, air was forcibly withdrawn from the proximal end of the tubing through a special petri plate assembly . The plates were cultured and the colonies were counted . Immediately after insufflation of organisms, air withdrawn from the proximal tubing had counts similar to the air sampled at the distal end . After a 1-min delay, the proximal samples contained only rare organisms . No organisms were recovered from proximal air samples after a delay of 5 or 10 min after insufflation of organisms . The absence of detectable aerosolized E . coli after delays of 5 and 10 min after insufflation of organisms into spirometry tubing supports the hypothesis that a significant transfer of aerosolized organisms does not occur during routine pulmonary function testing as long as an interval of 5 min or more is allowed between tests. Am J Respir Crit Care Med, 1999 Feb, 159(2), 563 - 70 Response to inhaled nitric oxide in acute lung injury depends on distribution of pulmonary blood flow prior to its administration; Gust R et al.; Responses to inhaled nitric oxide (iNO) in acute lung injury (ALI), as evidenced by improvements in oxygenation, are variable . We hypothesized that the effect of iNO may be related to the pre-iNO distribution of pulmonary blood flow (PBF) . In the present study we evaluated the effect of iNO on PBF in normal healthy dogs and in a canine model of ALI induced by oleic acid (OA) . In Group "OA only" (n = 5), ALI was induced by central venous injection of 0.08 ml/kg OA . In Group "E+OA" (n = 5), hypoxic pulmonary vasoconstriction after ALI was blocked with low-dose endotoxin (15 microg/kg of Escherichia coli endotoxin) administered 30 min before giving the same dose of OA . Measurements of regional PBF and lung water concentration (LWC) using positron emission tomography (PET) and H215O were performed before and after OA or placebo, and then again at concentrations of 10, 40, and 0 ppm iNO . One hundred twenty minutes after OA injury, PaO2/FIO2 fell significantly in Group OA only, from 567 +/- 32 to 437 +/- 67 mm Hg . In these animals, PBF redistributed from the dorsal edematous regions of the lungs to the nondependent zones, thus partially preserving normal ventilation/ perfusion relationships . As in the normal animals, in Group OA only, iNO did not significantly change either PBF or oxygenation . In Group E+OA, the administration of low-dose endotoxin eliminated perfusion redistribution from the dorsal edematous lung regions . As a result, PaO2/FIO2 fell from 558 +/- 70 to 119 +/- 53 mm Hg, a decrease that was significantly greater than that in Group OA only . In Group E+OA, administration of iNO restored perfusion redistribution to a similar level as in Group OA only, which was associated with a significant improvement in PaO2/FIO2, from 119 +/- 53 to 251 +/- 159 (10 ppm iNO), and 259 +/- 165 mm Hg (40 ppm iNO) . We conclude that the effect of iNO on oxygenation after ALI depends on the pre-iNO perfusion pattern, which may help explain the variable response to iNO often observed in patients with acute respiratory distress syndrome. Endocrinology, 1999 Feb, 140(2), 568 - 74 Characteristics of a highly labile human type 5 17beta-hydroxysteroid dehydrogenase; Dufort I et al.; 17Beta-hydroxysteroid dehydrogenases (17betaHSDs) play an essential role in the formation of active intracellular sex steroids . Six types of 17betaHSD have been described to date, which only share approximately 20% homology . Human type 5 17betaHSD complementary DNA is unique among the 17betaHSDs because it belongs to the aldo-keto reductase family, whereas the others are members of the short chain alcohol dehydrogenases . The characteristics of human type 5 17betaHSD were investigated in human embryonic (293) cells stably transfected with human and mouse type 5 17betaHSD, as well as human type 3 3alphaHSD . Using intact transfected cells, type 5 17betaHSD shows a substrate specificity pattern comparable to those of human type 3 17betaHSD and mouse type 5 17betaHSD . These enzymes catalyze more efficiently the transformation of androstenedione (4-dione) to testosterone, whereas the transformation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol is much lower . In contrast, type 3 3alphaHSD catalyzes more efficiently the transformation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol, whereas the transformation of 4-dione to testosterone represents only 7% of the 3alphaHSD activity . However, upon homogenization, human type 5 17betaHSD activity decreases to approximately 10% of the activity in intact cells and remains stable at this level together with the 3alphaHSD activity . Under the same conditions, however, the mouse enzyme is not altered by homogenization . Indeed, using purified human 17betaHSD overexpressed in Escherichia coli, we could confirm that a much greater amount of protein is required to produce activity similar to the enzymatic activity measured in intact transfected cells . The present data provide the answer to the question of why previous researchers could hardly detect type 5 17betaHSD activity . Indeed, all previous publications used cell or tissue homogenates or purified enzymes . Under such conditions, only the low level, but stable, 3alphaHSD and 17betaHSD activities could be measured, whereas the high level, but highly unstable, 17betaHSD activity could not be measured . As type 5 17betaHSD shares 84%, 86%, and 88% amino acid identity with types 1 and 3 3alphaHSD and 20alphaHSDs, respectively, Northern blot analysis used in previous studies could not provide unequivocal information . In this report, we used a more specific ribonuclease protection assay and could thus show that human type 5 17betaHSD is expressed in the liver, adrenal, and prostate; in prostatic cancer cell lines DU-145 and LNCaP; as well as in bone carcinoma (MG-63) cells . By analogy with type 3 17betaHSD, which is responsible for the formation of androgens in the testis, the expression of type 5 17betaHSD in the prostate and bone cells suggests that this enzyme is involved in the formation of active intracellular androgens in these tissues. Virus Genes, 1998, 17(3), 207 - 11 Sequence and expression in Escherichia coli of the coat protein gene of the dwarfing strain of soybean dwarf luteovirus; Smith OP et al.; The nucleotide sequence of the coat protein gene of the dwarfing (D) strain of soybean dwarf luteovirus (SbDV) was determined from cloned cDNA . The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated molecular mass of 22.2 kDa . A major portion of the coat protein open reading frame (ORF) was expressed in Escherichia coli as a pET fusion protein and the product was detected by western blot analysis using SbDV-D polyclonal antibodies . Comparison of the deduced coat protein amino acid sequence to that from the yellowing (Y) strain of SbDV demonstrated 88% identity. Acta Anaesthesiol Scand, 1999 Jan, 43(1), 56 - 63 Intestinal and hepatic perfusion and metabolism in hypodynamic endotoxic shock . Effects of nitric oxide synthase inhibition; Dahm PL et al.; BACKGROUND: Inhibition of nitric oxide synthase (NOS) has been claimed to be beneficial in septic shock . We investigated the overall and regional effects of a NOS-inhibitor on perfusion and metabolism during severe endotoxic shock . METHODS: Nineteen anaesthetised pigs were catheterised and ultrasonic flow-probes were placed around the portal vein, the hepatic artery, and the superior mesenteric artery . Thirteen animals were given a 3-h infusion of endotoxin; in 6 of these an infusion of NG-nitro-L-arginine-methyl-ester (L-NAME) was started an hour after the start of endotoxin while 7 animals served as controls and received endotoxin only . Six animals were sham operated with no further intervention . RESULTS: Endotoxin produced a hypodynamic shock with pulmonary hypertension . L-NAME did not increase arterial blood pressure, but deepened the fall in cardiac output and enhanced the increase in systemic and pulmonary vascular resistance . The infusion of endotoxin caused a decrease in flows in all regions . The addition of L-NAME induced a further decrease in the mesenteric artery flow only . L-NAME had no additional effect on hepatic artery flow ratio, while a transient decrease was seen in mesenteric flow ratio . Portal flow ratio decreased in the control group only . Global as well as regional oxygen extraction increased in both groups, more so in the L-NAME group . Lactate levels increased with no differences between the groups . CONCLUSION: In hypodynamic endotoxic shock, L-NAME infusion enhanced pulmonary vasoconstriction and increased left ventricular afterload . The resulting hypoperfusion caused an increase in mortality . The effects of L-NAME on global and mesenteric blood flow and metabolism were similar, while L-NAME had no additional effects on hepatic hypoperfusion or oxygen extraction . Thus, nitric oxide does not seem to be a major factor in the preservation of hepatic perfusion during unresuscitated endotoxic shock. J Mol Biol, 1999 Feb 5, 285(5), 1977 - 91 Classification of Arabidopsis thaliana gene sequences: clustering of coding sequences into two groups according to codon usage improves gene prediction; Mathe C et al.; While genomic sequences are accumulating, finding the location of the genes remains a major issue that can be solved only for about a half of them by homology searches . Prediction methods are thus required, but unfortunately are not fully satisfying . Most prediction methods implicitly assume a unique model for genes . This is an oversimplification as demonstrated by the possibility to group coding sequences into several classes in Escherichia coli and other genomes . As no classification existed for Arabidopsis thaliana, we classified genes according to the statistical features of their coding sequences . A clustering algorithm using a codon usage model was developed and applied to coding sequences from A . thaliana, E . coli, and a mixture of both . By using it, Arabidopsis sequences were clustered into two classes . The CU1 and CU2 classes differed essentially by the choice of pyrimidine bases at the codon silent sites: CU2 genes often use C whereas CU1 genes prefer T . This classification discriminated the Arabidopsis genes according to their expressiveness, highly expressed genes being clustered in CU2 and genes expected to have a lower expression, such as the regulatory genes, in CU1 . The algorithm separated the sequences of the Escherichia-Arabidopsis mixed data set into five classes according to the species, except for one class . This mixed class contained 89 % Arabidopsis genes from CU1 and 11 % E . coli genes, mostly horizontally transferred . Interestingly, most genes encoding organelle-targeted proteins, except the photosynthetic and photoassimilatory ones, were clustered in CU1 . By tailoring the GeneMark CDS prediction algorithm to the observed coding sequence classes, its quality of prediction was greatly improved . Similar improvement can be expected with other prediction systems . J Mol Biol, 1999 Feb 5, 285(5), 1935 - 50 Trans-splicing ribozymes for targeted gene delivery; Kohler U et al.; Ribozymes are potential tools for genetic manipulation, and various naturally occurring catalytic RNAs have been dissected and used as the basis for the design of new endoribonuclease activities . While such cleaving ribozymes may work well in vitro, they have not proved to be routinely effective in depleting living cells of the chosen target RNA . Recently, trans-splicing ribozymes have been employed to repair mutant mRNAs in vivo . We have designed modified trans-splicing ribozymes with improved biological activity . These allow accurate splicing of a new 3' exon sequence into a chosen site within a target RNA, and in frame fusion of the exon can result in expression of a new gene product . These trans-splicing ribozymes contain catalytic sequences derived from a self-splicing group I intron, which have been adapted to a chosen target mRNA by fusion of a region of extended complementarity to the target RNA and precise alteration of the guide sequences required for substrate recognition . Both modifications are required for improved biological activity of the ribozymes . Whereas cleaving ribozymes must efficiently deplete a chosen mRNA species to be effective in vivo, even inefficient trans-splicing can allow the useful expression of a new gene activity, dependent on the presence of a chosen RNA . We have targeted trans-splicing ribozymes against mRNAs of chloramphenicol acetyltransferase, human immunodeficiency virus, and cucumber mosaic virus, and demonstrated trans-splicing and delivery of a marker gene in Escherichia coli cells . The improved trans-splicing ribozymes may be tailored for virtually any target RNA, and provide a new tool for triggering gene expression in specific cell types . Appl Environ Microbiol, 1999 Feb, 65(2), 859 - 61 Enhancement of solar water pasteurization with reflectors; Safapour N et al.; A simple and reliable method that could be used in developing countries to pasteurize milk and water with solar energy is described . A cardboard reflector directs sunshine onto a black jar, heating water to pasteurizing temperatures in several hours . A reusable water pasteurization indicator verifies that pasteurization temperatures have been reached. Appl Environ Microbiol, 1999 Feb, 65(2), 409 - 14 Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells; Cha HJ et al.; We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate Escherichia coli cellular stress responses in quiescent or resting cells . Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor sigma32, the protease subunit ClpB, and the chaperone DnaK, to the reporter gene gfpuv . When perturbed by a chemical or physical stress (such as a heat shock, nutrient {amino acid} limitation, or addition of IPTG {isopropyl-beta-D-thiogalactopyranoside}, acetic acid, ethanol, phenol, antifoam, or salt {osmotic shock}), the E . coli cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence . Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded. J Mol Cell Cardiol, 1998 Nov, 30(11), 2261 - 8 Molecular cloning of rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein); Kannan S et al.; Rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein), a membrane-bound enzyme requiring millimolar concentrations of Ca2+ or Mg2+ for maximal hydrolysis of ATP, has been purified to apparent homogeneity . Tryptic digestion and amino acid sequencing was used to design an oligonucleotide probe for screening a rat heart cDNA library; this produced a partial cDNA clone (pND2.1), and sequencing of a 400 base pair portion revealed a 100% homology to human platelet CD36 . Northern blotting with pND2.1 detected a 3.1 kb transcript in rat heart but not in other tissues . Interspecies expression analysis (cardiac tissue total RNA blot probed with pND2.1) detected a approximately 2.0 kb transcript in canine, rabbit and porcine heart, whereas transcripts of a 4.1 kb, approximately 3.0 kb and 2.1 kb were observed in human cardiac tissue . A rat genomic DNA Southern blot, probed with pND2.1, indicated that there was a single copy of the gene in the rat genome . Expression of the pND2.1 cDNA in E . coli produced an 89 kDa polypeptide recognized by anti-human CD36 antibody but not by anti-rat Ca2+/Mg2+ ectoATPase antibody . It is concluded that rat cardiac Ca2+/Mg2+ ectoATPase is tightly associated with a protein highly homologous to the adhesion molecule CD36. J Vet Diagn Invest, 1999 Jan, 11(1), 20 - 6 Seroneutralization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the GP5 major envelope glycoprotein; Gonin P et al.; To determine the structural protein of the porcine reproductive and respiratory syndrome virus (PRRSV) involved in the production of neutralizing antibodies following clinical infection, correlation was studied between virus neutralization capability of convalescent pig sera and antibody response to the open reading frames (ORFs) 3-, 4-, 5-, and 7-encoded proteins GP3, GP4, GP5, and N, respectively . Individual virus genes were cloned into the pGEX-4T-1 vector, and the recombinant viral proteins were expressed in Escherichia coli fused to the glutathione S-transferase (GST) protein . The resulting GST-ORF3, GST-ORF4, GST-ORF5, and GST-ORF7 recombinant fusion proteins were purified by electroelution and used as antigens for serologic testing by indirect enzyme-linked immunosorbent assay and western immunoblotting . The overall antibody (IgG and IgM) titers to PRRSV of pooled convalescent pig sera were first determined by indirect immunofluorescence, and then sera with specific IgG titers > 1:1,024 were tested for their specific virus neutralization activity and reactivity to individual recombinant fusion proteins . Except for the early immune response (as revealed by the presence of specific IgM), neutralizing titers were correlated with anti-GP5 titers but not with anti-GP3 and anti-GP4 titers . The correlation between virus neutralization and anti-GP5 titers was significant (r = 0.811, P < or = 0.001). Biochimie, 1998 Dec, 80(12), 987 - 1001 Selected phenotypes of ihf mutants of Escherichia coli; Bykowski T et al.; In attempts to identify subunit-specific phenotypes of ihf mutants we analyzed viability, thermoresistance and protein synthesis patterns in ihfA and ihfB mutants and their respective parental strains . Despite some detected differences in the two-dimensional protein patterns, no significant subunit-specific, physiological effects could be observed . Each mutant was less viable and less thermoresistant than the wild type strain . Moreover, in contrast to the wild type the mutants did not reduce global protein synthesis after prolonged culturing . Examination of expression of transcriptional fusions allowed us to demonstrate autoregulation of both genes by IHF . Additional IHF binding sites in the regulatory region of both ihf genes were footprinted. Int J Biochem Cell Biol, 1998 Dec, 30(12), 1379 - 88 Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence; Gao ZG et al.; Porcine brain pyridoxal kinase has been cloned . A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique . The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa . The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase . Expression of the cloned cDNA in E . coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain . With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain . Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain. Br J Anaesth, 1998 Oct, 81(4), 601 - 2 Thiopental attenuates relaxation and cyclic GMP production in vascular smooth muscle of endotoxin-treated rat aorta, independent of nitric oxide production; Kim SO et al.; As thiopental (thiopentone) suppresses cyclic GMP (cGMP) formation produced by nitric oxide donor drugs, we have tested if it suppresses cGMP formation and increases vascular tone after induction of calcium-calmodulin-independent nitric oxide synthase (iNOS) . Rat aortic rings were treated with Escherichia coli lipopolysaccharide (LPS) 1 microgram ml-1 for 4 h, and the effects of thiopental on tension, cGMP concentrations and nitrite accumulation were determined . Thiopental 0.3 mmol litre-1 reduced the tension of phenylephrine-precontracted aortic rings before LPS treatment, but caused no significant effects on tension in the presence of L-arginine 10 mumol litre-1 after LPS treatment . L-Arginine 1 mumol litre-1 to 1 mmol litre-1 increased concentrations of cGMP in LPS-treated aorta in a concentration-dependent manner . This was reduced by thiopental 0.3-1 mmol litre-1 . Treatment with L-arginine 1 mmol litre-1 increased concentrations of nitrite, the end product of nitric oxide; this was not affected by thiopental 1 mmol litre-1 . We conclude that thiopental suppressed cGMP formation in iNOS-induced vascular smooth muscle without affecting nitric oxide production. J Endocrinol, 1999 Feb, 160(2), 239 - 45 Isolation of radiochemically pure 125I-labeled human thyrotropin receptor and its use for the detection of pathological autoantibodies in sera from Graves' patients; Minich WB et al.; We report a method for the purification and radioactive labeling of human TSH receptor (TSHR) . The method is based on the construction of a fusion TSHR (TSHR-Xa-BIO) which consists of the N-terminal 725 amino acids of human TSHR linked to the 4-amino acid Xa protease cleavage site and the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase (the C-terminal domain directs the efficient posttranslational biotinylation of the protein) . TSHR-Xa-BIO was produced in HeLa cells using recombinant vaccinia virus . The expressed protein was fully functional and was biotinylated with an efficiency of about 90% . Streptavidin-agarose-immobilized TSHR-Xa-BIO was labeled with 125I using the chloramine T oxidation procedure and specifically eluted from the solid phase after cleavage with protease Xa . Isolated native radiochemically pure 125I-labeled TSHR specifically interacted with pathological autoantibodies in the sera of patients with Graves' disease, and thus could be useful for the detection of these autoantibodies by immunoprecipitation analysis. J Virol Methods, 1998 Dec, 76(1-2), 101 - 8 A novel expression system based on host-range expansion of baculovirus; Zhu Y et al.; A host range expanded recombinant Autographa californica multiple-nucleocapsid nucleopolyhedrosis virus AcMNPV/r2 was obtained by cotransfection of the bacmid DNA from Escherichia coli DH10Bac along with a plasmid pBmH-M containing HindIII M fragment of Bombyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA . A recombinant transposon vector carrying a mutant green fluorescent protein gene (GFP) and a polyhedrin gene was constructed . Transposition was carried out in both E . coli DH10Bac and E . coli DH10BmH, which contains AcMNPV/r2 and a helper plasmid . Recombinant DNAs were transfected into Sf-9 cells to generate recombinant virus AcMNPV/r3 and AcMNPV/r4 respectively . Viral stock of AcMNPV/r4 was then infected into Bombyx mori cells (BmN) and Bombyx mori larvae (silkworm) . Analysis shows that GFP was highly expressed in Bombyx mori larvae . This expression system, is practicable therefore for mass production of foreign gene products. J Virol Methods, 1998 Dec, 76(1-2), 51 - 8 An efficient way to introduce unique restriction endonuclease sites into a baculovirus genome; Yang S et al.; Recombinant baculoviruses which can be linearized at unique sites with restriction endonucleases can greatly facilitate the construction of other recombinants including baculovirus expression vectors and site-specific mutants . We designed a strategy to introduce unique restriction endonuclease sites at virtually any location in a baculovirus genome . The unique sites were first introduced onto a transfer plasmid which also contained in the vector portion of the plasmid an E . coli lacZ gene and a Sse8387I site, a sequence which is not found in the viral genome . Cotransfection of the transfer plasmid and circular viral DNA generated single-crossover recombinant viruses which could be distinguished as blue plaques in the presence of X-gal, a chromogenic indicator for lacZ . Single-crossover recombinants were purposefully isolated and propagated to generate double-crossover recombinants . Viral DNA isolated from the mixed virus population was digested with Sse8387I to linearize only the single-crossover viral DNA; double-crossover recombinants in the progeny viral population resulting from transfection with the Sse8387I-linearized viral DNA mixture were thus highly enriched, making the task of screening much easier . To demonstrate the feasibility of this approach, we introduced Bsu36I sites into the orf24 and the vlf-1 regions of Autographa californica multiple-nucleocapsid nuclear polyhedrosis virus (AcMNPV) to generate recombinant viruses vncBsuorf24 and vncBsuvlf1, respectively . Both recombinant viruses were obtained by screening only ten plaques . This method should also be applicable to other kinds of mutations and may be applicable to other double-stranded DNA viruses. Biochem Cell Biol, 1998, 76(2-3), 189 - 97 An NMR study of ligand binding by maltodextrin binding protein; Gehring K et al.; Proton NMR spectra of maltodextrin binding protein from Escherichia coli were used to monitor conformational changes that accompany ligand binding . Chemical shift changes associated with the binding of different maltodextrins to maltodextrin binding protein were studied using one-dimensional difference spectra . Line-shape analysis of an isolated upfield methyl resonance was used to measure the kinetics of maltose binding at several temperatures . Maltose and linear maltodextrins caused similar changes to the upfield protein spectrum with no detectable differences between alpha and beta sugar anomers . Binding of a cyclic ligand, beta-cyclodextrin, caused smaller chemical shift changes than binding of linear maltodextrins . Two maltodextrin derivatives were also studied . Both maltohexaitol and maltohexanoic acid gave one-dimensional difference spectra that were intermediate between those of linear maltodextrins and beta-cyclodextrin . The methyl resonances at -1 and -0.35 ppm were assigned to leucine 160 on the basis of homonuclear COSY and TOCSY experiments and theoretical chemical shift calculations using the X-ray crystal structure of maltodextrin binding protein. Matrix Biol, 1998 Dec, 17(8-9), 673 - 7 Epitope-specific monoclonal antibodies against human C-terminal procollagen alpha1(III)-propeptide; Burchardt ER et al.; We have generated monoclonal antibodies against recombinant C-terminal human procollagen alpha1(III) propeptide (PIIICP), produced in E . coli in high yields . The monoclonal antibodies were screened for epitope specificity using recombinant truncated PIIICP . Several antibodies were identified which recognized different regions of the PIIICP molecule . The ability of the antibodies to detect PIIICP antigens in human cell line lysates and supernatants was demonstrated . As PIIICP antigens are a key marker of extracellular matrix metabolism, the monoclonal antibodies described here should be of value for clinical and basic research. FEBS Lett, 1999 Jan 8, 442(1), 43 - 7 Impact of the lysine-188 and aspartic acid-189 inversion on activity of trypsin; Briand L et al.; The impact of the charge rearrangement on the specificity of trypsin was tested by an inversion of sequence K188D/D189K maintaining the integrity of the charges of the substrate binding pocket when switching their polarity . In native trypsin, aspartate 189 situated at the bottom of the primary substrate binding pocket interacts with arginine and lysine side chains of the substrate . The kinetic parameters of the wild-type trypsin and K188D/D189K mutant were determined with synthetic tetrapeptide substrates . Compared with trypsin, the mutant K188D/D189K exhibits a 1.5- to 6-fold increase in the Km for the substrates containing arginine and lysine, respectively . This mutant shows a approximately 30-fold decrease of its k(cat) and its second-order rate constant k(cat)/Km decreases approximately 40- and 150-fold for substrates containing arginine and lysine, respectively . Hence, trypsin K188D/D189K displays a large increase in preference for arginine over lysine. FEBS Lett, 1999 Jan 8, 442(1), 29 - 33 Evidence for the co-existence of glutathione reductase and trypanothione reductase in the non-trypanosomatid Euglenozoa: Euglena gracilis Z; Montrichard F et al.; Two NADPH-dependent disulfide reductases, glutathione reductase and trypanothione reductase, were shown to be present in Euglena gracilis, purified to homogeneity and characterized . The glutathione reductase (Mr 50 kDa) displays a high specificity towards glutathione disulfide with a KM of 54 microM . The amino acid sequences of two peptides derived from the trypanothione reductase (Mr 54 kDa) show a high level of identity (81% and 64%) with sequences of trypanothione reductases from trypanosomatids . The trypanothione reductase is able to efficiently reduce trypanothione disulfide (KM 30.5 microM) and glutathionylspermidine disulfide (KM 90.6 microM) but not glutathione disulfide, nor Escherichia coli thioredoxin disulfide, nor 5,5'-dithiobis(2-nitrobenzoate) (DTNB) . These results demonstrate for the first time (i) the existence of trypanothione reductase in a non-trypanosomatid organism and (ii) the coexistence of trypanothione reductase and glutathione reductase in E . gracilis. FEBS Lett, 1999 Jan 8, 442(1), 15 - 9 Cell-free production and stable-isotope labeling of milligram quantities of proteins; Kigawa T et al.; We have improved the productivity of an Escherichia coli cell-free protein synthesis system . First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized . Second, the E . coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis . Third, during the protein synthesis, the reaction mixture was dialyzed against a low-molecular-weight substrate solution to prolong the reaction . Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture . Stable-isotope labeling of a protein with 13C/15N-labeled amino acids for NMR spectroscopy was achieved by this method. FEBS Lett, 1999 Jan 8, 442(1), 7 - 10 Functional reconstitution of RNase P activity from a plastid RNA subunit and a cyanobacterial protein subunit; Pascual A et al.; The plastid (cyanelle) from the Glaucocystophyceae alga Cyanophora paradoxa contains an RNase P RNA subunit (P RNA) similar to the cyanobacterial P RNA . We have synthesized this RNA by in vitro transcription and analyzed its activity in the absence or presence of the RNase P protein subunit (P protein) from Escherichia coli and the cyanobacterium Synechocystis sp . PCC 6803 . In contrast to the bacterial P RNA, the cyanelle P RNA is not active in the absence of protein in any of the conditions tested . A functional enzyme could be reconstituted with the Synechocystis protein but not with the E . coli protein . This is the first demonstration of RNase P activity reconstitution from organellar and bacterial subunits. Res Virol, 1998 Nov-Dec, 149(6), 413 - 8 A recombinant Toscana virus nucleoprotein in a diagnostic immunoblot test system; Schwarz TF et al.; Sandfly fever, a vector-borne disease endemic in the Mediterranean region, is caused by Toscana virus (TOS) . The disease is increasingly important as a travel-related infection . Serological diagnosis is currently dependent on viral antigens derived from TOS-infected cell cultures . In this study, we report the cloning and expression of the TOS nucleoprotein (N) in Escherichia coli and evaluation of the recombinant (r) TOS N protein as an antigen for immunoblot assays . The TOS N gene was amplified by reverse-transcriptase polymerase chain reaction and cloned into the bacterial expression vector pTrcHis-A . Sera with known TOS antibody status were used to evaluate the immunoblot assay . The expressed rTOS N protein was purified and used as antigen for immunoblots . By recombinant immunoblot, the TOS antibody status (IgM and/or IgG) of the test panel was correctly identified . No cross-reactivity was detected . The rTOS N protein is useful as an antigen for immunoblot assays, and will enable more laboratories to perform TOS antibody diagnosis. Biochemistry, 1998 Dec 22, 37(51), 18074 - 80 Promoter recognition by Escherichia coli RNA polymerase: effects of the UP element on open complex formation and promoter clearance; Strainic MG Jr et al.; Escherichia coli promoters for transcription of ribosomal and tRNAs are greatly activated by an A+T-rich "UP" element upstream of the -35 region . These same promoters have also been found to otherwise deviate in several respects from the consensus promoter sequence . Here we present the results of a kinetic characterization of the interaction of Escherichia coli RNA polymerase with UP element-containing promoters which by virtue of consensus or near-consensus sequence features should be among the most optimal that can be encountered by Escherichia coli RNA polymerase . We show that for such promoters, (1) the second-order rate constant describing formation of the initial (closed) complex is close to that expected for a diffusion-limited process, (2) the extent of activation by the UP element is temperature-sensitive, (3) the UP element accelerates a process after DNA binding by RNA polymerase, and (4) the presence of the UP element delays promoter clearance upon addition of nucleoside triphosphates to preformed RNA polymerase-promoter complexes . Finally, we provide evidence in support of models which describe the DNA melting process accompanying open complex formation as initiating in the -10 promoter region and progressing in the downstream direction. Rapid Commun Mass Spectrom, 1999, 13(1), 73 - 8 Detection and identification of low-mass peptides and proteins from solvent suspensions of Escherichia coli by high performance liquid chromatography fractionation and matrix-assisted laser desorption/ionization mass spectrometry; Dai Y et al.; The application of high performance liquid chromatography (HPLC) to separate components in solvent suspension of Escherichia coli followed by off-line matrix-assisted laser desorption/ionization (MALDI) analysis of collected fractions results in the detection of over 300 peaks in the 2000-19,000 Da mass range, an order of magnitude increase in the number of components observed when compared with direct MALDI analysis of the entire solvent suspension . Mass measurements of these separated components using a time-lag focusing MALDI instrument are reported . MALDI analysis of the proteolytic digests of several collected fractions facilitates the identification of three components as specific proteins expected to be present in E . coli . The methodologies reported here should be very useful in searching for unique biomarkers for bacterial discrimination. Biotechnol Bioeng, 1999 Feb 20, 62(4), 455 - 60 Chemical treatment of Escherichia coli: 3 . Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells; Falconer RJ et al.; In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells . Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants . In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported . In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized beta-mercaptoethanol, to the permeabilization buffer (6 M urea + 3 mM ethylenediaminetetraacetate {EDTA}) . 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea . Contaminating proteins are separated from the inclusion-body fraction by centrifugation . In the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol {DTT}) into the permeabilization buffer . Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R3-IGF-I from inclusion bodies located in the cytoplasm of intact E . coli, at a purity of 46% (w/w) . This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization) . A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration . This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. Biotechnol Bioeng, 1999 Feb 20, 62(4), 392 - 401 Metabolite and isotopomer balancing in the analysis of metabolic cycles: II . Applications; Park SM et al.; In a previous paper (Klapa et al., 1999), we presented a model for the analysis of isotopomer distributions of the TCA cycle intermediates resulting from 13C (or 14C) labeling experiments . Results allow the rigorous determination of the degree of enrichment at specific carbon atoms of metabolites, of the molecular weight distribution of metabolite isotopomers, as well as of the fine structure of NMR spectra in terms of a small number of metabolic fluxes . In this paper we validate the model by comparing model predictions with experimental data and then apply it to the analysis of metabolic networks that have been investigated in previous studies . The results have allowed us to conclude that: (1) there is no evidence of propionyl-CoA carboxylase pathway in Escherichia coli; and (2) the possibility that acetone utilization in mammals occurs solely via the "lactate/methylglyoxal" pathway is consistent with available labeling data . The presented modeling framework provides additional constraints that must be satisfied by experimental data in a biochemical network structure and therefore enhances the power of labeling methods for resolving in vivo metabolic fluxes. Biochem Biophys Res Commun, 1999 Jan 27, 254(3), 632 - 5 Transfer of sulfur to biotin from biotin synthase (BioB protein); Gibson KJ et al.; In biotin synthase reactions carried out in vitro, we observed efficient transfer of 35S to biotin from partially purified E . coli biotin synthase (product of the bioB gene) labelled by biosynthetic incorporation of {35S}-cysteine . Mass spectrometry was consistent with covalent alteration of the protein in the assay . These results suggest that BioB protein is a reagent, not a catalyst . J Bacteriol, 1999 Feb, 181(3), 1039 - 42 Topology of RbsC, a membrane component of the ribose transporter, belonging to the AraH superfamily; Park Y et al.; RbsC of Escherichia coli is the hydrophobic membrane component of ribose uptake system classified as the ATP-binding cassette transporter . To understand the structure and function of RbsC, its transmembrane topology was investigated by using 64 RbsC-PhoA fusions isolated either specifically or randomly . In order to confirm the cytoplasmic location of the short C-terminal region (5 amino acids), inside-out or right-side-out membrane vesicles were generated, and the C-terminal region was found to be digested by carboxypeptidase A only in inside-out vesicles . This result is consistent with the model, based on the results of alkaline phosphatase fusions, in which the protein traverses the membrane six times and the N and C termini are exposed to the cytoplasm. J Bacteriol, 1999 Feb, 181(3), 1035 - 8 Archaeal nucleosome positioning by CTG repeats; Sandman K et al.; DNA shape recognition determines the preferred binding sites for sequence-independent DNA binding proteins, and here we document that archaeal histones assemble archaeal nucleosomes in vitro centered preferentially within (CTG)6 and (CTG)8 repeats, close to junctions with flanking mixed-sequence DNA . Archaeal nucleosomes were not positioned by (CTG)4-, (CTG)5-, or (CTG)3AA(CTG)3-containing DNA sequences . The features of CTG repeat-containing sequences that direct eucaryal nucleosome positioning may also be similarly recognized by archaeal histones. J Bacteriol, 1999 Feb, 181(3), 1030 - 4 The udhA gene of Escherichia coli encodes a soluble pyridine nucleotide transhydrogenase; Boonstra B et al.; The udhA gene of Escherichia coli was cloned and expressed in E . coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity . The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E . coli genome sequences due to a sequencing error . E . coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase. J Bacteriol, 1999 Feb, 181(3), 916 - 22 Recovery of DNA replication in UV-irradiated Escherichia coli requires both excision repair and recF protein function; Courcelle J et al.; After UV doses that disrupt DNA replication, the recovery of replication at replication forks in Escherichia coli requires a functional copy of the recF gene . In recF mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that recF function is needed to stabilize the disrupted replication forks and facilitate the process of recovery . We show here that the ability of recF to promote the recovery of replication requires that the disrupting lesions be removed . In the absence of excision repair, recF+ cells protect the nascent DNA at replication forks, but replication does not resume . The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells . However, mutations in either uvr or recF result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that recF and excision repair contribute to a common pathway in the recovery of replication. J Bacteriol, 1999 Feb, 181(3), 893 - 8 Cra-dependent transcriptional activation of the icd gene of Escherichia coli; Prost JF et al.; The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR . In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra . We observed that (i) the Cra factor binds to and activates transcription from a site centered at position -76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the alpha subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions. J Bacteriol, 1999 Feb, 181(3), 833 - 40 Immunochemical analysis of UMP kinase from Escherichia coli; Landais S et al.; Mono- and polyclonal antibodies directed against UMP kinase from Escherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis . As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant . Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively . These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping . One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180 . A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2) . Polyclonal antisera were used to identify UMP kinase in the bacterial proteome . The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa . Immunogold labeling of UMP kinase in whole E . coli cells shows a localization of the protein near the bacterial membranes . Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon . The specific localization of UMP kinase might also be related to its putative role in cell division. J Bacteriol, 1999 Feb, 181(3), 823 - 32 Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations; Mukherjee A et al.; FtsZ is an ancestral homologue of tubulin that polymerizes in a GTP-dependent manner . In this study, we used 90 degrees angle light scattering to investigate FtsZ polymerization . The critical concentration for polymerization obtained by this method is similar to that obtained by centrifugation, confirming that the light scattering is proportional to polymer mass . Furthermore, the dynamics of FtsZ polymerization could be readily monitored by light scattering . Polymerization was very rapid, reaching steady state within 30 s . The length of the steady-state phase was proportional to the GTP concentration and was followed by a rapid decrease in light scattering . This decrease indicated net depolymerization that always occurred as the GTP in the reaction was consumed . FtsZ polymerization was observed over the pH range 6.5 to 7.9 . Importantly, Mg2+ was not required for polymerization although it was required for the dynamic behavior of the polymers . It was reported that 7 to 25 mM Ca2+ mediated dynamic assembly of FtsZ (X . -C . Yu and W . Margolin, EMBO J . 16:5455-5463, 1997) . However, we found that Ca2+ was not required for FtsZ assembly and that this concentration of Ca2+ reduced the dynamic behavior of FtsZ assembly. J Bacteriol, 1999 Feb, 181(3), 791 - 8 Excretion of endogenous cadaverine leads to a decrease in porin-mediated outer membrane permeability; Samartzidou H et al.; The permeability of the outer membrane of Escherichia coli to hydrophilic compounds is controlled by porin channels . Electrophysiological experiments showed that polyamines inhibit ionic flux through cationic porins when applied to either side of the membrane . Externally added polyamines, such as cadaverine, decrease porin-mediated fluxes of beta-lactam antibiotics in live cells . Here we tested the effects of endogenously expressed cadaverine on the rate of permeation of cephaloridine through porins, by manipulating in a pH-independent way the expression of the cadBA operon, which encodes proteins involved in the decarboxylation of lysine to cadaverine and in cadaverine excretion . We report that increased levels of excreted cadaverine correlate with a decreased outer membrane permeability to cephaloridine, without any change in porin expression . Cadaverine appears to promote a sustained inhibition of porins, since the effect remains even after removal of the exogenously added or excreted polyamine . The cadaverine-induced inhibition is sufficient to provide cells with some resistance to ampicillin but not to hydrophobic antibiotics . Finally, the mere expression of cadC, in the absence of cadaverine production, leads to a reduction in the amounts of OmpF and OmpC proteins, which suggests a novel mechanism for the environmental control of porin expression . The results presented here support the notion that polyamines can act as endogenous modulators of outer membrane permeability, possibly as part of an adaptive response to acidic conditions. J Bacteriol, 1999 Feb, 181(3), 757 - 63 Structure of the ring cleavage product of 1-hydroxy-2-naphthoate, an intermediate of the phenanthrene-degradative pathway of Nocardioides sp . strain KP7; Adachi K et al.; 1-Hydroxy-2-naphthoate (compound I) is a metabolite of the phenanthrene-degradative pathway in Nocardioides sp . strain KP7 . This singly hydroxylated aromatic compound is cleaved by 1-hydroxy-2-naphthoate dioxygenase . In this study, the structure of the ring cleavage product generated by the action of homogeneous 1-hydroxy-2-naphthoate dioxygenase was determined upon separation by high-performance liquid chromatography at pH 2.5 by using nuclear magnetic resonance (NMR) and mass spectroscopic techniques . The ring cleavage product at this pH existed in equilibrium between two forms, 2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III) and 2,2-dihydroxy-3-(3-oxo-1, 3-dihydro-1-isobenzofuranyl)propanoate (compound IV) . After the pH of the solution was raised to 7.5, the structure of the major species became (E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II; common name, trans-2'-carboxybenzalpyruvate), which was in equilibrium with compound III . Direct monitoring of the enzymatic formation of the ring cleavage product by 1H-NMR in a deuterated potassium phosphate buffer (pH 7.5) detected only compound II as a product, and the proton on carbon 3 of compound II was not exchanged with deuterium . Thus, compound II is likely to be the first stable product of dioxygenation of 1-hydroxy-2-naphthoate. Biochemistry, 1998 Dec 22, 37(51), 18045 - 55 Mutations in the cytosolic DnaJ homologue, YDJ1, delay and compromise the efficient translation of heterologous proteins in yeast; Brodsky JL et al.; The Saccharomyces cerevisiae YDJ1 gene encodes a yeast homologue of DnaJ, an Escherichia coli molecular chaperone and regulator of Hsp70 function . We examined the function of Ydj1p in vivo by analyzing the activity and production of firefly luciferase (FFLux) and green fluorescent protein (GFP) after inducible expression in yeast strains containing a wild type or a mutant YDJ1 gene . Although FFLux and GFP mRNA levels were similar in the wild type and mutant strains, the FFLux protein was translated about half as efficiently in the ydj1-151 mutant compared to the wild type strain; the lower FFLux level was not the result of increased FFLux turnover in the mutant . In contrast, GFP translation was significantly delayed in the ydj1-151 mutant compared to the wild type strain . Surprisingly, we observed that FFLux and GFP mRNA bound efficiently to polysomes in the ydj1-151 mutant . Analysis of polysome profiles also revealed a modest increase in the amount of 60S ribosomal subunits in the ydj1-151 strain, consistent with a translation defect in the mutant, although the Ydj1 protein was not found to be associated with polysomes . To determine whether the inducible expression of an endogenous yeast protein was also less efficient in the ydj1-151 strain, we examined the inducible synthesis of the yeast TATA-binding protein (TBP) but observed no translation defect . Statistical analysis of the FFLux, GFP, and TBP encoding genes suggests that Ydj1p facilitates the expression of proteins that are poorly translated because both FFLux and GFP contain an abundance of codons that are rarely used in yeast. Biochemistry, 1998 Dec 22, 37(51), 18001 - 9 Pressure-denatured state of Escherichia coli ribonuclease HI as monitored by Fourier transform infrared and NMR spectroscopy; Yamasaki K et al.; Pressure denaturation of Escherichia coli ribonuclease HI (RNase HI) was studied by Fourier transform infrared (FTIR) and two-dimensional NMR spectroscopy at pD* 3.0 and 25 degrees C . A reversible transition in the pressure range of 0.1-1090 MPa was observed with second-derivative FTIR experiments . A cooperative and gradual denaturation, involving both the secondary and tertiary structures, was observed between 240 and 450 MPa . The two peaks at 1629 and 1652 cm(-1), due to beta-strands and alpha-helices, respectively, did not fully disappear after the denaturation, and are different from the spectra of the random coil peptides . The hydrogen-deuterium exchange rates of the individual backbone amide protons were determined by heteronuclear NMR combined with the pressure-jump technique at 500, 650, and 850 MPa . Although most of the amides protected in the native structure are also highly protected in the pressure-denatured state, the rate constants (0.048 +/- 0.007 min(-1)) for the amide protons at 500 MPa are similar regardless of their locations, which is an indication of the EX1 mechanism of hydrogen-deuterium exchange . The pressure-denatured state of RNase HI at 500 MPa represents a novel denatured state, which is different from a typical molten globule state at atmospheric pressure (0.1 MPa), from the viewpoint of the homogeneous rate constants . The observations at 650 MPa are essentially the same as those at 500 MPa . However, at 850 MPa, the amide exchange rates for the highly hydrophobic C-terminal half of alpha-helix I are significantly slower than those for the other part of the protein, which can be interpreted as a hydrophobic collapse centered at the C-terminal half of alpha-helix I. Biochemistry, 1998 Dec 22, 37(51), 17944 - 51 Quinol and cytochrome oxidases in the cyanobacterium Synechocystis sp . PCC 6803; Howitt CA et al.; The genome of Synechocystis sp . PCC 6803 contains three sets of genes for terminal respiratory oxidases: the previously identified cytochrome aa3-type cytochrome c oxidase (CtaI), a second putative oxidase (CtaII) that we interpret to be a cytochrome bo-type quinol oxidase, and a putative cytochrome bd quinol oxidase (Cyd) . Genes for the two putative oxidases were cloned, and deletion constructs were made . Strains that lack one, two, or all three of the oxidases were generated . Deletion of the respiratory oxidases had no effect on photoautotrophic or photomixotrophic growth . Strains that lack one oxidase respire at near-wild-type rates, whereas those that lack both CtaI and Cyd do not respire . Thus, CtaII does not play a significant role in cellular metabolism under the conditions tested . An expression construct containing cydAB from Synechocystis sp . PCC 6803 was able to restore aerobic growth in a strain of Escherichia coli that lacks the cytochrome bo oxidase and the cytochrome bd oxidase encoded by cydAB . These results show that the cydAB operon from Synechocystis sp . PCC 6803 encodes a functional quinol oxidase . Deletion of Cyd and/or CtaII in strains lacking photosystem I did not change the fluorescence decay kinetics after illumination, and therefore, these oxidases do not significantly utilize reducing equivalents in the thylakoid membrane . This, combined with our inability to delete CtaI from strains lacking photosystem I, suggests that CtaI is the major oxidase on the thylakoid membrane and that Cyd is localized mostly on the cytoplasmic membrane . Transcripts for ctaDI were detected under all growth conditions tested, while transcripts for cydA and ctaEII could only be detected in cells grown at low light intensity (5 microE m(-2) s(-1)). Biochemistry, 1998 Dec 22, 37(51), 17875 - 81 Mutations in the activation loop tyrosine of the oncoprotein v-Fps; Saylor P et al.; Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis . Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated . Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively . For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme . The slopes for the plots of relative turnover versus solvent viscosity {(k(cat))eta} are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively . These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme . The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type . The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type . Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme . The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal . Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A {Adams et al . (1995) Biochemistry 34, 2447-2454} upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes. Biochemistry, 1998 Dec 22, 37(51), 17680 - 91 Role of Arg100 and Arg264 from Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal NADP+ binding and electron transfer; Martinez-Julvez M et al.; Previous studies and the crystal structure of Anabaena PCC 7119 FNR suggest that the side chains of Arg100 and Arg264 may be directly involved in the proper NADP+/NADPH orientation for an efficient electron-transfer reaction . Protein engineering on Arg100 and Arg264 from Anabaena PCC 7119 FNR has been carried out to investigate their roles in complex formation and electron transfer to NADP+ and to ferredoxin/flavodoxin . Arg100 has been replaced with an alanine, which removes the positive charge, the long side chain, as well as the ability to form hydrogen bonds, while a charge reversal mutation has been made at Arg264 by replacing it with a glutamic acid . Results with various spectroscopic techniques indicate that the mutated proteins folded properly and that significant protein structural rearrangements did not occur . Both mutants have been kinetically characterized by steady-state as well as fast transient kinetic techniques, and the three-dimensional structure of Arg264Glu FNR has been solved . The results reported herein reveal important conceptual information about the interaction of FNR with its substrates . A critical role is confirmed for the long, positively charged side chain of Arg100 . Studies on the Arg264Glu FNR mutant demonstrate that the Arg264 side chain is not critical for the nicotinamide orientation or for nicotinamide interaction with the isoalloxazine FAD moiety . However, this mutant showed altered behavior in its interaction and electron transfer with its protein partners, ferredoxin and flavodoxin. Biochemistry, 1998 Dec 22, 37(51), 17659 - 63 Quaternary structure of V1 and F1 ATPase: significance of structural homologies and diversities; Svergun DI et al.; The V1 ATPase from the tobacco hornworm Manduca sexta and the Escherichia coli F1 ATPase were characterized by small-angle X-ray scattering (SAXS) . The radii of gyration (Rg) of the complexes were 6.2 +/- 0.1 and 4.7 +/- 0.02 nm, respectively . The shape of the M . sexta V1 ATPase was determined ab initio from the scattering data showing six masses, presumed to be the A and B subunits, arranged in an alternating manner about a 3-fold axis . A seventh mass with a length of about 11.0 nm extends perpendicularly to the center of the hexameric unit . This central mass is presumed to be the stalk that connects V1 with the membrane domain (V(O)) in the intact V1V(O)-ATPase . In comparison, the shape of the F1 ATPase from E . coli possesses a quasi-3-fold symmetry over the major part of the enzyme . The overall asymmetry of the structure is given by a stem, assumed to include the central stalk subunits . The features of the V1 and F1 ATPase reveal structural homologies and diversities of the key components of the complexes. Shock, 1999 Jan, 11(1), 64 - 71 Contractile function and myoplasmic free Ca2+ (Cam) in coronary and mesenteric arteries of endotoxemic guinea pigs; Jones JJ et al.; Endotoxin-induced vascular hyporesponsiveness could potentially involve alterations of vascular smooth muscle (VSM) myoplasmic free calcium (Ca(m)) mobilization mechanisms . Contractile function and Ca(m)(fura-2 microfluorometry) regulation were evaluated in vitro using coronary (COR) and mesenteric (MES) artery preparations (100-250 microm inner diameter) isolated from guinea pigs 16 h after intraperitoneal (i.p.) injection of either saline (control; CON) or Escherichia coli endotoxin lipopolysaccharide (LPS; 4 mg/kg) . Concentration-response relationships to K+ (5-100 mM) were significantly enhanced in both COR and MES arteries isolated from LPS-treated animals . In contrast, contractile responses to prostaglandin F2alpha (PGF2alpha; 1-100 microM) were markedly impaired in COR and MES arteries from LPS-treated animals, while endothelin-1 (ET; 1-100 nM)-mediated contractile responses of these arteries were enhanced at the maximal dose (100 nM) . In COR arteries, PGF2alpha (1-100 microM) and ET (1-100 nM) produced biphasic increases in Ca(m) in both CON and LPS groups . No significant differences were observed in either the initial transient peak or secondary sustained Ca(m) responses between groups, suggesting a lack of effect of LPS upon intracellular Ca2+ release or Ca2+ influx mechanisms in COR arteries . Exposure of MES arteries to PGF2alpha and ET produced concentration-dependent increases in Ca(m) in both groups . However, Ca(m) responses of MES arteries lacked initial peak responses, suggesting potential differences in Ca(m) mobilization between COR and MES arteries . Ca(m) responses to K+ (80 mM) and PGF2alpha (1-100 microM) were similar in MES arteries from both groups; however, ET-mediated increases in Ca(m) were significantly blunted in LPS compared with CON MES arteries . Thus, endotoxemia produced differential effects upon depolarization (K4) and receptor (PGF2alpha, ET)-mediated contractile responses in both COR and MES arteries . Reductions in VSM Ca(m) mobilization appear unlikely as a mechanism for LPS-induced impairment of contractile function of COR and MES arteries; other mechanisms (i.e., decreased Ca2+ sensitivity of contractile proteins) may be involved in effects of LPS upon VSM function of COR and MES arteries. Braz J Med Biol Res, 1998 Nov, 31(11), 1363 - 74 Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense; Passaglia LM et al.; NifA protein activates transcription of nitrogen fixation operons by the alternative sigma 54 holoenzyme form of RNA polymerase . This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA . NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA . However, the purified NifA was able to induce the production of specific anti-A . brasilense NifA-antiserum that recognized NifA from A . brasilense but not from K . pneumoniae . Both GST-NifA and NifA expressed from the E . coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A . brasilense background . In order to investigate the mechanism that regulates NifA binding capacity we have used E . coli total protein extracts expressing A . brasilense nifA in mobility shift assays . DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size . These data show that the binding activity present in the C-terminal domain of A . brasilense NifA protein is still functional even in the presence of oxygen. J Biol Chem, 1999 Feb 5, 274(6), 3865 - 77 Expansion and deletion of triplet repeat sequences in Escherichia coli occur on the leading strand of DNA replication; Iyer RR et al.; Expansions and deletions of triplet repeat sequences that cause human hereditary neurological diseases were previously suggested to be mediated by the formation of DNA hairpins on the lagging strand during replication . The replication properties of CTG.CAG, CGG.CCG, and TTC.GAA repeats were studied in Escherichia coli using an in vivo phagemid system as a model for continuous leading strand synthesis . The repeats were substantially deleted when the CTG, CGG, and GAA repeats were the templates for rolling circle replication from the f1 phage origin . The deletions may be mediated by hairpins formed by these repeat tracts . The distributions of the deletion products of the CTG.CAG and CGG.CCG tracts indicated that hairpins of discrete sizes mediate deletions during complementary strand synthesis . Deletions during rolling circle synthesis are caused by larger hairpins of specific sizes . Thus, most deletion products were of defined lengths, suggesting a preference for specific hairpin intermediates . Small expansions of the CTG.CAG and CGG.CCG repeats were also observed, presumably due to the formation of CTG and CGG hairpins on the nascent complementary strand . Since rolling circle replication has been established in vitro as a model for leading strand synthesis, we conclude that triplet repeat instability can also occur on the leading strand of DNA replication. J Biol Chem, 1999 Feb 5, 274(6), 3859 - 64 Pokeweed antiviral protein accesses ribosomes by binding to L3; Hudak KA et al.; Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein, catalytically removes an adenine residue from the conserved alpha-sarcin loop of the large rRNA, thereby preventing the binding of eEF-2.GTP complex during protein elongation . Because the alpha-sarcin loop has been placed near the peptidyltransferase center in Escherichia coli ribosomes, we investigated the effects of alterations at the peptidyltransferase center on the activity of PAP . We demonstrate here that a chromosomal mutant of yeast, harboring the mak8-1 allele of peptidyltransferase-linked ribosomal protein L3 (RPL3), is resistant to the cytostatic effects of PAP . Unlike wild-type yeast, ribosomes from mak8-1 cells are not depurinated when PAP expression is induced in vivo, indicating that wild-type L3 is required for ribosome depurination . Co-immunoprecipitation studies show that PAP binds directly to L3 or Mak8-1p in vitro but does not physically interact with ribosome-associated Mak8-1p . L3 is required for PAP to bind to ribosomes and depurinate the 25 S rRNA, suggesting that it is located in close proximity to the alpha-sarcin loop . These results demonstrate for the first time that a ribosomal protein provides a receptor site for an ribosome-inactivating protein and allows depurination of the target adenine. J Biol Chem, 1999 Feb 5, 274(6), 3851 - 8 3'-Azido-3'-deoxythymidine-resistant mutants of DNA polymerase beta identified by in vivo selection; Kosa JL et al.; We developed an in vivo selection to identify 3'-azido-3'-deoxythymidine (AZT)-resistant mutants of rat DNA polymerase beta (pol beta) . The selection utilizes pol beta's ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I . pol beta allows SC18-12 cells to grow, but they depend on pol beta activity, so inhibition of pol beta by AZT kills them . We screened a library of randomly mutated pol beta cDNA for complementation of the pol I defect in the presence of AZT, and identified AZT-resistant mutants . We purified two enzymes with nonconservative mutations in the palm domain of the polymerase . The substitutions D246V and R253M result in reductions in the steady-state catalytic efficiency (Kcat/Km) of AZT-TP incorporation . The efficiency of dTTP incorporation was unchanged for the D246V enzyme, indicating that the substantial decrease in AZT-TP incorporation is responsible for its drug resistance . The R253M enzyme exhibits significantly higher Km(dTTP) and Kcat(dTTP) values, implying that the incorporation reaction is altered . These are the first pol beta mutants demonstrated to exhibit AZT resistance in vitro . The locations of the Asp-246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket. J Biol Chem, 1999 Feb 5, 274(6), 3705 - 10 Mismatch extension by Escherichia coli DNA polymerase III holoenzyme; Pham PT et al.; The in vitro fidelity of Escherichia coli DNA polymerase III holoenzyme (HE) is characterized by an unusual propensity for generating (-1)-frameshift mutations . Here we have examined the capability of HE isolated from both a wild-type and a proofreading-impaired mutD5 strain to polymerize from M13mp2 DNA primer-templates containing a terminal T(template).C mismatch . These substrates contained either an A or a G as the next (5') template base . The assay allows distinction between: (i) direct extension of the terminal C (producing a base substitution), (ii) exonucleolytic removal of the C, or (iii), for the G-containing template, extension after misalignment of the C on the next template G (producing a (-1)-frameshift) . On the A-containing substrate, both HEs did not extend the terminal C (<1%); instead, they exonucleolytically removed it (>99%) . In contrast, on the G-containing substrate, the MutD5 HE yielded 61% (-1)-frameshifts and 6% base substitutions . The wild-type HE mostly excised the mispaired C from this substrate before extension (98%), but among the 2% mutants, (-1)-frameshifts exceeded base substitutions by 20 to 1 . The preference of polymerase III HE for misalignment extension over direct mismatch extension provides a basis for explaining the in vitro (-1)-frameshift specificity of polymerase III HE. J Biol Chem, 1999 Feb 5, 274(6), 3315 - 22 Architecture of a complex between the sigma70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide . Luminescence resonance energy transfer study; Heyduk E et al.; We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to sigma70 holoenzyme . Five single reactive cysteine mutants of sigma70 (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor) . The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor) . The energy transfer was observed upon complex formation between the donor-labeled sigma70 holoenzyme and the acceptor-labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide . The oligonucleotide was bound in one preferred orientation . This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by sigma70: sequence specificity and strand polarity specificity . The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of sigma70 is located in an alpha-helix containing residue 442 . The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix. J Biol Chem, 1999 Feb 5, 274(6), 3294 - 9 Directed mutagenesis studies of the metal binding site at the subunit interface of Escherichia coli inorganic pyrophosphatase; Efimova IS et al.; Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase) have identified three Mg2+ ions/enzyme hexamer in water-filled cavities formed by Asn24, Ala25, and Asp26 at the trimer-trimer interface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A . A., and Goldman, A . (1996) Biochemistry 35, 4670-4677) . Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium . Mg2+ markedly decelerates the dissociation of enzyme hexamer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D26S and D26N variants, when little or no effect is seen . The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants . The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active site . It is concluded that the metal ion binding site found at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg2+ greatly stabilizes the enzyme hexamer but has little effect on catalysis. J Biol Chem, 1999 Feb 5, 274(6), 3279 - 84 Interaction of Escherichia coli DNA polymerase I (Klenow fragment) with primer-templates containing N-acetyl-2-aminofluorene or N-2-aminofluorene adducts in the active site; Dzantiev L et al.; DNA adducts formed by aromatic amines such as N-acetyl-2-aminofluorene (AAF) and N-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication . To understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primer-templates modified with an AAF or AF adduct . The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA . More specifically, it was found that the binding is 5-10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site . In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide . The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower . In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present. Blood, 1999 Feb 1, 93(3), 942 - 51 Immunoglobulin M-enriched human intravenous immunoglobulin prevents complement activation in vitro and in vivo in a rat model of acute inflammation; Rieben R et al.; An important antiinflammatory mechanism of intravenous immunoglobulin preparations (IVIG) is their ability to block complement activation . The purpose of this study was to compare the complement-inhibitory activity of four IVIG preparations differing in isotype composition . The preparations were: (1) IVIgG (48 g/L IgG, 2 g/L IgA; Intraglobin F); (2) Pentaglobin (38 g/L IgG, 6 g/L IgM, 6 g/L IgA); (3) IVIgM (35 g/L IgM, 12 g/L IgA, 3 g/L IgG); and (4) IVIgA (41 g/L IgA, 9 g/L IgG), all from Biotest Pharma GmbH, Dreieich, Germany . Their complement inhibitory activity was assessed in vitro by measurement of the blocking of C1q-, C4-, and C3 deposition on solid-phase aggregated rabbit IgG by enzyme-linked immunosorbent assay (ELISA) . Complement inhibition in this ELISA was best for IVIgM, followed by Pentaglobin and IVIgG; IVIgA did not exhibit an inhibitory effect . Control experiments with excess concentrations of C1q as well as with C1q-depleted serum showed that the inhibitory effects of IVIG were not caused by complement activation and thus, consumption, but that C4 and C3 were scavenged by IgM and to a lesser extent by IgG . These results were confirmed in vivo in the rat anti-Thy 1 nephritis model, in which a single dose of 500 mg/kg of IVIgM prevented C3-, C6-, and C5b-9 deposition in the rat glomeruli, whereas the effect of IVIgG was much less pronounced . Reduction of complement deposition was paralleled by a diminished albuminuria, which was completely absent in the IVIgM-treated rats . IVIgM and to a lesser extent IVIgG also prevented rat C3 deposition on cultured rat glomerular mesangial cells in vitro, but did not influence anti-Thy 1 binding . Neither IVIgM nor Pentaglobin nor IVIgG negatively affected in vitro phagocytosis of Escherichia coli (E coli) by human granulocytes . In conclusion, we have shown that IgM enrichment of IVIG preparations enhances their effect to prevent the inflammatory effects of complement activation. Biochem Biophys Res Commun, 1999 Jan 27, 254(3), 647 - 50 Radiation sensitivity of an Escherichia coli mutant lacking NADP+-dependent isocitrate dehydrogenase; Lee SM et al.; Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in radiation damage . NADP+-dependent isocitrate dehydrogenase (ICDH) in Escherichia coli produces NADPH, an essential reducing equivalent for the antioxidant system . The protective role of ICDH against ionizing radiation in E . coli was investigated in wild-type and ICDH-deficient strains . Upon exposure to ionizing radiation, the viability was lower and the lipid peroxidation was higher in mutant cells compared to wild-type cells . Activities of key antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase, and glucose-6-phosphate dehydrogenase were decreased by irradiation in both cells . Results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against ionizing radiation . Biochem Biophys Res Commun, 1999 Jan 8, 254(1), 253 - 8 Functional divergence of the plastid and cytosolic forms of the 54-kDa subunit of signal recognition particle; Schuenemann D et al.; Chloroplast and cytoplasmic signal recognition particles (cpSRP and cySRP) each contain a similar subunit, SRP54 . The chloroplast homologue binds to cpSRP43, which is absent from cytosolic SRP, and cySRP54 binds to SRP-RNA, which appears to be absent from cpSRP . In the presence of cpSRP43, cpSRP54 posttranslationally forms a soluble targeting intermediate, transit complex, with the major light harvesting protein of the thylakoid membrane . In contrast, cySRP54 functions cotranslationally . In this study we investigated whether cytosolic and chloroplast forms of SRP54 were interchangeable in three types of functional assays: complementation of an Escherichia coli SRP54 mutant, formation of the transit complex, and heterologous binding between the SRP54 subunits, cpSRP43, and SRP-RNA . In no cases were the 54-kDa subunits able to substitute for each other suggesting that the two proteins are fundamentally different . Biochem Biophys Res Commun, 1999 Jan 8, 254(1), 147 - 51 Use of benzyl mercaptan for direct preparation of long polypeptide benzylthio esters as substrates of subtiligase; Welker E et al.; Subtiligase, a double mutant of subtilisin, has been shown to be capable of joining together two unprotected peptide fragments, namely an activated peptide ester and a second peptide with a free N-terminal amino group . Inside cells, inteins are know to join peptide chains (exteins) by self-extrusion . The SC VMA1 intein was modified to undergo only in vitro N-terminal cleavage in the presence of small nucleophilic compounds, releasing the N-terminal extein . With a proper choice of the nucleophilic compounds it is shown that it is possible to generate long polypeptides, by molecular biology expression, with such an attached reactive ester which is an excellent substrate of the enzyme, subtiligase . This approach can successfully extend the current limit of the subtiligase-catalyzed fragment condensation method as well as provide another application of the recently discovered intein chemistry . Mutat Res, 1998 Nov 9, 422(1), 107 - 12 Glucose and related catabolite repressors are powerful inhibitors of pKM101-enhanced UV mutagenesis in Escherichia coli; Ambrose M et al.; When stationary phase Escherichia coli K12 trp (amber) cells were exposed to UV doses ranging from 180-540 J m(-2), we found that we could not recover any induced Trp+ revertants unless the irradiated cultures were first supplied with the Muc+ mutation-enhancing IncP plasmid pKM101 (by conjugation) . We also found that the numbers of UV-induced Trp+ revertants recovered from pKM101+ cultures varied quite dramatically depending upon which of several commonly-used carbon sources were present in the post-irradiation plating medium, e.g., there were always significantly fewer revertants on minimal glucose plates than on minimal glycerol plates . More importantly, there were also fewer UV-induced revertants on glycerol + glucose plates than on 'glycerol-only' plates . We then tested two glucose-related compounds which are known to depress intracellular cyclic AMP (cAMP) levels even more effectively than glucose (glucose-6-phosphate and the non-utilisable methyl-alpha-D-glucopyranoside) and found that they too were able to exert powerfully antimutagenic effects in UV-treated pKM101-containing bacteria . Taken together, these results provide strong additional support for our working hypothesis that at least one component of the mutational pathway which operates in UV-irradiated pKM101-containing cells is extremely sensitive to classical cAMP-mediated catabolite repression. Biochim Biophys Acta, 1998 Dec 10, 1448(2), 227 - 35 Membrane domain formation by calcium-dependent, lipid-binding proteins: insights from the C2 motif; Hinderliter AK et al.; We propose a novel role in cellular function for some membrane-binding proteins and, specifically, the C2 motif . The C2 motif binds phospholipid in a manner that is modulated by Ca2+ and is thought to confer membrane-binding ability on a wide variety of proteins, primarily proteins involved in signal transduction and membrane trafficking events . We hypothesize that in the absence of Ca2+ the C2 motif couples the free energy of binding to a bilayer membrane comprised of zwitterionic and negatively charged lipids to the formation of a domain enriched in the negative lipids . This in turn leads to the dynamic clustering of bound homologous or heterologous proteins incorporating the C2 motif, or other acidic lipid-binding motifs . In the presence of Ca2+, the protein clusters may be further stabilized . In support of this hypothesis we present evidence for membrane domain formation by the first C2 domain of synaptotagmin in the absence of Ca2+ . Fluid state phospholipid mixtures incorporating a pyrene-labeled phospholipid probe exhibited a change in pyrene excimer/monomer fluorescence ratio consistent with domain formation upon binding the C2 domain . In addition, we present the results of simulations of the interaction of the C2 domain with the membrane that indicate that protein clusters and lipid domains form in concert. Biochim Biophys Acta, 1998 Dec 8, 1429(1), 249 - 58 Threonine 82 in the regulatory chain is important for nucleotide affinity and for the allosteric stabilization of Escherichia coli aspartate transcarbamoylase; Williams MK et al.; The three-dimensional structure of Escherichia coli aspartate transcarbamoylase complexed with the allosteric effector CTP, shows an interaction between the hydroxyl of Thr-82 in the regulatory chain (Thr-82r) with the gamma-phosphate of CTP (R.P . Kosman, J.E . Gouaux, W.N . Lipscomb, Crystal structure of CTP-ligated T state aspartate transcarbamoylase at 2.5 A resolution: implications for aspartate transcarbamoylase mutants and the mechanism of negative cooperativity, Proteins Struct . Funct . Genet . 15 (1993) 147-176) . In order to determine whether the Thr-82r interaction with the gamma-phosphate of CTP is important for either binding of the nucleotide effectors or their function, site-specific mutagenesis was employed . The mutant enzyme in which Thr-82r was replaced by Ala had almost the identical maximal observed specific activity as the wild-type enzyme; however, the mutant enzyme had a significantly increased {Asp}0.5, the aspartate concentration at one-half the maximal observed specific activity, as well as slightly increased homotropic cooperativity . The mutant enzyme was also activated more by ATP and inhibited less by CTP as compared to the wild-type enzyme . In addition, the nucleotide concentration required for one-half maximal effect was increased approx . 3-fold as compared to the corresponding values for the wild-type enzyme . The maximal inhibition of the mutant enzyme, in the presence of UTP and CTP was similar to that observed for the wild-type enzyme; however, higher concentrations of the nucleotides were required to achieve this level of inhibition . The reduced affinity of CTP, UTP and ATP induced by the mutation indicates that the hydrogen bonding interaction between the gamma-phosphate of the nucleotide effector and the side-chain hydroxyl of Thr-82r is important for the binding of the nucleotide effectors to the allosteric site . Furthermore, this interaction is important for the discrimination between CTP and CDP . Finally, the greater homotropic cooperativity, greater {Asp}0.5, diminished CTP inhibition and greater ATP activation of the mutant enzyme correlates with the X-ray structure of the mutant enzyme which shows that the unligated enzyme is in an 'extreme' T-state . These findings add support to the theory that the global stabilization of the enzyme is critical for both the homotropic and heterotropic properties of aspartate transcarbamoylase. Biochim Biophys Acta, 1998 Dec 8, 1429(1), 83 - 92 6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species; Rippa M et al.; The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei . The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate . Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate . 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators . The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate . The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed. Biochim Biophys Acta, 1998 Dec 8, 1429(1), 69 - 73 Hydrophilic Thr can replace the hydrophobic and absolutely conservative A3Val in insulin; Chen H et al.; A mutant insulin, {A3Thr}human insulin, was obtained by means of site-directed mutagenesis . The {A3Thr}human insulin retains 50% receptor-binding potency and nearly total in vivo biological activity compared with native insulin, and can be crystallized using the same condition of native insulin . The results demonstrate that the absolutely conservative and hydrophobic valine at A3 can be substituted by hydrophilic threonine. J Med Microbiol, 1999 Jan, 48(1), 41 - 9 Virulence properties of atypical EPEC strains; Pelayo JS et al.; Virulence properties of 31 atypical enteropathogenic Escherichia coli (EPEC) strains isolated from cases of diarrhoea were examined . All except two strains adhered to HEp-2 cells in a localised adherence-like (LAL) pattern . With the exception of two strains, all were fluorescent actin staining (FAS) positive . Gentamicin HEp-2 invasion assay studies showed that all strains were invasive . Transmission electron microscopy of infected HEp-2 cells showed the characteristic attaching and effacing lesion and invasion of the cultured cells . Of the nine strains that hybridised with a DNA probe for alpha-haemolysin, five were haemolytic within 3 h of incubation, while the remaining strains were haemolytic only after incubation for 24 h . Three strains produced enterohaemolysin on blood agar . None of the 31 strains of E . coli induced fluid accumulation in the rabbit intestinal loop assay or displayed cytotoxic effects in HeLa and Vero cells . All the strains belonging to serotypes O26:H11, O26:H- and 0119:H2 expressed intimin beta, whereas all the strains from serotype O55:H7 expressed intimin gamma . The strains belonging to serogroup O111 expressed a non-typable intimin . The participation of intimin in LAL was supported by adhesion inhibition experiments in which antibodies to intimin significantly reduced the level of LAL. Exp Parasitol, 1999 Jan, 91(1), 59 - 69 Mosquito-Plasmodium interactions in response to immune activation of the vector; Lowenberger CA et al.; During the development of Plasmodium sp . within the mosquito midgut, the parasite undergoes a series of developmental changes . The elongated ookinete migrates through the layers of the midgut where it forms the oocyst under the basal lamina . We demonstrate here that if Aedes aegypti or Anopheles gambiae, normally susceptible to Plasmodium gallinaceum and P . berghei, respectively, are immune activated by the injection of bacteria into the hemocoel, and subsequently are fed on an infectious bloodmeal, there is a significant reduction in the prevalence and mean intensity of infection of oocysts on the midgut . Only those mosquitoes immune activated prior to, or immediately after, parasite ingestion exhibit this reduction in parasite development . Mosquitoes immune activated 2-5 days after bloodfeeding show no differences in parasite burdens compared with naive controls . Northern analyses reveal that transcriptional activity for mosquito defensins is not detected in the whole bodies of Ae . aegypti from 4 h to 10 days after ingesting P . gallinaceum, suggesting that parasite ingestion, passage from the food bolus through the midgut, oocyst formation, and subsequent release of sporozoites into the hemolymph do not induce the production of defensin . However, reverse transcriptase-PCR of RNA isolated solely from the midguts of Ae . aegypti indicates that transcription of mosquito defensins occurs in the midguts of naive mosquitoes and those ingesting an infectious or noninfectious bloodmeal . Bacteria-challenged Ae . aegypti showed high levels of mature defensin in the hemolymph that correlate with a lower prevalence and mean intensity of infection with oocysts . Because few oocysts were found on the midgut of immune-activated mosquitoes, the data suggest that some factor, induced by bacterial challenge, kills the parasite at a preoocyst stage. Methods Enzymol, 1999, 301, 70 - 8 Assay of isoforms of Escherichia coli-expressed nitric oxide synthase; Martasek P et al.; The techniques described herein have added to our repertoire of experimental approaches for the characterization of the NOSs . These procedures have reinforced our conviction that the NOSs are structurally suited to perform unique functions in their cellular milieux and that these differences have physiological consequences. Biochem Genet, 1998 Oct, 36(9-10), 329 - 50 Regulation of the expression of the sn-glycerol-3-phosphate dehydrogenase gene in Drosophila melanogaster; Bartoszewski S et al.; P element-mediated transformation has been used to investigate the regulation of expression of the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster . A 13-kb construct containing the eight exons and associated introns, 5 kb of the 5' region, and 3 kb downstream from the structural gene produced normal levels of enzyme activity and rescued the poor viability of flies lacking the enzyme . All the regulatory elements essential for normal enzyme expression were located in a fragment that included the exons and introns and 1-kb upstream noncoding sequence . Deletions of the 1.6-kb second intron reduced activity to 25% . Transformants with fusion constructs between the sn-glycerol-3-phosphate dehydrogenase gene and the beta-galactosidase gene from E . coli revealed three elements that affected expression . A (CT)9 repeat element at the 5' end of the second intron increased expression in both larvae and adults, particularly at emergence . A second regulatory element, which includes a (CT)7 repeat, was located 5' to the TATA box and had similar effects on the gene's expression . A third, undefined, enhancer was located in the second intron, between 0.5 and 1.8 kb downstream of the translation initiation codon . This element increases enzyme activity to a similar extent in larvae and adults but has little effect when the enhancer at the 5' end of the intron is present. Virology, 1999 Jan 20, 253(2), 155 - 61 Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome; Miyake H et al.; Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent trans-activator of specific sets of target genes, and on the other hand, it is a trans-repressor of other sets of genes . It is also an inhibitor of the tumor suppressor protein p16(INK4a) and thus has been thought to contribute to induction of adult T-cell leukemia (ATL) . We examined the mutagenic effects of Tax on a cellular gene, hypoxanthine guanine phosphoribosyltransferase (hprt), and LacI gene in lambda shuttle vector exogenously integrated in Big Blue Rat-2 (BBRat-2) cells . Expression of Tax in BBRat-2 cells enhanced the frequency of HPRT(-) phenotype severalfold . Tax-expressing cell clones, BBTax-1 and -2 established from BBRat-2 cells, gave rise to an average mutation frequency of 5.9 x 10(-5) in LacI gene, but Tax-negative cell clones, BBRat-C1 and -C2, showed 2 . 1 x 10(-5) . The 2.8-fold increase in mutation frequency in the presence of Tax indicates that Tax expression enhanced mutation frequency in chromosomal DNA . However, neither the mutation spectrum of base transitions, transversions, and deletions/insertions nor the loci of the mutations were significantly affected by Tax expression . These findings indicate that Tax has the capability to induce random mutations and suggest that Tax would be able to modulate cellular phenotypes through mutation of the cellular genome . Biochem Biophys Res Commun, 1999 Jan 19, 254(2), 306 - 10 A dehydroalanyl residue can capture the 5'-deoxyadenosyl radical generated from S-adenosylmethionine by pyruvate formate-lyase-activating enzyme; Wagner AF et al.; The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is produced post-translationally by pyruvate formate-lyase-activating enzyme (PFL activase), employing adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates . Previous 2H-labelings found incorporation of the pro-S hydrogen of Gly-734 into the 5'-deoxyadenosine co-product, indicating that a deoxyadenosyl radical intermediate, generated by reductive cleavage of AdoMet, serves as the actual H atom abstracting species in this system . We have now examined an octapeptide (Suc-Arg-Val-Pro-DeltaAla-Tyr-Ala-Val-Arg-NH2) that is analogous to the Gly-734 site of the PFL polypeptide but contains a dehydroalanyl residue (DeltaAla) in the glycyl position . Applied to the PFL activase reaction, this peptide becomes C-adenosylated at the olefinic beta carbon of DeltaAla . The modified peptide was isolated in micromol-quantities and characterized, after chymotryptic truncation, by MS and 2D NMR . PFL activase functions catalytically (kcat >/= 1 min-1) in the peptide modification reaction, which occurs with stoichiometric consumption of AdoMet . The mechanism appears to involve addition of the nucleophilic deoxyadenosyl radical to the electrophilic CC double bond of DeltaAla, followed by quenching of the peptide backbone-centered adduct radical by the buffer medium . The trapping-property of the DeltaAla residue should be exploitable in investigating of how the Fe4S4 protein PFL activase generates the highly reactive deoxyadenosyl radical . Biochem Biophys Res Commun, 1998 Dec 30, 253(3), 756 - 60 Expression of rat interleukin-5 and generation of neutralizing antiserum: a comparative study of rat IL-5 produced in Escherichia coli and insect cells; Pierrot C et al.; A cDNA coding for rat IL-5 was obtained by RT-PCR from total spleen RNA . With the exception of a single a.a . replacement at position 85 (L-P), it is identical to the published sequence obtained by retroviral gene transfer . This cDNA was used to express biologically active recombinant IL-5 in E . coli and in insect cells using a baculovirus system . Rat IL-5 is more active on B13, an IL-5 dependent cell line, when produced in insect cells (specific activity 1.47 x 10(11)UI/mg compared to 4.28 x 10(6)UI/mg) . This increased activity seems to be associated with the presence of IL-5 homodimers in recombinant protein preparations . A rabbit antiserum raised against recombinant bacterial IL-5 specifically inhibited B13 proliferation induced by bacterial and baculoviral IL-5 . The availability of such reagents should facilitate studying the role of IL-5 in different infectious diseases, experimental allergic encephalomyelitis and in transplantation biology where the rat represents a more suitable model than mice. Biochem Biophys Res Commun, 1998 Dec 30, 253(3), 648 - 52 Interaction between mitochondrial precursor proteins and cytosolic soluble domains of mitochondrial import receptors, Tom20 and Tom70, measured by surface plasmon resonance; Iwata K et al.; We studied the interaction between mitochondrial precursor proteins and postulated mitochondrial surface receptor proteins, Tom20 and Tom70, by using a methodology of surface plasmon resonance . For these studies, import-competent mitochondrial precursor proteins, pCOXIV-DHFR and pSu9-DHFR, and cytosolic domains of the two receptor proteins were separately expressed in and purified from E . coli cells as a soluble form . By measuring surface plasmon resonance, both of the purified precursor proteins were found to specifically bind to either of the cytosolic domains of import receptors immobilized on a sensor chip . On the other hand, import-incompetent SynB2-DHFR and DHFR itself were shown to possess little or no binding abilities to the sensor chip, respectively . Using this system, we could demonstrate that the proposed carboxy-terminal acidic bristle domain of Tom20 is not essential for the precursor binding . Chemical modification of the acidic amino acid residues of either cytosolic domain on the sensor chip partially inhibited the binding of pSu9-DHFR, whereas the binding of pCOXIV-DHFR was almost unaffected . These results suggest that distinct set of amino acid residues of the receptor proteins might be responsible for the binding of different precursor proteins. Biochem Biophys Res Commun, 1998 Dec 30, 253(3), 582 - 7 Analysis of active site residues of the antiviral protein from summer leaves from Phytolacca americana by site-directed mutagenesis; Poyet JL et al.; The summer leaf isoform of the pokeweed (Phytolacca americana) antiviral protein, PAP II, was produced in high yields from inclusion bodies in recombinant E . coli . On the basis of its sequence similarity with the spring leaf isoform (PAP I) and with the A chain of ricin, a three-dimensional model of the protein was constructed as an aid in the design of active site mutants . PAP II variants mutated in residues Asp 88 (D88N), Tyr 117 (Y117S), Glu 172 (E172Q), Arg 175 (R175H) and a combination of Asp 88 and Arg 175 (D88N/R175H) were produced in E . coli and assayed for their ability to inhibit protein synthesis in a rabbit reticulocyte lysate . All of these mutations had effects deleterious to the enzymatic activity of PAP II . The results were interpreted in the light of three reaction mechanisms proposed for ribosome-inactivating proteins (RIPs) . We conclude that none of the proposed mechanisms is entirely consistent with the data presented here. J Mol Biol, 1999 Jan 22, 285(3), 1245 - 56 Crystal structure of intact elongation factor EF-Tu from Escherichia coli in GDP conformation at 2.05 A resolution; Song H et al.; The crystal structure of intact elongation factor Tu (EF-Tu) from Escherichia coli in GDP-bound conformation has been determined using a combination of multiple isomorphous replacement (MIR) and multiwavelength anomalous diffraction (MAD) methods . The current atomic model has been refined to a crystallographic R factor of 20.3 % and free R-factor of 26.8 % in the resolution range of 10-2.05 A . The protein consists of three domains: domain 1 has an alpha/beta structure; while domain 2 and domain 3 are beta-barrel structures . Although the global fold of the current model is similar to those of published structures, the secondary structural assignment has been improved due to the high quality of the current model . The switch I region (residues 40-62) is well ordered in this structure . Comparison with the structure of EF-Tu in GDP-bound form from Thermus aquaticus shows that although the individual domain structures are similar in these two structures, the orientation of domains changes significantly . Interactions between domains 1 and 3 in our E . coli EF-Tu-GDP complex are quite different from those of EF-Tu with bound GTP from T . aquaticus, due to the domain rearrangement upon GTP binding . The binding sites of the Mg2+ and guanine nucleotide are revealed in detail . Two water molecules that co-ordinate the Mg2+ have been identified to be well conserved in the GDP and GTP-bound forms of EF-Tu structures, as well as in the structure of Ras p21 with bound GDP . Comparisons of the Mg2+ binding site with other guanine nucleotide binding proteins in GDP-bound forms show that the Mg2+ co-ordination patterns are well preserved among these structures . J Mol Biol, 1999 Jan 22, 285(3), 965 - 75 The phenotype of mutations of G2655 in the sarcin/ricin domain of 23 S ribosomal RNA; Macbeth MR et al.; The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA forms a part of the site for the association of the elongation factors with the ribosome and hence is critical for the binding of aminoacyl-tRNA and for translocation . The domain is also the site of action of the eponymous toxins which catalyze covalent modification of single nucleotides that inactivate the ribosome . The conformation of the conserved guanosine at position 2655 is an especially prominent feature of the structure of the SRD: the nucleotide is bulged out of a helix and forms a base-triple with A2665 and U2656 . G2655 in 23 S rRNA is protected from chemical modification when the elongation factors, EF-Tu and EF-G, are bound to ribosomes and the analog of G2655 in oligoribonucleotides is critical for recognition by the toxin sarcin and by EF-G . The contribution of G2655 to the function of the ribosome has been evaluated by constructing mutations in the nucleotide and determining the phenotype . Constitutive expression of a plasmid-encoded rrnB operon with a deletion of, or transversions in, G2655 is lethal to E . coli cells, whereas a defect in the growth of cells with a G2655A transition is observed only in competition with wild-type cells . The sedimentation profiles of ribosomes with mutations in G2655 are altered; most markedly by deletion or transversion of the nucleotide, less severely by transition to adenosine . Mutations of G2655 confer resistance to sarcin on ribosomes . Ribosomes with G2655Delta, G2655C, or G2655U mutations in 23 S rRNA are not active in protein synthesis, whereas those with the G2655A transition mutation suffer decreased activity . Anal Biochem, 1999 Feb 1, 267(1), 234 - 5 Phage recovery by electroporation of naked DNA into host cells avoids the use of packaging extracts; Planelles L et al.; In this paper, we describe the application of electroporation to deliver phage DNA into bacterial cells in order to recover it as phage particles . The methodology represents a quicker and cheaper alternative to the use of packaging extracts to rescue phage clones stored as naked DNAs . Furthermore, our data demonstrate that there were not rearrangements or recombinations between phage DNAs when a mixture of different DNAs was electroporated, suggesting the use of electroporation as a reliable method for construction of gene libraries . Anal Biochem, 1999 Feb 1, 267(1), 169 - 84 Spontaneous alpha-N-6-phosphogluconoylation of a "His tag" in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins; Geoghegan KF et al.; Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-{His}6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da . The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications . As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case . Phosphatase treatment converted +258-Da protein into +178-Da protein . The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry . As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like . Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation . NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical . Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group . A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) . Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation . Anal Biochem, 1999 Feb 1, 267(1), 114 - 20 Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein; Tolosa L et al.; We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry . The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26 . When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 microM glucose . The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose . A modulation sensor was created by combining ANS26-GGBP with a long-lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette . Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz . Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration . These results suggest an approach to glucose sensing with simple devices . Anal Biochem, 1999 Feb 1, 267(1), 104 - 13 Quantitative determination of globotriaosylceramide by immunodetection of glycolipid-bound recombinant verotoxin B subunit; Zeidner KM et al.; An assay for the neutral glycosphingolipid, globotriaosylceramide (Galalpha1-4Galbeta1-4Glcbeta1-1Cer; GL-3), was developed based on the B subunit of Escherichia coli verotoxin (VTB) . The VTB gene was isolated, overexpressed in E . coli, and purified by a single immunoaffinity chromatographic step using a monoclonal anti-VTB IgG-agarose column . Purified recombinant VTB was used to develop an enzyme-linked immunosorbent assay (ELISA) to determine the GL-3 concentrations in plasma and tissue extracts from normal individuals and patients and mice with alpha-galactosidase A deficiency (human Fabry disease) . The mean (+/-1 SD) plasma GL-3 concentrations in affected male and female heterozygotes with Fabry disease were 12.6 +/- 3.7 and 1.1 +/- 0.7 microg/ml, respectively, whereas normal individuals had 0.9 +/- 0.4 microg/ml . In 5- to 6-month-old mice with alpha-galactosidase A deficiency, the average GL-3 concentrations in spleen, kidney, liver, heart, and plasma were 2790 +/- 400, 1100 +/- 93, 378 +/- 67, and 196 +/- 28 ng/mg wet wt and 5 . 1 +/- 2.0 microg/ml, respectively, whereas tissues from wild-type mice contained very low or undetectable GL-3 levels . This ELISA assay should prove useful for determining the GL-3 levels, as well as for monitoring the effectiveness of therapeutic endeavors in patients with Fabry disease . Acta Biochim Pol, 1998, 45(3), 755 - 68 7-Deazapurine 2'-deoxyribofuranosides are noncleavable competitive inhibitors of Escherichia coli purine nucleoside phosphorylase (PNP); Bzowska A et al.; A series of 7-deazapurine 2'-deoxyribofuranosides were synthesized according to already known procedures and their substrate and inhibitor properties with purified E . coli purine nucleoside phosphorylase were examined . In agreement with previous findings, substrate activity was not detected for any of the compounds tested . Most of the nucleosides showed weak inhibition in the preliminary screening, i.e . at a concentration of about 100 microM . However some combinations of 6-chloro, 6-amino or 6-methoxy substituents with bulky hydrophobic groups at position 7 of the base and/or chloro, amino, methoxy or methylthio group at position 2 markedly enhanced affinity of such modified nucleosides for the E . coli enzyme . The most potent inhibition was observed for two nucleosides: 6-chloro- and 2-amino-6-chloro-7-deazapurine 2'-deoxyribofuranosides that show inhibition constants Ki = 2.4 and 2.3 microM, respectively . Several other compounds were also found to be good inhibitors, with inhibition constants in the range 5-50 microM . In all instances the inhibition was competitive vs . the nucleoside substrate 7-methylguanosine . Inhibition constants for 7-deazapurine nucleosides are in general several-fold lower than those observed for their purine counterparts . Therefore 7-deaza modification together with substitutions at positions 2, 6 and 7 of the base is a very promising approach to obtain competitive noncleavable inhibitors of E . coli PNP that may bind to the enzyme with inhibition constants in the microM range. Acta Biochim Pol, 1998, 45(3), 677 - 90 Hydrogen peroxide effects in Escherichia coli cells; Asad NR et al.; We analyzed DNA lesions produced by H2O2 under low iron conditions, the cross adaptive response and the synergistic lethal effect produced by iron chelator-o-phenanthroline, using different Escherichia coli mutants deficient in DNA repair mechanisms . At normal iron levels the lesions produced by H2O2 are repaired mainly by the exonuclease III protein . Under low iron conditions we observed that the Fpg and UvrA proteins as well as SOS and OxyR systems participate in the repair of these lesions . The lethal effect of H2O2 is strengthened by o-phenanthroline if both compounds are added simultaneously to the culture medium . This phenomenon was observed in the wild type cells and in the xthA mutant (hypersensitive to H2O2) . E . coli cells treated with low concentrations of H2O2 (micromolar) acquire resistance to different DNA damaging agents . Our results indicate also that pretreatment with high (millimolar) H2O2 concentrations protects cells against killing, by UV and this phenomenon is independent of the SOS system, but dependent on RecA and UvrA proteins . H2O2 induces protection against lethal and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . H2O2 also protects the cells against killing by cumene hydroperoxide, possibly with the participation of Ahp protein. Antisense Nucleic Acid Drug Dev, 1998 Dec, 8(6), 499 - 506 Unusual interactions between cleavage products of a cis-cleaving hammerhead ribozyme; Castanotto D et al.; We have synthesized and tested a cis-cleaving ribozyme designed to have thermodynamically stable stem-loop structures . This cis-ribozyme cleaves very efficiently in vitro, with a cleavage rate of about 0.5/min . Surprisingly, during the course of in vitro transcription and cleavage of our ribozyme, a product of unusual mobility accumulates and coincides with a sharp decline in the rate of formation of cleavage products . Analyses of this electrophoretic variant demonstrated that it is formed by interactions of the cleavage products . Despite the fact that the products and ribozyme transcript are of identical sequence, the cleavage products interact only with one another and not with the uncleaved precursor . This suggests a significant structural difference between the cleaved and uncleaved ribozyme transcripts . Testing of this cis-ribozyme in both yeast and mammalian cells shows no significant cleavage activity in vivo . We conclude that the structure of the ribozyme flanking sequences is important for optimizing the rate of ribozyme cleavage, but this enhanced rate does not necessarily correlate with enhanced in vivo function. Nutrition, 1999 Jan, 15(1), 23 - 8 Effects of glutamine administration on liver regeneration following hepatectomy; Ito A et al.; The intestine is now known to be an important site of protein production in the body, and glutamine (GLN) stimulates both secreted and non-secreted protein synthesis in the small bowel . The purpose of the present study was to evaluate the effect of GLN-supplemented parenteral nutrition on liver regeneration after hepatectomy . Animals were divided into two groups: a sham-operated control group (Group A) and a 70%-hepatectomy group (Group B) . Postoperatively, one-third of the animals in each group were maintained on intravenous 10% glucose solution, on 10% glucose with 2% standard amino acid solution, or 10% glucose supplemented with 2% glutamine for 24 h . GLN administration after hepatectomy significantly promoted liver regeneration . In addition, assessment of amino acid metabolism showed that GLN administration activated GLN metabolism in the intestine and promoted alanine uptake by the remnant liver . This metabolic response also enhanced both secreted and non-secreted protein synthesis in intestinal epithelial cells, especially in cells isolated from the crypts . The proteins produced are important as a portal production factor for liver regeneration and intestinal cell proliferation . Bacterial and endotoxin translocation, on the other hand, was significantly reduced . Thus, the results of this study suggest that intravenous administration of GLN after hepatectomy significantly promoted liver regeneration. Ann N Y Acad Sci, 1998 Sep 29, 856, 69 - 75 Role of IL-10 in inflammation . Studies using cytokine knockout mice; Leon LR et al.; Interleukin-10 (IL-10) inhibits the synthesis of proinflammatory cytokines known to be involved in fever, including IL-1, IL-6, and tumor necrosis factor-alpha . We hypothesized that IL-10 modulates lipopolysaccharide (LPS)-induced fever in mice . Body temperature was measured by biotelemetry . Swiss Webster mice injected with recombinant murine IL-10 (rmuIL-10) were resistant to fever induced by a low dose of LPS (100 micrograms/kg, i.p.) and to the hypothermic and febrile effects of a high (septic-like) dose of LPS (2.5 mg/kg, i.p.) . Injection of rmuIL-10 alone had no effect on afebrile body temperature of Swiss Webster mice . IL-10 knockout mice showed an exacerbated and prolonged fever in response to a low dose of LPS (50 micrograms/kg, i.p.) compared to their wild-type counterparts . These data support the hypothesis that IL-10 acts as an endogenous cryogen during LPS-induced fever in mice. Ann N Y Acad Sci, 1998 Sep 29, 856, 33 - 47 IL-6 and IL-1 beta in fever . Studies using cytokine-deficient (knockout) mice; Kozak W et al.; Previous data support the hypothesis that during inflammation, interleukin (IL)-1 beta and IL-6 are involved in fever, in activation of the hypothalamic-pituitary-adrenal (HPA) axis, and in the induction of eicosanoids . Most of the pathophysiologic effects of IL-1 beta and Il-6 are mediated by prostaglandins (PGs), modulated by other cytokines, and antagonized by glucocorticoids (GC), a final product of the HPA axis . To further test these relationships, we measured changes in body temperature using biotelemetry in mice deficient in genes for IL-1 beta and/or IL-6 (IL-1 beta knockout {KO} and IL-6 KO) following injection with lipopolysaccharide (LPS) to induce systemic inflammation or turpentine to induce local abscess . Circulating IL-6, tumor necrosis factor alpha (TNF-alpha), GC, and PGE2 were measured in these mice after treatment . IL-1 beta KO mice responded with reduced fever and IL-6 KO mice with normal fever to a high dose of LPS . In contrast, neither type of KO mice produced fever to turpentine . PGE2 levels (measured in the circulation) were suppressed in both types of KO mice injected with turpentine . IL-1 beta KO mice showed deficiency in IL-6 following turpentine, but not LPS, injection . LPS-induced increases in TNF-alpha did not differ between IL-1 beta KO mice and their wild-type counterparts, whereas IL-6 KO mice showed exacerbated LPS-induced circulating TNF-alpha . No differences were noted in plasma elevations of GC between KO and wild-type mice following injection of LPS or turpentine, indicating that IL-1 beta and IL-6 are not required for activation of the HPA axis during inflammation . Our data demonstrate that in the mouse, IL-1 beta and IL-6 are critical for the induction of fever during local inflammation, whereas in systemic inflammation they appear only to contribute to fever. J Mol Biol, 1999 Jan 29, 285(4), 1869 - 86 Stability and folding of the tumour suppressor protein p16; Tang KS et al.; The tumour suppressor p16 is a member of the INK4 family of inhibi tors of the cyclin D-dependent kinases, CDK4 and CDK6, that are involved in the key growth control pathway of the eukaryotic cell cycle . The 156 amino acid residue protein is composed of four ankyrin repeats (a helix-turn-helix motif) that stack linearly as two four-helix bundles resulting in a non-globular, elongated molecule . The thermodynamic and kinetic properties of the folding of p16 are unusual . The protein has a very low free energy of unfolding, Delta GH-2O/D-N, of 3.1 kcal mol-1 at 25 degreesC . The rate-determining transition state of folding/unfolding is very compact (89% as compact as the native state) . The other unusual feature is the very rapid rate of unfolding in the absence of denaturant of 0.8 s-1 at 25 degreesC . Thus, p16 has both thermodynamic and kinetic instability . These features may be essential for the regulatory function of the INK4 proteins and of other ankyrin-repeat-containing proteins that mediate a wide range of protein-protein interactions . The mechanisms of inactivation of p16 by eight cancer-associated mutations were dissected using a systematic method designed to probe the integrity of the secondary structure and the global fold . The structure and folding of p16 appear to be highly vulnerable to single point mutations, probably as a result of the protein's low stability . This vulnerability provides one explanation for the striking frequency of p16 mutations in tumours and in immortalised cell lines . J Mol Biol, 1999 Jan 29, 285(4), 1831 - 43 Folding, heterodimeric association and specific peptide recognition of a murine alphabeta T-cell receptor expressed in Escherichia coli; Pecorari F et al.; In a systematic study of the murine T-cell receptor UZ3-4, expressed and refolded from inclusion bodies in Escherichia coli, it was found that functional molecules can be obtained only under a very narrow set of conditions . The refolded T-cell receptor UZ3-4 specifically recognizes its cognate peptide (from mycobacterial Hsp60) in the context of H-2Db, but not another peptide bound to H-2Db, and the dissociation constant was determined by BIAcore as 10(-4) M . Using T-cell receptor constructs comprising all extracellular domains (ValphaCalpha and VbetaCbeta), found to be necessary for stability of the final product, significant amounts of native molecules were obtained only if the intermolecular Calpha-Cbeta disulfide bridge bond was deleted, even though the interaction between the complete alpha and beta-chain was determined to be very weak and fully reversible (KD approximately 10(-7) to 10(-6) M) . Fusion of Jun and Fos to the constant domains also decreased the folding yield, because of premature association of intermediates leading to aggregation . Furthermore, only in a very narrow set of concentrations of oxidized and reduced glutathione, native disulfide bonds dominated . This shows that T-cell receptor domains are very prone to aggregation and misassociation during folding, compounded by incorrect disulfide bond formation . Once folded, however, the heterodimeric molecule is very stable and could be concentrated to millimolar concentration . J Mol Biol, 1999 Jan 29, 285(4), 1801 - 10 Membrane assembly of the Escherichia coli outer membrane protein OmpA: exploring sequence constraints on transmembrane beta-strands; Koebnik R; The eight-stranded antiparallel beta-barrel domain of the OmpA protein from Escherichia coli serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins . Previous studies have shown that neither the periplasmic turns nor the surface-exposed loops contain topogenic information . Consequently, the question of whether any structural constraint is imposed onto individual transmembrane beta-strands is now addressed . To this end, amino acid sequences of beta-strands 4, 6 and 8 were randomized . In vivo membrane assembly of mutant proteins was assayed and 288 variants were sequenced . Three parameters were found to be important for efficient membrane assembly . (i) At least four of five randomized residues with side-chains pointing towards the lipid bilayer must be hydrophobic and none of the three central residues must be charged . (ii) Side-chains pointing into the beta-barrel interior must not be enlarged too much, possibly because of packing constraints . (iii) Proline residues are, in general, hardly tolerated in the transmembrane beta-strands . J Mol Biol, 1999 Jan 29, 285(4), 1777 - 88 The chaperonin GroEL binds to late-folding non-native conformations present in native Escherichia coli and murine dihydrofolate reductases; Clark AC et al.; Dihydrofolate reductases from mouse (MuDHFR) or Escherichia coli (EcDHFR) are shown to refold via several intermediate forms, each of which can bind to the chaperonin GroEL . When stable complexes with GroEL are formed, they consist of late-folding intermediates . In addition, we find that late-folding intermediates that are present in the native enzyme bind to GroEL . For the E . coli and murine proteins, the extent of protein bound increases as the temperature is increased from 8 degreesC to 42 degreesC, at which temperature either protein is completely bound as the last (EcDHFR) or the last two (MuDHFR) folding intermediate(s) . Thus for EcDHFR, the binding is transient at low temperature (<30 degreesC) and stable at high temperature (>35 degreesC) . For MuDHFR, complex formation appears less temperature dependent . In general, the data demonstrate that the overall binding free energy for the interaction of GroEL with native DHFR is the sum of the free energy for the first step in DHFR unfolding, which is unfavorable, and the free energy of binding the non-native conformation, which is favorable . For EcDHFR, this results in an overall binding free energy that is unfavorable below 30 degreesC . Over the temperature range of 8 degreesC to 42 degreesC, GroEL binds MuDHFR more tightly than EcDHFR, due partially to a small free energy difference between two pre-existing non-native conformations of MuDHFR, resulting in binding to more than one folding intermediate . J Mol Biol, 1999 Jan 29, 285(4), 1765 - 76 Native Escherichia coli and murine dihydrofolate reductases contain late-folding non-native structures; Clark AC et al.; We have examined the equilibrium and kinetic folding properties of two structurally homologous dihydrofolate reductases, Escherichia coli DHFR (EcDHFR) and murine DHFR (MuDHFR), as a function of temperature and ligand concentration . Conformational heterogeneity in native DHFR is well documented, and the results demonstrate that the non-native form(s) represents late intermediate(s) in the folding process . We have measured the concentrations of native and non-native forms and the rate constants for their interconversion over a temperature range of 3 degreesC to 49 degreesC, allowing characterization of the thermodynamic as well as the kinetic properties of the final folding step(s) relative to the overall folding reaction . Differences in ligand binding suggest that the intermediate structures for these two proteins may be different during refolding . J Mol Biol, 1999 Jan 29, 285(4), 1655 - 66 A dimeric form of Escherichia coli succinyl-CoA synthetase produced by site-directed mutagenesis; Bailey DL et al.; Succinyl-CoA synthetase (SCS) catalyzes the substrate-level phosphorylation step of the citric acid cycle . The enzyme from Escherichia coli is an (alphabeta)2-heterotetramer with two active sites, one in each alphabeta-dimer . To determine whether the two active sites could function independently, mutations were made to split the tetramer into alphabeta-dimers . Because two choices for the tetramer (I and II) were possible from the X-ray crystallographic analyses, mutations were made at two different interfaces . All mutations based on tetramer I resulted in an intact tetramer . Of the two mutants based on tetramer II, one was insoluble and the other, where M156beta, Y158beta, R161beta and E162beta were changed to D, D, E and R, respectively, was a dimer . This quaternary structure was confirmed by fast protein liquid chromatography, blue native PAGE and ultracentrifugation . The DDER mutant has kinetic parameters similar to the tetrameric E . coli enzyme . Like the tetrameric enzyme, it shows ATP-facilitated dethiophosphorylation, proving that this property is a single-site effect . The ATP-facilitated dethiophosphorylation is inhibited by phosphate . It is concluded that dimerization of alphabeta-dimers is not a prerequisite for catalytic competency nor for alternating sites cooperativity in the tetramer . The rationale behind the dimer-of-dimers in E . coli SCS is still not known, but increased solubility, increased stability and in vivo interactions of the tetramer with other proteins are still possibilities . J Mol Biol, 1999 Jan 29, 285(4), 1633 - 53 A detailed structural description of Escherichia coli succinyl-CoA synthetase; Fraser ME et al.; Succinyl-CoA synthetase (SCS) carries out the substrate-level phosphorylation of GDP or ADP in the citric acid cycle . A molecular model of the enzyme from Escherichia coli, crystallized in the presence of CoA, has been refined against data collected to 2.3 A resolution . The crystals are of space group P4322, having unit cell dimensions a=b=98.68 A, c=403.76 A and the data set includes the data measured from 23 crystals . E . coli SCS is an (alphabeta)2-tetramer; there are two copies of each subunit in the asymmetric unit of the crystals . The crystal packing leaves two choices for which pair of alphabeta-dimers form the physiologically relevant tetramer . The copies of the alphabeta-dimer are similar, each having one active site where the phosphorylated histidine residue and the thiol group of CoA are found . CoA is bound in an extended conformation to the nucleotide-binding motif in the N-terminal domain of the alpha-subunit . The phosphoryl group of the phosphorylated histidine residue is positioned at the amino termini of two alpha-helices, one from the C-terminal domain of the alpha-subunit and the other from the C-terminal domain of the beta-subunit . These two domains have similar topologies, despite only 14 % sequence identity . By analogy to other nucleotide-binding proteins, the binding site for the nucleotide may reside in the N-terminal domain of the beta-subunit . If this is so, the catalytic histidine residue would have to move about 35 A to react with the nucleotide . J Mol Biol, 1999 Jan 29, 285(4), 1525 - 36 A mutant of BamHI restriction endonuclease which requires N6-methyladenine for cleavage; Whitaker RD et al.; Amino acid residues Asn116 and Ser118 of the restriction endonuclease BamHI make several sequence-specific and water-bridged contacts to the DNA bases . An in vivo selection was used to isolate BamHI variants at position 116, 118 and 122 which maintained sequence specificity to GGATCC sites . Here, the variants N116H, N116H/S118G and S118G were purified and characterized . The variants N116H and N116H/S118G were found to have lost their ability to cleave unmethylated GGATCC sequences by more than two orders of magnitude, while maintaining nearly wild-type levels of activity on the N6-methyladenine-containing sequence, GGmATCC . In contrast, wild-type BamHI and variant S118G have only a three- to fourfold lower activity on unmethylated GGATCC sequences compared with GGmATCC sequences . The N116 to H116 mutation has effectively altered the specificity of BamHI from an endonuclease which recognizes and cleaves GGATCC and GGmATC, to an endonuclease which only cleaves GGmATCC . The N116H change of specificity is due to the lowered binding affinity for the unmethylated sequence because of the loss of two asparagine-DNA hydrogen bonds and the introduction of a favorable van der Waals contact between the imidazole group of histidine and the N6-methyl group of adenine . J Mol Biol, 1999 Jan 29, 285(4), 1515 - 23 Genetic analysis of the base-specific contacts of BamHI restriction endonuclease; Dorner LF et al.; Here, we investigate the highly specific interaction of the BamHI endonuclease with its cognate recognition sequence GGATCC by determining which amino acid residues can be substituted at the DNA interface while maintaining specificity . Mutational studies, together with the structural determination of the restriction endonuclease BamHI have revealed the amino acid residues which are involved in DNA catalysis and those which play a role in the specific binding of the enzyme to its cognate DNA recognition sequence . Amino acid residues N116, S118, R122, D154 and R155 are involved in DNA sequence recognition and are located in the major groove in close proximity to the nucleotide bases comprising the recognition sequence . Cassette mutagenesis of these amino acids, together with in vivo transcriptional interference selection, was used to identify an array of substitutions which maintain site-specific binding to the cognate GGATCC sequence . This approach has demonstrated the extent of acceptable variation among amino acid residues which are directly involved in site-specific binding . One variant, double mutant N116H, S118G was found to cleave DNA only when the adenine base in the recognition site is methylated . J Mol Biol, 1999 Jan 29, 285(4), 1503 - 13 Membrane topology of the Rickettsia prowazekii ATP/ADP translocase revealed by novel dual pho-lac reporters; Alexeyev MF et al.; Here, we report the construction and characterization of dual reporters, consisting of both an Escherichia coli alkaline phosphatase (AP) gene and an alpha-fragment of the beta-galactosidase (BG) gene, for studying membrane protein topology by the gene fusion approach . Each of the reporters, when fused to periplasmic domains of polytopic proteins, produces fusions with high AP activity and, when fused to cytoplasmic domains, produces fusions with high BG activity in E . coli strains capable of alpha-complementation . The dual nature of these reporters simplifies interpretation of data obtained with poorly expressed fusions and allows one to evaluate the reliability of topological data . Deleterious effects resulting from the cell's attempt to export the full-length BG are eliminated in this approach . We describe dual indicator plates that allow for discrimination between colonies bearing cytoplasmic fusions, periplasmic fusions, and no fusions . We have generated a set of fusions to the topologically well-studied lactose permease of E . coli and demonstrated that topological information generated by these new reporters is in good agreement with the existing model . We used this new methodology for the determination of membrane topology of the Rickettsia prowazekii ATP/ADP translocase (Tlc) . Our results were in agreement with the proposed in silico topological model in which Tlc traverses the cytoplasmic membrane of E . coli 12 times with its N and C termini facing the cytoplasm . J Mol Biol, 1999 Jan 29, 285(4), 1485 - 501 Inversion/dimerization of plasmids mediated by inverted repeats; Lyu YL et al.; In contrast with earlier studies on the lambda and Escherichia coli genomes, recombination between inverted repeats on plasmids is highly efficient and shown to be recA-independent . In addition, the recombination product is exclusively a head-to-head inverted dimer . Here, we show that this recombination/rearrangement event can occur on different plasmid replicons and is not specific to the particular sequence within the inverted repeats . Transcription readthrough into the inverted repeats has little effect on this event . Genetic analysis has also indicated that most known recombination enzymes are not involved in this process . Specifically, single or double mutants defective in Holliday junction resolution systems (RuvABC and/or RecG/RusA) do not abolish this recombination/rearrangement event . This result does not support the previous models (i.e . the reciprocal-strand-switching and the cruciform-dumbbell models) in which intermediates containing Holliday junctions are proposed . Further analysis has demonstrated that the recombination/rearrangement frequency is dramatically (over 1000-fold) reduced if mismatches (2.8 %) are present within the inverted repeats . Mutations in dam, mutH and mutL genes partially or completely restored the recombination/rearrangement frequency to the level exhibited by the perfect inverted repeats, suggesting the formation of heteroduplexes during recombination/rearrangement . Sequencing analysis of the recombination/rearrangement products have indicated that the majority of the products do not involve crossing-over . We discuss a possible mechanism in which blockage of the lagging strand polymerase by a hairpin triggers recombination/rearrangement mediated by inverted repeats . J Mol Biol, 1999 Jan 29, 285(4), 1475 - 83 Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23 S ribosomal RNA; Gregory ST et al.; Evidence is presented for the participation of the P loop (nucleotides G2250-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidyl transferase center . Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interaction with the CCA end of P site-bound tRNA . These mutations altered the pattern of reactivity of RNA to chemical probes in a structural subdomain encompassing the P loop and extending roughly from G2238 to A2433 . Most of the effects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indicating that these mutations perturb the tertiary structure of this region of RNA . Changes in chemical modification were also seen in a subdomain composed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U2552-C2556), the latter a site of interaction with the CCA end of A site-bound tRNA . Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity . These results indicate that, in addition to its direct role in peptide bond formation, the P loop contributes to the tertiary structure of the peptidyl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites . J Mol Biol, 1999 Jan 29, 285(4), 1457 - 73 Degradation of FinP antisense RNA from F-like plasmids: the RNA-binding protein, FinO, protects FinP from ribonuclease E; Jerome LJ et al.; Transfer of F-like plasmids is regulated by the FinOP system, which controls the expression of traJ, a positive regulator of the transfer operon . F FinP is a 79 base antisense RNA, composed of two stem-loops, complementary to the 5' untranslated leader of traJ mRNA . Binding of FinP to the traJ leader sequesters the traJ ribosome binding site, preventing its translation and repressing plasmid transfer . The FinO protein binds stem-loop II of FinP and traJ mRNA and promotes duplex formation in vitro . FinO stabilizes FinP, increasing its effective concentration in vivo . To determine how FinO protects FinP from decay, the degradation of FinP was examined in a series of ribonuclease-deficient strains . Using Northern blot analysis, full-length FinP was found to be stabilized sevenfold in an RNase E-deficient strain . The major site of RNase E cleavage was mapped on synthetic FinP, to the single-stranded region between stem-loops I and II . A secondary site near the 5' end ( approximately 10 bases) was also observed . A GST-FinO fusion protein protected FinP from RNase E cleavage at both sites in vitro . Two duplexes between FinP and traJ mRNA were detected in an RNase III-deficient strain . The larger duplex resulted from extension of the FinP transcript at its 3' end, suggesting readthrough at the terminator that corresponds to FinP stem-loop II . A point mutant of finP (finP305; C30U) that is unable to repress traJ in the presence of FinO was also characterized . The pattern of RNase E digestion of finP305 RNA differed from FinP, and GST-FinO did not protect finP305 RNA from cleavage in vitro . The half-life of finP305 RNA decreased more than tenfold in vivo, such that the steady-state levels of finP305 RNA, in the presence of FinO, were insufficient to significantly reduce the level of traJ mRNA available for translation, allowing derepressed levels of transfer . Arch Biochem Biophys, 1999 Feb 1, 362(1), 131 - 8 Analysis of purified maize starch synthases IIa and IIb: SS isoforms can be distinguished based on their kinetic properties; Imparl-Radosevich JM et al.; Since starch synthases IIa (SSIIa) and SSIIb have not been purified from plant tissue, their structure-function relationships have not been well characterized . Therefore, we have expressed these SS genes in Escherichia coli, purified them to apparent homogeneity, and studied their kinetic properties . In addition, the N-terminally truncated forms of these enzymes were studied in an attempt to understand the function of the diverse N-terminal sequences in SS . Our results show that, like SSI, the N-terminal extensions of SSIIa and SSIIb are not essential for catalytic activity and no extensive changes in their kinetic properties are observed upon their N-terminal truncation . Each isoform of SS can be distinguished based on its kinetic properties . Maize SSI and maize SSIIb exhibit higher Vmax with glycogen as a primer, while the converse is true for SSIIa . However, the specific activity of SSIIb is at least two- to threefold higher than that for either SSI or SSIIa . Although SSIIb exhibits the highest maximal velocity of the isoforms compared, its apparent affinity for primer is twofold lower than the affinity of SSI and SSIIa for primer . Perhaps these differences in primer affinity, primer preference, and maximal velocities all contribute in some way to the different structure(s) of starch during its synthesis . Expression and purification of maize SS has now provided us a useful tool to address the role(s) of SS in starch synthesis and starch structure . Arch Biochem Biophys, 1999 Feb 1, 362(1), 67 - 78 S-nitrosylation and S-glutathiolation of protein sulfhydryls by S-nitroso glutathione; Ji Y et al.; The modification of reactive protein sulfhydryls by S-nitrosoglutathione and other NO donors has been studied by gel isoelectric focusing . S-nitrosylated, unmodified, and S-glutathiolated protein forms are differentiated by this method . With specific antibodies for the protein of interest, both S-nitrosylation and S-glutathiolation of the protein were analyzed in mixtures obtained as soluble tissue or cell extracts . The effect of S-nitrosoglutathione (GSNO) on purified phosphorylase b, on carbonic anhydrase III in an extract from rat liver, and on H-ras expressed in Escherichia coli was examined . When fresh GSNO reacted with pure phosphorylase b, only S-nitrosylated forms of the protein were observed . Likewise the NO donors, amyl nitrite, spermine NONOate, and diethylamine NONOate, all generated S-nitrosylated phosphorylase b . When crude mixtures of proteins from rat liver (containing carbonic anhydrase III) or from E . coli (containing an overexpressed form of H-ras) were exposed to fresh GSNO, both the S-nitrosylated and the S-glutathiolated forms of the proteins were observed . It is suggested that reactive intermediates from the breakdown of GSNO are responsible for the observed S-glutathiolation . These experiments show that both S-nitrosylated and S-glutathiolated forms of proteins may be generated by the addition of GSNO to mixtures containing proteins with reactive sulfhydryls . These protein modifications may exhibit metabolic consequences independent of the release of nitric oxide . Infect Immun, 1999 Feb, 67(2), 675 - 80 Protective immunization with a novel membrane protein of Plasmodium yoelii-infected erythrocytes; Burns JM Jr et al.; Immunization with a particulate fraction of blood-stage antigens was shown previously to protect mice against Plasmodium yoelii malaria . To identify antigens inducing the protective response, sera from immunized mice were used to screen a P . yoelii cDNA expression library . Sequence analysis of one 2.6-kb cDNA clone indicated that the identified gene, pypag-1, encoded a novel plasmodial antigen . Two nonoverlapping regions of pypag-1 were expressed in Escherichia coli . The first recombinant antigen, pAg-1N, contained the N-terminal 337 residues, which included a putative transmembrane domain and a region relatively rich in tryptophan residues . The second recombinant antigen, pAg-1C, contained the remaining C-terminal 211 residues, which included 31 copies of a 5-amino-acid degenerative repeat . Immunoblot studies using rabbit antiserum raised against recombinant pAg-1N showed that the native pypAg-1 protein migrated at approximately 98 kDa, considerably slower than its predicted molecular mass of 66 kDa . Immunofluorescence studies localized the expression of the native pypAg-1 protein both to the cytoplasm and at the surface of P . yoelii-infected erythrocytes . Immunization with either pAg-1N or pAg-1C induced a four- to sevenfold reduction in P . yoelii blood-stage parasitemia . As such, pypAg-1 appears to contain at least two distinct protective epitopes . To our knowledge, this is the first characterization of a protective antigen of P . yoelii that is associated with the erythrocyte membrane. J Biol Chem, 1999 Jan 29, 274(5), 3067 - 75 Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment); Minnick DT et al.; To investigate the interactions that determine DNA polymerase accuracy, we have measured the fidelity of 26 mutants with amino acid substitutions in the polymerase domain of a 3'-5'-exonuclease-deficient Klenow fragment . Most of these mutant polymerases synthesized DNA with an apparent fidelity similar to that of the wild-type control, suggesting that fidelity at the polymerase active site depends on highly specific enzyme-substrate interactions and is not easily perturbed . In addition to the previously studied Y766A mutator, four novel base substitution mutators were identified; they are R668A, R682A, E710A, and N845A . Each of these five mutator alleles results from substitution of a highly conserved amino acid side chain located on the exposed surface of the polymerase cleft near the polymerase active site . Analysis of base substitution errors at four template positions indicated that each of the five mutator polymerases has its own characteristic error specificity, suggesting that the Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845 side chains may contribute to polymerase fidelity in a variety of different ways . We separated the contributions of the nucleotide insertion and mismatch extension steps by using a novel fidelity assay that scores base substitution errors during synthesis to fill a single nucleotide gap (and hence does not require mismatch extension) and by measuring the rates of polymerase-catalyzed mismatch extension reactions . The R682A, E710A, Y766A, and N845A mutations cause decreased fidelity at the nucleotide insertion step, whereas R668A results in lower fidelity in both nucleotide insertion and mismatch extension . Relative to wild type, several Klenow fragment mutants showed substantially more discrimination against extension of a T.G mismatch under the conditions of the fidelity assay, providing one explanation for the anti-mutator phenotypes of mutants such as R754A and Q849A. Cancer Gene Ther, 1998 Nov-Dec, 5(6), 350 - 6 Gene transfer into human dendritic antigen-presenting cells by vaccinia virus and adenovirus vectors; Di Nicola M et al.; In a search for means to deliver exogenous gene(s) into human dendritic cells (DCs) from the perspective of tumor-specific vaccination, we have evaluated two recombinant viruses, both of which carry a reporter gene which is namely a modified vaccinia virus Ankara (MVA) and an adenovirus, as possible expression vectors . The recombinant MVA-P11 LZ vector carries the Escherichia coli lacZ gene coding for the enzyme beta-galactosidase, and the recombinant Ad-MFG-AP vector carries a modified membrane-exposed alkaline phosphatase (AP) gene . DCs were generated ex vivo in the presence of tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, stem cell factor, and flk-2/flt-3 ligand taken from CD34+ hematopoietic progenitors that were mobilized into the peripheral blood of cancer patients treated with high-dose cyclophosphamide and filgrastim . The target cells used for gene delivery were either CD34+ cells that had been subsequently induced to differentiate into mature DCs or DCs transduced after ex vivo generation from CD34+ cells . The results showed that: (a) infection of CD34+ cell derived-DCs (mature DCs) with either viral vector resulted in the efficient synthesis of recombinant protein, and (b) CD34+ cells were permissive for the expression of the recombinant reporter gene after infection with Ad-MFG-AP but not after infection with MVA-P11 LZ . In conclusion, these results suggest that vaccinia and adenovirus vectors are candidate to act as vehicles in genetically engineering human DCs. RNA, 1999 Jan, 5(1), 139 - 46 Maturation of 23S ribosomal RNA requires the exoribonuclease RNase T; Li Z et al.; Ribosomal RNAs are generally synthesized as long, primary transcripts that must be extensively processed to generate the mature, functional species . In Escherichia coli, it is known that the initial 30S precursor is cleaved during its synthesis by the endonuclease RNase III to generate precursors to the 16S, 23S, and 5S rRNAs . However, despite extensive study, the processes by which these intermediate products are converted to their mature forms are poorly understood . In this article, we describe the maturation of 23S rRNA . Based on Northern analysis of RNA isolated from a variety of mutant strains lacking one or multiple ribonucleases, we show that maturation of the 3' terminus requires the action of RNase T, an enzyme previously implicated in the end turnover of tRNA and in the maturation of small, stable RNAs . Although other exoribonucleases can participate in shortening the 3' end of the initial RNase III cleavage product, RNase T is required for removal of the last few residues . In the absence of RNase T, 23S rRNA products with extra 3' residues accumulate and are incorporated into ribosomes, with only small effects on cell growth . Purified RNase T accurately and efficiently converts these immature ribosomes to their mature forms in vitro, whereas free RNA is processed relatively poorly . In vivo, the processing defect at the 3' end has no effect on 5' maturation, indicating that the latter process proceeds independently . We also find that a portion of the 23S rRNA that accumulates in many RNase T- cells becomes polyadenylated because of the action of poly(A) polymerase I . The requirement for RNase T in 23S rRNA maturation is discussed in relation to a model in which only this enzyme, among the eight exoribonucleases present in E . coli, is able to efficiently remove nucleotides close to the double-stranded stem generated by the pairing of the 5' and 3' termini of most stable RNAs. RNA, 1999 Jan, 5(1), 102 - 16 Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA; Heide C et al.; We have identified by nucleotide analog interference mapping (NAIM) exocyclic NH2 groups of guanosines in RNase P RNA from Escherichia coli that are important for tRNA binding . The majority of affected guanosines represent phylogenetically conserved nucleotides . Several sites of interference could be assigned to direct contacts with the tRNA moiety, whereas others were interpreted as reflecting indirect effects on tRNA binding due to the disruption of tertiary contacts within the catalytic RNA . Our results support the involvement of the 2-NH2 groups of G292/G293 in pairing with C74 and C75 of tRNA CCA-termini, as well as formation of two consecutive base triples involving C75 and A76 of CCA-ends interacting with G292/A258 and G291/G259, respectively . Moreover, we present first biochemical evidence for two tertiary contacts (L18/P8 and L8/P4) within the catalytic RNA, whose formation has been postulated previously on the basis of phylogenetic comparative analyses . The tRNA binding interference data obtained in this and our previous studies are consistent with the formation of a consecutive nucleotide triple and quadruple between the tetraloop L18 and helix P8 . Formation of the nucleotide triple (G316 and A94:U104 in wild-type E . coli RNase P RNA) is also supported by mutational analysis . For the mutant RNase P RNA carrying a G94:C104 double mutation, an additional G316-to-A mutation resulted in a restoration of binding affinity for mature and precursor tRNA. RNA, 1999 Jan, 5(1), 93 - 101 Core sequence in the RNA motif recognized by the ErmE methyltransferase revealed by relaxing the fidelity of the enzyme for its target; Hansen LH et al.; Under physiological conditions, the ErmE methyltransferase specifically modifies a single adenosine within ribosomal RNA (rRNA), and thereby confers resistance to multiple antibiotics . The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated efficiently, and with equally high fidelity, in rRNAs from phylogenetically diverse bacteria . However, the fidelity of ErmE is reduced when magnesium is removed, and over twenty new sites of ErmE methylation appear in E . coli 16S and 23S rRNAs . These sites show widely different degrees of reactivity to ErmE . The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA . This suggests that methylation at the new sites results from changes in the RNA substrate rather than the methyltransferase . Chemical probing confirms that the rRNA structure opens upon magnesium depletion, exposing potential new interaction sites to the enzyme . The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclusively at adenosines (underlined); these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are slight preferences for the nucleotides shown in lower case) . This consensus is believed to represent the core of the motif that Erm methyltransferases recognize at their canonical A2058 site . The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recognition. RNA, 1999 Jan, 5(1), 82 - 92 Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy; Sette M et al.; Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional (15N,1H)-HSQC spectra . In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids . At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible . Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected . Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction . Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and alpha-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain . The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor. RNA, 1999 Jan, 5(1), 1 - 13 Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron; Roman J et al.; Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes . Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site . To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E . coli . Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA . Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected . The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA . The intron is spliced 10-fold less efficiently in E . coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site. J Immunol, 1999 Jan 15, 162(2), 1113 - 9 Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors; Tanghe A et al.; Using culture filtrate Ag-specific mAbs generated from mycobacteria-infected H-2b haplotype mice, we have previously identified three genes in the Mycobacterium tuberculosis genome, encoding proteins homologous to the periplasmic ATP-binding cassette phosphate-binding receptor PstS of the phosphate-specific transport system of E . coli . To define the potential vaccinal properties of these phosphate-binding proteins, female C57BL/6 mice were injected i.m . with plasmid DNA encoding PstS-1, PstS-2, or PstS-3 proteins from M . tuberculosis and immunogenicity and protective efficacy against i.v . challenge with M . tuberculosis H37Rv was analyzed . Significant levels of highly Ag-specific Abs and Th1-type cytokines IL-2 and IFN-gamma could be detected following vaccination with each of the three genes . However, only mice vaccinated with PstS-3 DNA demonstrated significant and sustained reduction in bacterial CFU numbers in spleen and lungs for 3 mo after M . tuberculosis challenge, as compared with CFU counts in mice vaccinated with control DNA . Vaccination with PstS-2 DNA induced a modest reduction in CFU counts in spleen only, whereas vaccination with PstS-1 DNA was completely ineffective in reducing bacterial multiplication . In conclusion, our results indicate that DNA vaccination is a powerful and easy method for comparative screening of potentially protective Ags from M . tuberculosis and that the PstS-3 protein is a promising new subunit vaccine candidate. J Immunol, 1999 Jan 15, 162(2), 704 - 10 T cell responses to heat-shock protein 60: differential responses by CD4+ T cell subsets according to their expression of CD45 isotypes; Ramage JM et al.; We demonstrate that human T lymphocytes proliferate in vitro to highly purified human heat-shock protein 60 (Hu.hsp60) . The response to this self Ag was confined to the CD45RA+ RO- T cell subset, with minimal responses by adult CD45RA- RO+ T cells . Experiments using keyhole limpet hemocyanin as a prototypic novel Ag, or tetanus toxoid as a recall Ag, were consistent with the notion that CD45RA+ RO- and CD45RA- RO+ T cell subsets can be designated as naive and memory cells, respectively; thus, responses to Hu.hsp60 were confined to the putative naive subset . In contrast, both CD45RA+ RO- and CD45RA- RO+ T cell populations proliferated to bacterial hsp60 from Mycobacterium leprae, Escherichia coli, or Chlamydia trachomatis . However, only CD45RA- RO+ (memory) T cells responded to a mycobacterial hsp60-derived peptide previously defined as a major bacteria-specific epitope . Experiments with cord blood T cells, which are CD45RA+ RO- and can be considered truly naive, showed that the peptide could elicit responses from naive T cells in vitro; cord blood cells also responded to Hu.hsp60 . Since bacterial hsp60 Ags contain both conserved and nonconserved epitopes, we speculate that in vivo challenge with bacterial hsp60 will activate T cells capable of seeing either type of epitope, but only those that see nonconserved epitopes maintain the CD45RA- RO+ memory phenotype . However, T cells recognizing conserved epitopes, while not apparently being recruited to the memory pool, may nevertheless play a role in immunoregulation, particularly in the context of inflammation, when expression of Hu.hsp60 is increased. J Clin Invest, 1999 Jan, 103(2), 167 - 74 Compartmentalization of extracellular cGMP determines absorptive or secretory responses in the rat jejunum; Jin XH et al.; We examined potential mechanisms by which angiotensin subtype-2 (AT2) receptor stimulation induces net fluid absorption and serosal guanosine cyclic 3',5'-monophosphate (cGMP) formation in the rat jejunum . L-arginine (L-ARG) given intravenously or interstitially enhanced net fluid absorption and cGMP formation, which were completely blocked by the nitric oxide (NO) synthase inhibitor, N-nitro-L-arginine methylester (L-NAME), but not by the specific AT2 receptor antagonist, PD-123319 (PD) . Dietary sodium restriction also increased jejunal interstitial fluid cGMP and fluid absorption . Both could be blocked by PD or L-NAME, suggesting that the effects of sodium restriction occur via ANG II at the AT2 receptor . L-ARG-stimulated fluid absorption was blocked by the soluble guanylyl cyclase inhibitor 1-H-{1,2,4}oxadiazolo{4, 2-alpha}quinoxalin-1-one (ODQ) . Cyclic GMP-specific phosphodiesterase in the interstitial space decreased extracellular cGMP content and prevented the absorptive effects of L-ARG . Angiotensin II (ANG II) caused an increase in net Na+ and Cl- ion absorption and 22Na+ unidirectional efflux (absorption) from the jejunal loop . In contrast, intraluminal heat-stable enterotoxin of Escherichia coli (STa) increased loop cGMP and fluid secretion that were not blocked by either L-NAME or ODQ . These findings suggest that ANG II acts at the serosal side via AT2 receptors to stimulate cGMP production via soluble guanylyl cyclase activation and absorption through the generation of NO, but that mucosal STa activation of particulate guanylyl cyclase causes secretion independently of NO, thus demonstrating the opposite effects of cGMP in the mucosal and serosal compartments of the jejunum. Infect Immun, 1999 Feb, 67(2), 986 - 8 Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide dismutase protects mice against virulent B . abortus; Onate AA et al.; Vaccination of mice with Escherichia coli expressing Brucella Cu/Zn superoxide dismutase (SOD) {E . coli(pBSSOD)} induced a significant level of protection against virulent Brucella abortus challenge, although this level was not as high as the one reached with B . abortus vaccine strain RB51 . In addition, vaccination with E . coli(pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein. Infect Immun, 1999 Feb, 67(2), 942 - 5 Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein; Komatsuzawa H et al.; The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure . DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa . The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4 . This monoclonal antibody reacted specifically with Omp29 of A . actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA . This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated . The N-terminal amino acid sequence of the protein expressed in E . coli perfectly matched those deduced from the purified Omp29 of strain Y4 . The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A . actinomycetemcomitans serotype c (NCTC 9710) . Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34. Infect Immun, 1999 Feb, 67(2), 745 - 53 Increased type 1 fimbrial expression among commensal Escherichia coli isolates in the murine cecum following catabolic stress; Hendrickson BA et al.; Although indigenous bacteria intimately colonize the intestinal mucosa, under normal conditions the intestinal epithelial cell is free of adherent bacteria . Nonetheless, commensal bacteria such as Escherichia coli adhere to and translocate across the intestinal epithelium in association with a number of pathologic states including hemorrhagic shock, immunosuppression, traumatic tissue injury, and lack of enteral feedings . The adhesins involved in the adherence of indigenous E . coli to the intestinal epithelium in vivo following catabolic stress are unknown . We have developed a mouse model to study the bacterial adhesins which mediate the increased intestinal adherence of E . coli after partial hepatectomy and short-term starvation . Our studies demonstrated that hepatectomy and starvation in the mouse were associated with a 7,500-fold increase in the numbers of E . coli bacteria adhering to the cecum . In addition, erythrocyte agglutination studies, as well as immunostaining of fimbrial preparations and electron micrographs of the bacteria, revealed that surface type 1 fimbriae were more abundant in the commensal E . coli harvested from the ceca of the stressed mice . These E . coli isolates adhered to a mouse colon cell line and injected cecal loops in a mannose-inhibitable manner, which suggests a role for type 1 fimbriae in the adherence of the E . coli isolates to the cecum in vivo following host catabolic stress. Infect Immun, 1999 Feb, 67(2), 694 - 9 Characterization of a novel methyl-accepting chemotaxis gene, dmcB, from the oral spirochete Treponema denticola; Li H et al.; Immediately downstream from the previously isolated Treponema denticola ATCC 35405 prtB gene coding for a chymotrypsinlike protease activity, an open reading frame, ORF3, was identified which shared significant homology with the highly conserved domains (HCDs) of bacterial methyl-accepting chemotaxis proteins (MCPs) . Nucleotide sequencing of this ORF revealed that the gene would code for a protein with a size of approximately 41 kDa . In addition, this sequence contained a domain which was virtually identical to the HCD of a recently characterized MCP, DmcA, of strain 35405 . Therefore, this ORF was named dmcB . Northern blot analysis suggested that dmcB was part of an operon structure containing prtB . Insertional inactivation of dmcB utilizing an ermF-ermAM cassette resulted in a mutant with decreased chemoattraction toward nutrient supplements . In addition, the mutant displayed an altered pattern of methylated proteins under conditions of chemotaxis . Inactivation of the dmcB gene also attenuated the methylation of the DmcA protein . These results suggest that the dmcB gene codes for an MCP in T . denticola which may interact with other MCPs in these organisms. Infect Immun, 1999 Feb, 67(2), 496 - 503 Deamidation of Cdc42 and Rac by Escherichia coli cytotoxic necrotizing factor 1: activation of c-Jun N-terminal kinase in HeLa cells; Lerm M et al.; Recently, Escherichia coli cytotoxic necrotizing factor 1 (CNF1) was shown to activate the low-molecular-mass GTPase RhoA by deamidation of Gln63, thereby inhibiting intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (G . Schmidt, P . Sehr, M . Wilm, J . Selzer, M . Mann, and K . Aktories, Nature 387:725-729, 1997; G . Flatau, E . Lemichez, M . Gauthier, P . Chardin, S . Paris, C . Fiorentini, and P . Boquet, Nature 387:729-733, 1997) . Here we report that in addition to RhoA, Cdc42 and Rac also are targets for CNF1 in vitro and in intact cells . Treatment of HeLa cells with CNF1 induced a transient formation of microspikes and formation of membrane ruffles . CNF1 caused a transient 10- to 50-fold increase in the activity of the c-Jun N-terminal kinase . Tryptic peptides of Cdc42 obtained from CNF1-treated cells by immunoprecipitation exhibited an increase in mass of 1 Da compared to control peptides, indicating the deamidation of glutamine 61 by the toxin . The same increase in mass was observed with the respective peptides obtained from CNF1-modified recombinant Cdc42 and Rac1 . Modification of recombinant Cdc42 and Rac1 by CNF1 inhibited intrinsic and GAP-stimulated GTPase activities and retarded binding of 2'(3')-O-(N-methylanthraniloyl)GDP . The data suggest that recombinant as well as cellular Cdc42 and Rac are substrates for CNF1. Biophys J, 1999 Feb, 76(2), 1024 - 33 Oriented, active Escherichia coli RNA polymerase: an atomic force microscope study; Thomson NH et al.; Combining a system for binding proteins to surfaces (Sigal, G . B., C . Bamdad, A . Barberis, J . Strominger, and G . M . Whitesides . 1996 . Anal . Chem . 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P . Wagner, and G . Semenza . 1993 . Surface Sci . 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM) . Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols . One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion . AFM images showed that these two alkanethiols phase-segregate . Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell . The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts . These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used. J Biol Chem, 1999 Jan 29, 274(5), 2978 - 87 Complex formation between Azotobacter vinelandii ferredoxin I and its physiological electron donor NADPH-ferredoxin reductase; Jung YS et al.; In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPH-ferredoxin reductase (FPR) . In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined . AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI . The Lys in FPR has been identified as Lys258, a residue that forms a salt bridge with one of the phosphate oxygens of FAD in the absence of FdI . UV-Vis and circular dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region close to the FAD . Cytochrome c reductase assays and electron paramagnetic resonance data show that electron transfer between the two proteins is pH-dependent and that the {3Fe-4S}+ cluster of FdI is specifically reduced by NADPH via FPR, suggesting that the {3Fe-4S} cluster is near FAD in the complex . To further investigate the FPR:FdI interaction, the electrostatic potentials for each protein were calculated . Strongly negative regions around the {3Fe-4S} cluster of FdI are electrostatically complementary with a strongly positive region overlaying the FAD of FPR, centered on Lys258 . These proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are physiological redox partners. J Biol Chem, 1999 Jan 29, 274(5), 2637 - 44 Transient ADP-ribosylation of a 2'-phosphate implicated in its removal from ligated tRNA during splicing in yeast; Spinelli SL et al.; The last step of tRNA splicing in yeast is catalyzed by Tpt1 protein, which transfers the 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1"-2"-cyclic phosphate (Appr>p) . Structural and functional TPT1 homologs are found widely in eukaryotes and, surprisingly, also in Escherichia coli, which does not have this class of tRNA splicing . To understand the possible roles of the Tpt1 enzymes as well as the unusual use of NAD, the reaction mechanism of the E . coli homolog KptA was investigated . We show here that KptA protein removes the 2'-phosphate from RNA via an intermediate in which the phosphate is ADP-ribosylated followed by a presumed transesterification to release the RNA and generate Appr>p . The intermediate was characterized by analysis of its components and their linkages, using various labeled substrates and cofactors . Because the yeast and mouse Tpt1 proteins, like KptA protein, can catalyze the conversion of the KptA-generated intermediate to both product and the original substrate, these enzymes likely use the same reaction mechanism . Step 1 of this reaction is strikingly similar to the ADP-ribosylation of proteins catalyzed by a number of bacterial toxins. Science, 1999 Jan 22, 283(5401), 546 - 9 Silencing of genes flanking the P1 plasmid centromere; Rodionov O et al.; Partition modules stabilize bacterial plasmids and chromosomes by actively promoting their segregation into daughter cells . The partition module of plasmid P1 is typical and consists of a centromere site, parS, and genes that encode proteins ParA and ParB . We show that ParB can silence genes flanking parS (to which ParB binds), apparently by polymerizing along the DNA from a nucleation site at parS . Wild-type ParB contacts an extensive region of P1 DNA; silencing-defective ParB proteins, which were found to be partition-defective, are less able to spread . Hence, the silenced structure appears to function in partitioning. Res Vet Sci, 1998 Nov-Dec, 65(3), 201 - 4 Efficacy of phosphomycin in the control of Escherichia coli infection of broiler chickens; Fernandez A et al.; Seventy-five 25-day-old broilers were divided into three groups: group I unmedicated and challenged with E . coli O78:K80; group F infected and treated with 150 ppm of phosphomycin in their drinking water, and group C acted as a control . Their weights, feed intake, clinical signs, macroscopic lesions, E . coli reisolation, and serum biochemistry were compared . Group F showed fewer symptoms and gross lesions than those from group I while the average daily gain, bodyweight, and feed intake were similar to the control group . E . coli was reisolated in 32 per cent of the livers and spleens from group I, compared with 4 per cent of liver and 8 per cent of spleens from group F . There was an increase in the levels of total protein and globulins in group I but not in group F . These results provide evidence of the therapeutic efficacy of phosphomycin in the control of an experimental E . coli infection in broiler chickens. J Chromatogr A, 1998 Dec 11, 827(2), 337 - 44 New approaches to the isolation of DNA by ion-exchange chromatography; Levison PR et al.; The performance of different anion-exchange media have been compared for the isolation of plasmid DNA and genomic DNA from bacterial cells and human whole blood . Whatman DEAE-Magarose, based on an agarose bead containing a paramagnetic component, has been compared with prepacked gravity-flow columns containing a derivatised silica matrix . In each case the DNA isolation at various scales of operation was similar both in terms of yield and quality . The magnetic susceptibility of DEAE-Magarose is very high, facilitating the use of this separation technique for rapid flexible batch chromatographic processes, a limitation of the prepacked column techniques. Eur J Biochem, 1999 Jan, 259(1-2), 543 - 50 Depletion of Escherichia coli 4.5S RNA leads to an increase in the amount of protein elongation factor EF-G associated with ribosomes; Nakamura K et al.; In Escherichia coli, 4.5S RNA is found in complexes with both protein translocation protein, Ffh (a bacterial homolog of mammalian SRP54) and protein synthesis elongation factor G (EF-G) . To analyze the function of 4.5S RNA in translation, we initially assessed the sensitivity of the association of 4.5S RNA with the ribosome after treatment with antibiotics that affect various stages of protein synthesis . Fusidic acid and viomycin caused 4.5S RNA to cosediment with the 70S ribosomal fraction, indicating that 4.5S RNA enters the ribosome before ribosomal translocation and release of EF-G-GDP from the ribosome . On the other hand, depletion of 4.5S RNA led to the retention of a significant amount of EF-G on 70S ribosomes . In addition, 4.5S RNA shares a conserved decanucleotide sequence (58GAAGCAGCCA67) motif with the characterized EF-G-binding site at positions 1068-1077 on 23S RNA . We therefore examined by gel mobility-shift assay whether or not mutations in the domain-IV region of 4.5S RNA, including this conserved motif, disturb the binding of EF-G to 23S RNA . Any mutation at the C62, G64 or A67 residues within this motif abolished competition activity . Therefore, we propose that 4.5S RNA is concerned with the mode of association of EF-G with the ribosomes . Moreover, this function depends on the secondary structure of 4.5S RNA as well as a ten-base sequence conserved between the two RNAs. Eur J Biochem, 1999 Jan, 259(1-2), 519 - 27 Replacement of terminal cysteine with histidine in the metallothionein alpha and beta domains maintains its binding capacity; Romero-Isart N et al.; To generate novel forms of metal-binding proteins, six mutant mouse metallothionein (MT) 1 fragments, in which a terminal cysteine residue was replaced by histidine, were expressed in Escherichia coli . The spectroscopic and analytical results showed that the alphaMT (C33H, C36H, C41H, C57H) and betaMT (C5H, C13H) mutant forms bound 4 and 3 Zn(II) atoms per molecule of protein to the nearest integer, even though in C41H and C5H, species of lower stoichiometry were also detected . In Cd(II) titrations, all the Zn(II) ions bound to the mutant proteins were displaced from the binding sites, giving rise to Cd-mutated MT forms with 4 and 3 Cd(II), respectively . However, although Cys-to-His substitutions maintained the binding capacity of the MT fragments, they caused structural changes with respect to the wild-type proteins . While C13H, C36H and C57H seem to contain Zn(II)-aggregates that are closely related to those of the wild-type proteins, only C41H and C57H gave rise to Cd(II)-aggregates similar to those of Cd4-alphaMT, where the His residue plays the role of the substituted Cys . Despite the structural implications of the Cys-to-His replacement, the dissociation constants showed no major decrease in the Cd-binding affinity in any of the mutants assayed compared with the wild-type. Eur J Biochem, 1999 Jan, 259(1-2), 441 - 8 Domain structure, GTP-hydrolyzing activity and 7S RNA binding of Acidianus ambivalens ffh-homologous protein suggest an SRP-like complex in archaea; Moll R et al.; In this study we provide, for the first time, experimental evidence that a protein homologous to bacterial Ffh is part of an SRP-like ribonucleoprotein complex in hyperthermophilic archaea . The gene encoding the Ffh homologue in the hyperthermophilic archaeote Acidianus ambivalens has been cloned and sequenced . Recombinant Ffh protein was expressed in E . coli and subjected to biochemical and functional studies . A . ambivalens Ffh encodes a 50.4-kDa protein that is structured by three distinct regions: the N-terminal hydrophilic N-region (N), the GTP/GDP-binding domain (G) and a C-terminal located C-domain (C) . The A . ambivalens Ffh sequence shares 44-46% sequence similarity with Ffh of methanogenic archaea, 34-36% similarity with eukaryal SRP54 and 30-34% similarity with bacterial Ffh . A polyclonal antiserum raised against the first two domains of A . ambivalens Ffh reacts specifically with a single protein (apparent molecular mass: 46 kDa, termed p46) present in cytosolic and in plasmamembrane cell fractions of A . ambivalens . Recombinant Ffh has a melting point of tm = 89 degreesC . Its intrinsic GTPase activity obviously depends on neutral pH and low ionic strength with a preference for chloride and acetate salts . Highest rates of GTP hydrolysis have been achieved at 81 degreesC in presence of 0.1-1 mm Mg2+ . GTP hydrolysis is significantly inhibited by high glycerol concentrations, and the GTP hydrolysis rate also markedly decreases by addition of detergents . The Km for GTP is 13.7 microm at 70 degreesC and GTP hydrolysis is strongly inhibited by GDP (Ki = 8 microm) . A . ambivalens Ffh, which includes an RNA-binding motif in the C-terminal domain, is shown to bind specifically to 7S RNA of the related crenarchaeote Sulfolobus solfataricus . Comparative sequence analysis reveals the presence of typical signal sequences in plasma membrane as well as extracellular proteins of hyperthermophilic crenarchaea which strongly supposes recognition events by an Ffh containing SRP-like particle in these organisms. Eur J Biochem, 1999 Jan, 259(1-2), 339 - 46 Affinity chromatography using trypanocidal arsenical drugs identifies a specific interaction between glycerol-3-phosphate dehydrogenase from Trypanosoma brucei and Cymelarsan; Denise H et al.; A 36-kDa protein was isolated by affinity chromatography using Cymelarsan, an arsenical drug currently used in African trypanosomiasis treatment, as ligand . This protein was identified as glycerol-3-phosphate dehydrogenase . Trypanosomal glycerol-3-phosphate was bound covalently, whereas its counterpart from rabbit muscle bound by ionic interaction . Arsenical drugs inhibit the enzyme in a dose-dependent manner . Oxidation of cysteine residues protects against inactivation without significantly diminishing enzymic activity . Drug concentrations giving 50% inhibition of the dehydrogenase activity were determined for the enzyme from both Trypanosoma brucei and rabbit and indicate a higher sensitivity of the trypanosomal enzyme to arsenical drugs and thiol reagents . MS was used to identify residues of glycerol-3-phosphate dehydrogenase bound by Cymelarsan; they are not conserved in the mammalian enzyme. Eur J Biochem, 1999 Jan, 259(1-2), 310 - 9 Physicochemical and immunological studies of the N-terminal domain of the Torpedo acetylcholine receptor alpha-subunit expressed in Escherichia coli; Alexeev T et al.; The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of alpha2 beta gamma delta subunits . Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail . In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1-209 of the Torpedo californica AChR alpha-subunit in Escherichia coli . Two vectors coding for a (His)6 tag, either preceding or following the 1-209 sequence, were used and the recombinant proteins obtained (designated alpha1-209pET and alpha1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column . The chemical structure of both proteins was verified by Edman degradation and mass spectrometry . The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents . The two proteins bound {125I}alpha-bungarotoxin with equal potency (KD approximately 130 nm, Bmax approximately 10 nmol.mg-1) . Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR . The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly beta-structure (50-56%), the fraction of alpha-helices amounting to 32-35% . Nonionic (beta-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of beta-structure or increased the alpha-helicity, respectively . The predominance of beta-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR alpha-subunit expressed in the mammalian cells {West et al . (1997) J . Biol . Chem . 272, 25 468} . Thus, expression in E . coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR alpha-subunit and appears to share with the latter a similar secondary structure . The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis. Eur J Biochem, 1999 Jan, 259(1-2), 281 - 8 I-NjaI, a nuclear intron-encoded homing endonuclease from Naegleria, generates a pentanucleotide 3' cleavage-overhang within a 19 base-pair partially symmetric DNA recognition site; Elde M et al.; Different species of the amoebo-flagellate Naegleria harbor optional group I introns in the nuclear ribosomal DNA that contain open reading frames . Intron proteins from Naegleria jamiesoni, Naegleria andersoni, and Naegleria italica (named I-NjaI, I-NanI and I-NitI, respectively) were expressed in Escherichia coli and found to be isoschizomeric homing endonucleases that specifically recognize and cleave intron-lacking homologous alleles of ribosomal DNA . The I-NjaI endonuclease was affinity purified, characterized in more detail, and found to generate five-nucleotide 3' staggered ends at the intron insertion site which differs from the ends generated by all other known homing endonucleases . The recognition site was delimited and found to cover an approximately 19 base-pair partially symmetric sequence spanning both the cleavage site and the intron insertion site . The palindromic feature was supported by mutational analysis of the target DNA . All single-site substitutions within the recognition sequence were cleaved by the purified I-NjaI endonuclease, but at different efficiencies . The center of symmetry and cleavage was found to be completely degenerate in specificity, which resembles that of the subclass IIW bacterial restriction enzymes. Eur J Biochem, 1999 Jan, 259(1-2), 96 - 103 Affinity of the periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and non-native outer membrane proteins . Role of Skp in the biogenesis of outer membrane protein; De Cock H et al.; The Skp protein of Escherichia coli has been proposed to be a periplasmic molecular chaperone involved in the biogenesis of outer membrane proteins . In this study, evidence is obtained that Skp exists in two different states characterized by their different sensitivity to proteases . The conversion between these states can be modulated in vitro by phospholipids, lipopolysaccharides and bivalent cations . Skp is able to associate with and insert into phospholipid membranes in vitro, indicating that it may associate with phospholipids in the inner and/or outer membrane in vivo . In addition, it interacts specifically with outer membrane proteins that are in their non-native state . We propose that Skp is required in vivo for the efficient targeting of unfolded outer membrane proteins to the membrane. Eur J Biochem, 1999 Jan, 259(1-2), 79 - 87 Characterization of human T-cell leukemia virus type I integrase expressed in Escherichia coli; Muller B et al.; The C-terminal part of the pol gene of the human T-cell leukemia virus type I (HTLV-I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells . We expressed the putative IN from an infectious molecular clone of HTLV-I in Escherichia coli . Comparison with protein resulting from coexpression of HTLV-I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR-Pol precursor by proteolytic cleavage . HTLV-I IN was purified from E . coli under native conditions . The protein behaved like a dimer in size-exclusion chromatography . It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5-derived vs . U3-derived sequences in the processing and strand-transfer reactions . In the disintegration reaction, HTLV-I IN not only accepted the double-stranded branched substrate corresponding to the product of a strand-transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single-stranded or a single adenosine overhang. Arch Microbiol, 1999 Jan, 171(2), 127 - 30 Expression of bvgAS of Bordetella pertussis represses flagellar biosynthesis of Escherichia coli; Han YW et al.; BvgAS is a two-component system of Bordetella pertussis involved in the reciprocal regulation of the virulence genes and the flagellar biosynthesis . In this study, we found that expression of bvgAS in Escherichia coli also results in reduced motility . The repression was relieved by the addition of known chemical modulators of BvgAS such as MgSO4 and nicotinic acid, indicating that functional BvgAS proteins are required for the negative control of E . coli motility . In addition, BvgAS repressed the transcription of the flhDC master operon of E . coli, which consequently caused non-flagellation on the cell surface . However, expression of BvgAS had no effect on stress-resistant motile mutants of E . coli . These data suggest that E . coli may have BvgA-like protein(s) involved in the regulatory interactions between the stress response and the flagellar biosynthesis. Biochem J, 1999 Feb 1, 337 ( Pt 3), 585 - 90 L-ATP is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity? Verri A, Montecucco A, Gosselin G, Boudou V, Imbach JL, Spadari S, Focher F. We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids . l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase . l-ATP strongly inhibits (Ki 80 microM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase alpha, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected . Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 microM), suggesting that it interacts with the ATP-binding site of the enzyme . Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction . On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates . Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy. Biochem J, 1999 Feb 1, 337 ( Pt 3), 425 - 31 A novel 17beta-hydroxysteroid dehydrogenase in the fungus Cochliobolus lunatus: new insights into the evolution of steroid-hormone signalling; Lanisnik Rizner T et al.; 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) catalyses the reduction of steroids and of several o- and p-quinones . After purification of the enzyme, its partial amino acid sequence was determined . A PCR fragment amplified with primers derived from peptide sequences was generated for screening the Coch . lunatus cDNA library . Three independent full-length cDNA clones were isolated and sequenced, revealing an 810-bp open reading frame encoding a 270-amino-acid protein . After expression in Escherichia coli and purification to homogeneity, the enzyme was found to be active towards androstenedione and menadione, and was able to form dimers of Mr 60000 . The amino acid sequence of the novel 17beta-HSD demonstrated high homology with fungal carbonyl reductases, such as versicolorin reductase from Emericella nidulans (Aspergillus nidulans; VerA) and Asp . parasiticus (Ver1), polyhydroxynaphthalene reductase from Magnaporthe grisea, the product of the Brn1 gene from Coch . heterostrophus and a reductase from Colletotrichum lagenarium, which are all members of the short-chain dehydrogenase/reductase superfamily . 17beta-HSDcl is the first discovered fungal 17beta-hydroxysteroid dehydrogenase belonging to this family . The primary structure of this enzyme may therefore help to elucidate the evolutionary history of steroid dehydrogenases. Biochem J, 1999 Feb 1, 337 ( Pt 3), 415 - 23 Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the alpha subunit of RNA polymerase at class I promoters; Law EC et al.; The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters . At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) . Since alphaCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions . We previously showed that, at these promoters, the CRP-alphaCTD interaction and the CRP-UP element interaction contribute independently and additively to transcription initiation . In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5 . This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two alphaCTDs of RNA polymerase are not optimally positioned . Two experiments to test this hypothesis are presented. Mol Cells, 1998 Dec 31, 8(6), 709 - 16 Maltose binding protein (MBP) fusion proteins with low or no affinity to amylose resins can be single-step purified using a novel anti-MBP monoclonal antibody; Park JH et al.; The maltose binding protein (MBP) fusion protein expression system is a powerful tool to produce and isolate recombinant proteins in E . coli . Whereas the conventional isolation technique for MBP-fusion proteins takes advantage of the binding affinity of MBP to maltose, this method is limited insofar as the biological activity of MBP has to be fully conserved for a successful purification . In this study, a novel monoclonal antibody (mAb) specific for MBP, termed HAM-19, was generated and its application in the purification and detection of MBP-fusion proteins determined . Using anti-MBP immunoaffinity columns, even recombinant MBP fusion products with lowered or impaired binding affinity to maltose were purified in a single step procedure . In comparison to amylose resins, HAM-19 immunoaffinity columns showed a higher binding capacity and affinity to MBP-fusion proteins . Furthermore, the mAb HAM-19 also provides a technical improvement over polyclonal antisera for the detection and analysis of MBP-fusion proteins which are under use in various forms in the fields of molecular and cellular biology. Biol Chem, 1998 Dec, 379(12), 1449 - 52 The influence of Ala205 on the specificity of cathepsin L produced by dextran sulfate assisted activation of the recombinant proenzyme; Barlic-Maganja D et al.; Human procathepsin L has been expressed in E . coli in the form of inclusion bodies . The recombinant protein was isolated, refolded and processed at pH 5.5 by the addition of dextran sulfate which increased the overall yield of cathepsin L almost 10-fold . After the auto-activation of the 38 kDa procathepsin L at least three processing sites were determined by N-terminal amino acid sequencing . After replacing the Ala205 residue by glutamic acid, cathepsin B-like specificity was introduced into cathepsin L . This mutation resulted in a 15-fold increased activity toward the substrate Z-Arg-Arg-AMC and in a 29-fold decreased activity toward Z-Phe-Arg-AMC . Residue 205 is thereby confirmed experimentally to be critical for the specificity of cathepsins B and L. Infect Dis Obstet Gynecol, 1998, 6(5), 230 - 4 The effects of Escherichia coli STa (heat stable) toxin on the contractility of isolated human myometrium in vitro; de Carrera AL et al.; OBJECTIVE: The purpose of the study was to assess the effects of Escherichia coli STa (heat stable) toxin on isolated human myometrial response to oxytocin . METHODS: One hundred and sixteen muscle strips were obtained from the lower uterine segment of 42 women undergoing cesarean section at term . Amniotic membranes and decidua were excluded . Uterine contractility in response to cumulative doses of E . coli STa toxin was recorded, as well as uterine response to cumulative doses of oxytocin before and after incubation with STa toxin or vehicle . The 50th percentile effective oxytocin concentration (EC50) of muscle strips with and without spontaneous activity before and after the incubation with STa toxin or vehicle was calculated . A paired t test was used for comparison . RESULTS: Muscle strips with and without spontaneous activity responded to cumulative doses of oxytocin before and after the incubation with STa toxin or vehicle . No differences in contraction force, duration, or frequency were noted between the groups (P > 0.05) . Furthermore, this toxin was not able to induce uterine contractility when tested alone . CONCLUSIONS: The inability of this toxin to affect myometrial response to oxytocin in this study may be due to the absence of amnion cells, chorion, or decidua . Other possible explanations for the lack of response are discussed. Biochemistry, 1999 Jan 19, 38(3), 1136 - 43 Thermal denaturation of Escherichia coli thioredoxin studied by hydrogen/deuterium exchange and electrospray ionization mass spectrometry: monitoring a two-state protein unfolding transition; Maier CS et al.; Thermally denatured oxidized Escherichia coli thioredoxin (TRX) in 2% acetic acid was examined by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism . Conformational dynamics during thermal unfolding were probed by hydrogen/deuterium (H/D) exchange-in experiments . ESI-MS was used to determine the H/D ratios . TRX shows only a marginal change in negative ellipticity at 222 nm during thermal unfolding, but in the near-UV circular dichroism (240-350 nm) a clear transition is observed (Tm = 61 degrees C), and unfolding goes to completion . ESI mass spectra were recorded as a function of temperature, and the observed bimodal charge state distributions were analyzed assuming a two-state unfolding mechanism which allowed an estimation of the midpoint temperature, Tm = 64 degrees C . Under conditions at which the compact, folded conformational state is only marginally stable (80 degrees C, 2% acetic acid-d1), H/D exchange-in experiments in combination with ESI-MS resulted in mass spectra differing in the number of incorporated deuteriums which indicates the presence of two distinct populations of molecules after short incubation periods . As the exchange-in time increases, the population representing the unfolded state increases and the population which is protected against exchange decreases . The rate of conversion was used to estimate the rate constant of unfolding which was 2.1 +/- 0.2 min-1 . The results presented here indicate that thermally denatured TRX under the conditions used may represent a collapsed unfolded state with properties often attributed to molten globule-like states, such as pronounced secondary structure but absence of rigid tertiary structure and, hence, lack of protection against H/D exchange. Biochemistry, 1999 Jan 19, 38(3), 1087 - 94 Crystal structures of rat thymidylate synthase inhibited by Tomudex, a potent anticancer drug; Sotelo-Mundo RR et al.; Two crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex (ZD1694) have been determined to resolutions of 3.3 and 2.6 A . Tomudex is one of several new antifolates targeted to TS and the first to be approved for clinical use . The structures represent the first views of any mammalian TS bound to ligands and suggest that the rat protein undergoes a ligand-induced conformational change similar to that of the Escherichia coli protein . Surprisingly, Tomudex does not induce the "closed" conformation in rat TS that is seen on binding to E . coli TS, resulting in inhibitor atoms that differ in position by more than 1.5 A . Several species-specific differences in sequence may be the reason for this . Phe 74 shifts to a new position in the rat complex and is in van der Waals contact with the inhibitor, while in the E . coli protein the equivalent amino acid (His 51) hydrogen bonds to the glutamate portion of the inhibitor . Amino acids Arg 101, Asn 106, and Met 305 make no contacts with the inhibitor in the open conformation, unlike the equivalent residues in the E . coli protein (Thr 78, Trp 83, and Val 262) . dUMP binding is similar in both proteins, except that there is no covalent adduct to the active site cysteine (Cys 189) in the rat structures . Two insertions in the rat protein are clearly seen, but the N-termini (residues 1-20) and C-termini (residues 301-307) are disordered in both crystal forms. Biochemistry, 1999 Jan 19, 38(3), 1040 - 9 Structural and functional properties of human hemoglobins reassembled after synthesis in Escherichia coli; Hui HL et al.; Human hemoglobin produced in the Escherichia coli coexpression system of Hernan et al . {(1992) Biochemistry 31, 8619-8628} has been transformed into a functionally homogeneous protein whose properties closely approximate those of normal hemoglobin A . Both of the alpha and beta chains of this hemoglobin contain a valine-methionine substitution at position 1 in order to accommodate the difference in specificity of the protein-processing enzymes of procaryotes . Despite extensive purification, functional homogeneity of the E . coli expressed hemoglobin was achieved only by the complete disassembly of the hemoglobin into its component alpha and beta globins and their reassembly in the presence of hemin . The kinetics of CO combination and the thermodynamics of O2 binding and cooperativity of the reassembled alphaV1M-betaV1M hemoglobin closely approximate those of HbA . The alpha globin obtained from the E . coli expressed hemoglobin was also combined with normal human beta chains and hemin to form the alphaV1M variant . The alpha+M variant of HbA, in which the normal N-terminal valine of the alpha chains is preceded by a methionine residue, was prepared by the same procedure . The kinetics of the reactions of CO with the alphaV1M and alpha+M variants are similar to those for HbA . The equilibria of oxygen binding to alphaV1M and HbA are similar whereas alpha+M exhibits a significantly higher oxygen affinity . The three-dimensional structures of alphaV1M and alpha+M offer an explanation for the latter affinity difference . Although the structures of alphaV1M and HbA, which have been determined by X-ray crystallography, are virtually indistinguishable except at the N-terminal residues, that of alpha+M indicates the displacement of a solvent molecule, possibly a chloride ion, from arginine 141alpha . Such an alteration in an anion binding site could result in increased oxygen affinity. Biochemistry, 1999 Jan 19, 38(3), 1018 - 29 Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels; Bilsel O et al.; A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism of the alpha-subunit of tryptophan synthase from Escherichia coli . A distinctive property of this 29 kDa alpha/beta barrel protein is the presence of two stable equilibrium intermediates, populated at approximately 3 and 5 M urea . The refolding process displays multiple kinetic phases whose lifetimes span the submillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the order of 10-100 s . In an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globally fit to various kinetic models . Refolding and unfolding experiments from various initial urea concentrations as well as forward and reverse double-jump experiments were critical for model discrimination . The simplest kinetic model that is consistent with all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure . This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathway intermediates that correspond to the 3 M urea equilibrium intermediate . Reequilibrations among these conformers act as rate-limiting steps in folding for a majority of the population . A fraction of the native conformation appears in less than 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface. Biochemistry, 1999 Jan 19, 38(3), 952 - 63 Kinetic mechanism of damage site recognition and uracil flipping by Escherichia coli uracil DNA glycosylase; Stivers JT et al.; The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes hydrolytic cleavage of the N-glycosidic bond of premutagenic uracil residues in DNA by flipping the uracil base from the DNA helix . The mechanism of base flipping and the role this step plays in site-specific DNA binding and catalysis by enzymes are largely unknown . The thermodynamics and kinetics of DNA binding and uracil flipping by UDG have been studied in the absence of glycosidic bond cleavage using substrate analogues containing the 2'-alpha and 2'-beta fluorine isomers of 2'-fluoro-2'-deoxyuridine (Ubeta, Ualpha) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP) . Activity measurements show that DNA containing a Ubeta or Ualpha nucleotide is a 10(7)-fold slower substrate for UDG (t1/2 approximately 20 h), which allows measurements of DNA binding and base flipping in the absence of glycosidic bond cleavage . When UDG binds these analogues, but not other DNA molecules, a 4-8-fold 2-AP fluorescence enhancement is observed, as expected for a decrease in 2-AP base stacking resulting from enzymatic flipping of the adjacent uracil . Thermodynamic measurements show that UDG forms weak nonspecific complexes with dsDNA (KDns = 1.5 microM) and binds approximately 25-fold more tightly to Ubeta containing dsDNA (KDapp approximately 50 nM) . Thus, base flipping contributes less than approximately 2 kcal/mol to the free energy of binding and is not a major component of the >10(6)-fold catalytic specificity of UDG . Kinetic studies at 25 degrees C show that site-specific binding occurs by a two-step mechanism . The first step (E + S left and right arrow ES) involves the diffusion-controlled binding of UDG to form a weak nonspecific complex with the DNA (KD approximately 1.5-3 microM) . The second step (ES left and right arrow E'F) involves a rapid step leading to reversible uracil flipping (kmax approximately 1200 s-1) . This step is followed closely by a conformational change in UDG that was monitored by the quenching of tryptophan fluorescence . The results provide evidence for an enzyme-assisted mechanism for uracil flipping and exclude a passive mechanism in which the enzyme traps a transient extrahelical base in the free substrate . The data suggest that the duplex structure of the DNA is locally destabilized before the base-flipping step, thereby facilitating extrusion of the uracil . Thus, base flipping contributes little to the free energy of DNA binding but contributes greatly to specificity through an induced-fit mechanism. Biochemistry, 1999 Jan 19, 38(3), 945 - 51 Directed hydroxyl radical probing of 16S ribosomal RNA in 70S ribosomes from internal positions of the RNA; Newcomb LF et al.; Directed hydroxyl radical probing of 16S ribosomal RNA from Fe(II) tethered to specific sites within the RNA was used to determine RNA-RNA proximities in 70S ribosomes . We have transcribed 16S ribosomal RNA in vitro as two separate fragments, covalently attached an Fe(II) probe to a 5'-guanosine-alpha-phosphorothioate at the junction between the two fragments, and reconstituted 30S subunits with the two separate pieces of RNA and the small subunit proteins . Reconstituted 30S subunits capable of association with 50S subunits were selected by isolation of 70S ribosomes . Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved sites in the 16S rRNA backbone that were close in three-dimensional space to the Fe(II), and a primer extension was used to identify these sites of cleavage . Two sets of 16S ribosomal RNA fragments, 1-360/361-1542 and 1-448/449-1542, were reconstituted into active 30S subunits . Fe(II) tethered to position 361 results in cleavage of 16S rRNA around nucleotides 34, 160, 497, 512, 520, 537, 552, and 615, as well as around positions 1410, 1422, 1480, and 1490 . Fe(II) tethered to position 449 induces cleavage around nucleotide 488 and around positions 42 and 617 . Fe(II) tethered to the 5' end of 16S rRNA induces cleavage of the rRNA around nucleotides 5, 601, 615, and 642 . These results provide constraints for the positioning of these regions of 16S rRNA, for which there has previously been only limited structural information, within the 30S subunit. Biochemistry, 1999 Jan 19, 38(3), 905 - 13 Functional and structural analysis of cis-proline mutants of Escherichia coli aspartate aminotransferase; Birolo L et al.; To elucidate the role of the two conserved cis-proline residues of aspartate aminotransferase (AspAT), one double and two single mutants of the enzyme from Escherichia coli (EcAspAT) were prepared: P138A, P195A and P138A/P195A in which the two prolines were replaced by alanine . The crystal structures of P195A and P138A/P195A have been determined at 2.3-2.1 A resolution . The wild-type geometry, including the cis conformation of the 194-195 peptide bond is retained upon substitution of proline 195 by alanine, whereas the trans conformation is adopted at the 137-138 peptide bond . Quite surprisingly, the replacement of each of the two prolines by alanine does not significantly affect either the activity or the stability of the protein . All the three mutants follow the same pathway as the wild type for unfolding equilibrium induced by guanidine hydrochloride {Herold, M., and Kirschner, K . (1990) Biochemistry 29, 1907-1913} . The kinetics of renaturation of P195A, where the alanine retains the wild-type cis conformation, is faster than wild type, whereas renaturation of P138A, which adopts the trans conformation, is slower . We conclude that cis-prolines seem to have been retained throughout the evolution of aspartate aminotransferase to possibly play a subtle role in directing the traffic of intermediates toward the unique structure of the native state, rather than to respond to the needs for a specific catalytic or functional role. Biochimie, 1998 Nov, 80(11), 895 - 8 Protein engineering on enzymes of the peptide elongation cycle in Sulfolobus solfataricus; Bocchini V et al.; The present article is a review of the work done on the elongation factors EF-1 alpha, EF-2 and EF-1 beta isolated from the hyperthermophilic archaeon Sulfolobus solfataricus . The molecular, physical and biochemical properties of the intact, truncated, mutant or chimeric forms are described and compared. Biochimie, 1998 Oct, 80(10), 813 - 20 Apoflavodoxin: structure, stability, and FMN binding; Maldonado S et al.; Flavodoxins are one domain alpha/beta electron transfer proteins that participate in photosynthetic reactions . All flavodoxins carry a molecule of flavin mononucleotide (FMN), non-covalently bound, that confers redox properties to the protein . There are two structurally distinct flavodoxins, short ones and long flavodoxins; the latter contain an extra loop with unknown function . We have undertaken the study of the stability and folding of the apoflavodoxin from Anabaena (a long flavodoxin) and the analysis of the interaction between the apoflavodoxin and FMN . Our studies indicate that apoflavodoxin folds in a few seconds to a form that is competent in FMN binding . The stability of this apoflavodoxin is low and its urea denaturation can be described by a two-state mechanism . The role of the different parts of the apoflavodoxin in the stability and structure of the whole protein is being investigated using mutagenesis and specific cleavage to generate apoflavodoxin fragments . The X-ray structure of apoflavodoxin is very similar to that of its complex with FMN, the main difference being the conformation of the two aromatic residues that sandwich FMN in the complex . In apoflavodoxin these groups interact with each other so closing the FMN binding site . Despite this fact, apoflavodoxin binds FMN tightly and rapidly, and the resulting holoflavodoxin displays a high conformational stability . We have found that one role of the aromatic residues that interact with FMN is to help to retain bound the reduced form of the cofactor whose complex with apoflavodoxin is otherwise too weak. Genes Cells, 1998 Oct, 3(10), 635 - 47 Function in vivo of separate segments of the beta subunit of Escherichia coli RNA polymerase; Trigwell S et al.; BACKGROUND: Transcription of genetic material is catalysed by the enzyme DNA-dependent, RNA polymerase . The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed beta and beta', are conserved throughout nature . The beta subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme . Several lines of evidence have suggested that the function of the beta subunit is not dependent upon the contiguity of the sequence blocks . In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the beta subunit of RNA polymerase . To this end, the beta structural gene rpoB was separated into four near-equal, non-overlapping segments (as well as 'half' genes) on the basis of 'split' genes in nature, known functional organization and sequence conservation . These segments were used to prepare sequence-specific antibodies against the four individual regions, as well as being expressed in vivo from a tight, lac-controlled high-copy number vector . RESULTS: Immunological probing of the holoenzyme in vitro suggested that the amino-terminal half of the beta polypeptide is buried within the enzyme complex . Of the four segments expressed in vivo, the extreme C-terminal segment was trans-dominant lethal (of the effect of large N-terminal amber fragments on cellular growth; Nene & Glass 1982) and this isolated region was shown to bind the translational elongation factor EF-Tu in vivo . CONCLUSIONS: These in vivo and in vitro studies, in conjunction with recent in vitro work (Severinov et al . 1995), unambiguously demonstrate that individual regions of beta may adopt structurally and functionally competent forms, and underline the possibility of in vivo investigation of separate regions of this massive polypeptide chain . A model is presented for the role of EF-Tu in stringent control. Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 604 - 9 Functional dissection of the transmitter module of the histidine kinase NtrB in Escherichia coli; Kramer G et al.; Signal transduction by two-component systems involves phosphorylation and thereby activation of the response regulator by the cognate histidine kinase . Bifunctional histidine kinases have two opposing activities: depending on the environmental stimuli they either promote phosphorylation or stimulate the rapid dephosphorylation of the response regulator . To determine the mechanism of this switch, we analyzed the domain organization of the bifunctional histidine kinase NtrB . Based on sequence alignments with other histidine kinases and a deletion analysis, we defined three separate subdomains of the transmitter module, the H domain (amino acids 123-221), the N domain (amino acids 221-269), and the G domain (amino acids 269-349) . The transmitter module, when separately expressed, exhibited a constitutive positive phenotype . In contrast, in the absence of the G domain, the H domain exhibits a constitutive negative phenotype . This negative regulatory activity of the H domain is inhibited by the G domain . The G domain could be physically uncoupled; when coexpressed with the H-N fragment, the constitutive positive phenotype of the transmitter was restored . We demonstrate, in vitro, that the constitutive negative phenotype of the fragments lacking the G domain is caused by stimulation of dephosphorylation of the response regulator NtrC-P . Based on our analysis, we suggest that the function of the sensor domain is to control the interaction of the H and G domains . If these subdomains interact, NtrB acts as a positive regulator; if they cannot interact, NtrB acts as a negative regulator. Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 418 - 23 Substrate recognition by class I lysyl-tRNA synthetases: a molecular basis for gene displacement; Ibba M et al.; Lysyl-tRNA synthetases (LysRSs) are unique amongst the aminoacyl-tRNA synthetases in being composed of unrelated class I and class II enzymes . To allow direct comparison between the two types of LysRS, substrate recognition by class I LysRSs was examined . Genes encoding both an archaeal and a bacterial class I enzyme were able to rescue an Escherichia coli strain deficient in LysRS, indicating their ability to functionally substitute for a class II LysRS in vivo . In vitro characterization showed lysine activation and recognition to be tRNA-dependent, an attribute of several class I, but not class II, aminoacyl-tRNA synthetases . Examination of tRNA recognition showed that class I LysRSs recognize the same elements in tRNALys as their class II counterparts, namely the discriminator base (N73) and the anticodon . This sequence-specific recognition of the same nucleotides in tRNALys by the two unrelated types of enzyme suggests that tRNALys predates at least one of the LysRSs in the evolution of the translational apparatus . The only observed variation in recognition was that the G2.U71 wobble pair of spirochete tRNALys acts as antideterminant for class II LysRS but does not alter class I enzyme recognition . This difference in tRNA recognition strongly favors the use of a class I-type enzyme to aminoacylate particular tRNALys species and provides a molecular basis for the observed displacement of class II by class I LysRSs in certain bacteria. J Med Virol, 1999 Feb, 57(2), 174 - 8 IgG immune response to B19 parvovirus VP1 and VP2 linear epitopes by immunoblot assay; Manaresi E et al.; Human B19 parvovirus recombinant capsid proteins VP1 and VP2 were expressed in E . coli and purified . Recombinant proteins were used to detect a specific IgG immune response against VP1 and VP2 linear epitopes by immunoblot assay . A total of 222 serum samples from 218 apparently immunocompetent subjects with different clinical conditions and laboratory evaluations with regards to B19 infection were analyzed . The sera had previously been tested for B19 DNA and for specific IgM and IgG against VP2 conformational antigens by ELISA assay . The data show that, during the active or very recent phase of infection, IgG anti-VP1 linear epitopes appear in concomitance and with the same frequency as IgG anti-VP2 conformational antigens . IgG against conformational VP2 antigens and against linear VP1 epitopes seem to persist for months or years in the majority of individuals . IgG against VP2 linear epitopes are generally present during the active or very recent phase of infection and during the convalescent phase, while they are present only in about 20% of subjects with signs of a past B19 infection. J Med Virol, 1999 Feb, 57(2), 104 - 10 Hepatitis C virus core protein fused to hepatitis B virus core antigen for serological diagnosis of both hepatitis C and hepatitis B infections by ELISA; Wu CL et al.; The sequence encoding the truncated core protein (amino acids 1-98) of hepatitis C virus (HCc) was expressed in E . coli for production of HCc(1-98), or fused with the truncated core antigen (HBcAg) and segments from the preS1 and preS2 regions from hepatitis B virus (HBV) for production of HBcPreS1PreS2HCc(1-98) . The HCc(1-98) and HBcPreS1PreS2HCc(1-98) proteins reacted with sera from HCV-infected individuals by immunoblot analyses, while the latter protein also exhibited HBV core antigenicity . They induced antibodies against HBcAg and/or HCV core protein in rabbits and in mice . Moreover, HBcPreS1PreS2HCc(1-98) is more immunogenic than HCc(1-98) in terms of anti-HCc induction . An ELISA that employed recombinant HCV core antigens of either HCc(1-98) or HBcPreS1PreS2HCc(1-98) to detect anti-HCc and/or anti-HBc antibodies was developed . Evaluation of serum samples with different status of HBV and HCV infections suggested that HCc(1-98) might be suitable for the determination of antibodies against HCV core protein, while HBcPreS1PreS2HCc(1-98) might be of value to detect HCV and/or HBV infection in donated blood in HBV low-prevalence countries. Pharm Res, 1998 Dec, 15(12), 1808 - 15 Improved folding yields of a model protein using protein disulfide isomerase; Du C et al.; PURPOSE: To study the effects of recombinant human protein disulfide isomerase (rhPDI) concentration, reduced glutathione:oxidized glutathione ratio (GSH:GSSG) and temperature on the efficiency of oxidative folding of a model protein, recombinant human interleukin 2 (C125A mutation) (C125A rhIL-2) . METHODS: C 125A rhIL-2 inclusion bodies were reduced and denatured by guanidium hydrochloride (Gdm.Cl) and 100 mM GSH . The solution was diluted 10 times into folding buffer, allowing C125A rhIL-2 to fold either in the absence or presence of rhPDI . The renatured and unfolded C125A rhIL-2 species were quantitated by reversed phase-HPLC . RESULTS: The initial folding rate of C125A rhIL-2 linearly increased with rhPDI:C125A rhIL-2 molar ratio in the first 2.5 minutes, and reached the highest rate when the rhPDI:C125A rhIL-2 ratio was 1:1 . The oxidative folding of C125A rhIL-2 linearly increased as the GSH:GSSG molar ratio decreased from 10:0 to 10:3 . The folding of C125A rhIL-2 was also dependent on temperature, and optimum folding was realized at 23 degrees C . CONCLUSIONS: These results demonstrate that under optimal redox potential and temperature, rhPDI enhances the oxidative folding of C125A rhIL-2 . In the oxidative folding of C125A rhIL-2, rhPDI exerts its effect on folding by the acceleration of thiol/disulfide interchange. J Neurooncol, 1998 Nov, 40(2), 137 - 50 Verotoxins inhibit the growth of and induce apoptosis in human astrocytoma cells; Arab S et al.; Verotoxin 1 (VT1) is an E . coli toxin comprising an A subunit with N-glycanase activity, and five smaller B subunits capable of binding to the functional receptor globotriaosylceramide (Galalpha1-4-Galbeta1-4-Glcceramide-Gb3) . VT is implicated in hemorrhagic colitis and the more serious hemolytic uremic syndrome . VT1 is active against various tumor cell lines in vitro and in vivo . To extend the anti-cancer spectrum of activity of VT to human brain tumors, in the present analysis we studied the effects of VT on the growth of 6 permanent human astrocytoma cell lines . All astrocytoma cell lines analyzed express Gb3 and were sensitive to VT-1 at a dose of 50 ng/ml, but sensitivity was not proportional to the relative Gb3 concentration . VT induced apoptosis in these cells was shown by electron microscopy . Morphological evidence (nuclear shrinkage and chromatin condensation) of apoptosis could be clearly distinguished 1.5 hrs after toxin addition . Ultrastructural preservation of organelles was observed in conjunction with blebbing of the plasma membrane, condensation of chromatin within the nucleus and nuclear shrinkage . Apoptosis was also induced by the recombinant toxin B subunit alone, suggesting that the ligation of Gb3 is the primary induction mechanism . These studies indicate that verotoxin/Gb3 targetting may provide a novel basis for the inhibition of astrocytoma tumour cell growth. FEBS Lett, 1998 Dec 28, 441(3), 458 - 62 Antiferritin single-chain antibody: a functional protein with incomplete folding? Martsev SP, Kravchuk ZI, Chumanevich AA, Vlasov AP, Dubnovitsky AP, Bespalov IA, Arosio P, Deyev SM. The pET(scF11) plasmid was constructed comprising the gene of a single-chain antibody against human ferritin . This plasmid encodes the leader peptide pelB followed by the heavy chain variable V(H) domain, (Gly4Ser)3 linker peptide, and light chain variable V(L) domain . The correctly processed scF11 antibody was expressed in Escherichia coli as an insoluble protein without the leader peptide . Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion-exchange chromatography . ScF11 demonstrated only a approximately 8-fold decrease in the affinity (Ka = 5.1 x 10(8) M(-1) in RIA and 1.8 x 10(8) M(-1) in ELISA) vs . the parent IgG2a/kappa monoclonal antibody F11 . The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein . However, scF11 demonstrated (i) the lack of denaturant-induced fluorescence 'dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS) recognizing partially structured states of a protein . These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core . This work provides the first indication that the functional single-chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding. FEBS Lett, 1998 Dec 28, 441(3), 407 - 12 Sequence analysis of a monoclonal antibody specific for the preS2 region of hepatitis B surface antigen, and the cloning, expression and characterisation of its single-chain Fv construction; Passafiume M et al.; The nucleotide sequence of the monoclonal antibody F124, specific for the preS2 region of the surface antigen of hepatitis B virus, has been determined and an single-chain Fv fragment (scFv) recombinant construction has been cloned and expressed into the periplasmic region of Escherichia coli . The recombinant antibody fragment contains a (Gly4Ser)3 linker connecting the C-terminus of the heavy chain variable region (V(L)) domain to the N-terminus of the light chain variable region (V(L)) domain . A 23-residue peptide segment, containing a c-myc marker for immunochemical detection of the scFv and hexahistidine tag to facilitate its purification, was added C-terminal to the V(L) domain . The scFv mimics the antibody in binding to the native antigen in the form of recombinant hepatitis B surface antigen (HBsAg) particles as well as to peptide fragments carrying the viral epitope. Anticancer Res, 1998 Nov-Dec, 18(6A), 4311 - 5 A humanized single-chain Fv fragment with high targeting potential against human malignant gliomas; Ohtomo T et al.; A humanized ONS-M21 antibody (hM21) against human medulloblastoma and glioma cells was engineered as a single-chain Fv fragment (scFv), and its ability to internalize into tumor cells was evaluated by conjugation with ricin A . The scFv of hM21 (schM21) was easily purified from E.coli by one-step affinity column chromatography . Purified schM21 bound to a medulloblastoma ONS-76 cell with almost equal antigen-binding activity of hM21-Fab fragment . Furthermore, the schM21-ricin A conjugate inhibited the growth of ONS-76 cells, but not that of antigen-negative hepatoma HuH-7 cells, suggesting that the schM21 can be internalized after binding to antigen-positive cells . Thus, schM21 could be expected to act as a novel carrier of diagnostic and therapeutic agents for brain tumors. Izv Akad Nauk Ser Biol, 1998 Nov-Dec, (6), 670 - 7 {Introduction and long-term storage of recombinant luminescent Escherichia coli strain Z905 in laboratory water microecosystems}; Popova LIu et al.; We studied preservation of recombinant Escherichia coli strain Z905 (AprLux+) in liquid microecosystems (LME) after the introduction . E . coli cells were shown to remain viable and preserve the ability to express the cloned lux genes for a long time (more than a year) in LME . The majority of the clones have reduced efficiency of the expression due to either changed regulation of the lux operon or decreased number of copies of the plasmid . These mechanisms could be realized either independently or simultaneously depending on LME conditions . We have exposed the major factors affecting the metabolic activity of the E . coli strain Z905 (AprLux+) introduced into model ecosystems and the level of expression of the cloned genes. Plant Cell Physiol, 1998 Nov, 39(11), 1145 - 55 Isolation and characterization of a cDNA clone of UDP-galactose: flavonoid 3-O-galactosyltransferase (UF3GaT) expressed in Vigna mungo seedlings; Mato M et al.; Four cDNA clones were isolated from Vigna mungo seedlings by the screening with cDNA encoding UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) of Antirrhinum majus as a probe; the product of the gene corresponding to one cDNA was more highly expressed in the first simple leaves than in stems . Nucleotide sequence analysis revealed 1,691 bp (including 326 bp non-reading) containing an open reading frame of 455 amino acids . The deduced amino acid sequence showed 42% and 23% identity with those of A . majus UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petunia hybrida UDP-rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase (RT), respectively . One region of the cDNA (amino acids 325 to 387) showed similarity to ceramide UDP-galactosyltransferases of mice, rats and humans . A crude extract from Escherichia coli, in which the protein was expressed from the cDNA, showed high UF3GaT activity but low UF3GT activity, and was similar in K(m), optimal pH and substrate specificity to UF3GaT from V . mungo . We conclude that we have obtained UDP-galactose:flavonoid 3-O-galactosyltransferase (UF3GaT) cDNA from V . mungo. Mikrobiologiia, 1998 Sep-Oct, 67(5), 601 - 6 {Role of putrescine and potassium transport in regulating the topological state of DNA during adaptation of Escherichia coli to temperature stress}; Tkachenko AG et al.; The effect of a temperature increase to 52 degrees C or the addition of ethanol (6%) to an exponential Escherichia coli culture on putrescine and potassium transport was studied . The first stage of heat shock was accompanied by a decrease in the extent of DNA supercoiling, due to the dissociation of the putrescine-DNA complex . The loss of potassium ions at this phase produced a synergistic effect . The second phase of the heat shock was characterized by a reversal in the direction of putrescine and potassium transport, which was accompanied by restoration of the prestress extent of DNA supercoiling . An increase in the ATP pool and cell energy charge resulting from the uncoupling of the energy metabolism and synthetic processes also played an important role in the restoration of the DNA initial topology at the second phase of the heat shock via the activation of the energy-dependent gyrase or the heat shock protein DnaK . A mechanism is suggested that explains the involvement of putrescine in the regulation of DNA topology, which is a universal regulator of gene expression under stress, heat shock in particular. Mikrobiologiia, 1998 Sep-Oct, 67(5), 594 - 600 {Hydrogen peroxide modulates intracellular levels of thiols and potassium in Escherichia coli cells}; Smirnova GV et al.; Exposure of growing Escherichia coli K12 cells to 2.0-11.0 mM H2O2 resulted in an increase in the intracellular level of low-molecular-weight thiols (LMWT), whereas exposure to 25 mM H2O2 resulted in its decrease . An inverse correlation between levels of LMWT and potassium was revealed . The treatment of E . coli delta oxyR cells, which are incapable of the adaptive response to H2O2, with 10 mM H2O2 caused a decrease in the LMWT level . In E . coli oxyR2 cells, which constitutively express oxyR-controlled proteins, the same treatment caused a 20% increase in the LMWT level . In response to treatment with the oxidant, delta oxyR mutants lost two times more potassium than wild-type cells (oxyR+) . A time course study of the levels of LMWT and potassium in mutants with an affected katG gene, which encodes the HPI catalase and is under the control of oxyR, showed that oxyR may regulate LMWT and potassium levels indirectly, through the regulation of catalase activity . A relationship between catalase activity and the LMWT level was revealed in hydrogen peroxide-treated E . coli cells. Mol Reprod Dev, 1999 Feb, 52(2), 216 - 24 Expression of recombinant mouse sperm protein sp56 and assessment of its potential for use as an antigen in an immunocontraceptive vaccine; Hardy CM et al.; Recombinant mouse sp56 protein was produced for testing as an antigen in an immunocontraceptive vaccine . The coding sequence for the mature sp56 protein was cloned into the bacterial expression system pFLAG using a PCR-based method on mouse testis cDNA . Polyclonal antisera were raised in mice against affinity purified recombinant sp56 fusion protein (sp56FLAG) or an artificial sp56 peptide fused to a carrier protein (KLH) and shown to cross-react to a protein band of 75 kD in detergent extracts of mouse sperm by Western immunoblot analysis under reducing conditions . The antisera to sp56FLAG also immunolocalized over the entire acrosome of mouse sperm . Female BALB/c mice were immunized intraperitoneally with sp56FLAG in a fertility trial with 20 microg sp56FLAG in Freund's Complete Adjuvant and boosted three to five times with 20 microg sp56FLAG in Freund's Incomplete Adjuvant . Litter sizes of sp56FLAG-treated mice were significantly smaller than control-treated animals after five boosts. J Biochem Mol Toxicol, 1999, 13(2), 63 - 9 Activities of conjugating and antioxidant enzymes following endotoxin exposure; Watson AM et al.; Endotoxin exposure elicits various responses in mammals including the acute phase response that has been shown to cause changes in the activity of several forms of cytochrome P450s and other enzymes . Therefore, the hepatic conjugating enzyme, glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UDPGT), the antioxidant enzymes, glutathione peroxidase (GSHPx), catalase, and superoxide dismutase (SOD), as well as lipid peroxidation were investigated following the administration of endotoxin to male Sprague-Dawley rats (8 mg/kg body weight) . Rats were euthanized at various times following endotoxin administration and the livers removed and processed to assess various enzyme activities . Glutathione S-transferase, UDPGT, and GSHPx activity showed statistically significant decreases after 24 hours and remained lower than controls for the duration of the study . Decreases in total SOD and catalase activities were seen at 24, 48, and 72 hours following endotoxin administration; however, only catalase activity showed statistically significant differences between control and treated samples at those time points, and total SOD activity showed a statistically significant decrease at 24 hours . No statistically significant changes were seen in the level of lipid peroxidation in the liver microsomes from endotoxin-treated animals . Changes in the conjugative enzymes and the free-radical scavenging enzymes following endotoxin exposure may alter the host's metabolism and response to free radicals. J Biol Chem, 1999 Jan 22, 274(4), 2263 - 70 Organization of open complexes at Escherichia coli promoters . Location of promoter DNA sites close to region 2.5 of the sigma70 subunit of RNA polymerase; Bown JA et al.; A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of sigma70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters . In this study we have engineered sigma70 to introduce a unique cysteine residue at a number of positions in region 2.5 . Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA.Fe (FeBABE) . RNA polymerase core reconstituted with tagged sigma derivatives was shown to be transcriptionally active . Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex . Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the -10 hexamer . This positioning is independent of promoter sequence . A model for the interaction of this region of sigma with promoter DNA is discussed. J Biol Chem, 1999 Jan 22, 274(4), 2255 - 62 Secondary structure mapping of an RNA ligand that has high affinity for the MetJ repressor protein and interference modification analysis of the protein-RNA complex; McGregor A et al.; The secondary structure of an RNA aptamer, which has a high affinity for the Escherichia coli MetJ repressor protein, has been mapped using ribonucleases and with diethyl pyrocarbonate . The RNA ligand is composed of a stem-loop with a highly structured internal loop . Interference modification showed that the bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ . Most of the terminal stem-loop could be removed with little effect on the binding . Ethylation interference suggests that none of the phosphate groups are absolutely essential for tight binding . The data suggest that the MetJ binding site on the aptamer is distinct from that of the natural DNA target, the 8-base pair Met box. J Biol Chem, 1999 Jan 22, 274(4), 2045 - 52 An additional electrostatic interaction between adrenodoxin and P450c27 (CYP27A1) results in tighter binding than between adrenodoxin and p450scc (CYP11A1); Pikuleva IA et al.; Mitochondrial cytochrome P450c27 (product of the CYP27A1 gene) is found to have significantly higher affinity for the common redox partner adrenodoxin than another mitochondrial P450, P450scc (product of the CYP11A1 gene) . To investigate the basis of the approximately 30-fold difference in adrenodoxin binding, two sets of P450c27 mutants were generated, expressed in Escherichia coli, and purified . Mutations of one set were within the putative adrenodoxin-binding site containing conserved lysine residues also crucial in P450scc for binding adrenodoxin . The second set included mutations within a sequence aligning with the "meander region" of P450BM-3 proposed to be a site of redox-partner interactions in P450s (Hasemann, C . A., Kurumbail, R . G., Boddupalli, S . S., Peterson, J . A., and Deisenhofer, J . (1995) Structure 3, 41-62) . Mutation of the P450c27 conserved lysines (K354A and K358A) led to a approximately 20-fold increase in apparent Ks for adrenodoxin, confirming that these two positively charged residues conserved in mitochondrial P450s are important for adrenodoxin binding . Mutation of Arg-418, conserved in the CYP27A1 family, to serine also decreased the affinity for adrenodoxin approximately 20-fold . This residue is predicted to be located in the meander region . A triple K354A/K358A/R418S mutation profoundly reduced adrenodoxin binding . Thus, in contrast to P450scc, where mutation of the two conserved positively charged residues results in virtually complete inhibition of adrenodoxin binding, in P450c27 there are three of such residues (Lys-354, Lys-358, and Arg-418) important for adrenodoxin interaction. J Biol Chem, 1999 Jan 22, 274(4), 1942 - 8 Transcriptional inhibition of the operon for the spermidine uptake system by the substrate-binding protein PotD; Antognoni F et al.; Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes . Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually found in the periplasm, and the inhibitory effect of PotD was increased by spermidine . Transcription was not affected by bovine serum albumin, PotA, or PotF, suggesting that the effects of PotD are specific to the PotD protein . In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1:500 of template DNA:PotD . It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene . Binding of PotD to the downstream site was stimulated by spermidine . Overexpression of PotD in Escherichia coli DH5alpha inhibited the uptake of spermidine, the synthesis of PotABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites . In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts . Our results indicate that PotD precursor can also inhibit spermidine transport . The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins . Thr-35 and Ser-85 of PotD were found to be important for this interaction . These results suggest that transcription of the spermidine transport (potABCD) operon is inhibited in vivo by PotD precursor rather than PotD through its binding to two regions close to the transcriptional initiation site of the operon. Biochemistry, 1999 Jan 5, 38(1), 496 - 508 Effects of hydration, ion release, and excluded volume on the melting of triplex and duplex DNA; Spink CH et al.; The stability of DNA duplex and triplex structures not only depends on molecular forces such as base pairing or tripling or electrostatic interactions but also is sensitive to its aqueous environment . This paper presents data on the melting of Escherichia coli and poly(dA).poly(dT) duplex DNA and on the poly(dT).poly(dA) . poly(dT) triplex in a variety of media to assess the contributions from the osmotic status and salt content of the media . The effects of volume exclusion on the stability of the DNA structures are also studied . From thermal transition measurements in the presence of low-molecular weight osmotic stressors, the number of water molecules released upon melting is found to be four waters per base pair for duplex melting and one water for the conversion of triplex to single-strand and duplex . The effects of Na+ counterion binding are also determined in ethylene glycol solutions so that the variation of counterion binding with water activity is evaluated . The data show that there is a modest decrease in the extent of counterion binding for both duplex and triplex as water activity decreases . Finally, using larger polyethylene glycol cosolutes, the effects on melting of volume exclusion by the solutes are assessed, and the results correlated with simple geometric models for the excluded volume . These results point out that DNA stability is sensitive to important conditions in the environment of the duplex or triplex, and thus, conformation and reactivity can be influenced by these solution conditions. Biochemistry, 1999 Jan 5, 38(1), 432 - 41 EPR mapping of interactions between spin-labeled variants of human carbonic anhydrase II and GroEL: evidence for increased flexibility of the hydrophobic core by the interaction; Persson M et al.; Human carbonic anhydrase II (HCA II) interacts weakly with GroEL at room temperature . To further investigate this interaction we used electron paramagnetic resonance (EPR) spectroscopy to study HCA II cysteine mutants spin-labeled at selected positions . From our results it is evident that protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements . HCA II needs to be unfolded to about the same extent as a GuHCl-induced molten-globule intermediate of the enzyme to interact with GroEL . The interaction with GroEL includes interactions with outer parts of the HCA II molecule, such as peripheral beta-strands and the N-terminal domain, which have previously been shown to be rather unstable . As a result of the interaction, the rigid and compact hydrophobic core exhibits higher flexibility than in the molten globule, which is likely to facilitate rearrangements of misfolded structure during the folding process . The degree of binding to GroEL and accompanying inactivation of the enzyme depend on the stability of the HCA II variant, and nonspecific hydrophobic interactions appear to be most important in stabilizing the GroEL-substrate complex. Biochemistry, 1999 Jan 5, 38(1), 415 - 22 Catalytic properties of hybrid complexes of the NAD(H)-binding and NADP(H)-binding domains of the proton-translocating transhydrogenases from Escherichia coli and Rhodospirillum rubrum; Fjellstrom O et al.; Transhydrogenase couples reversible hydride transfer from NADH to NADP+ to proton translocation across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria . The enzyme is composed of three parts . Domain I (dI) and domain III (dIII) are water soluble and contain the binding sites for NAD(H) and NADP(H), respectively; domain II (dII) spans the membrane . In the present investigation, dI from Rhodospirillum rubrum (rrI) and Escherichia coli (ecI), and dIII from R . rubrum (rrIII) and E . coli (ecIII) were overexpressed in E . coli and subsequently purified . Also, a preparation of a partially degraded E . coli transhydrogenase (ecbeta) was examined . Catalytic activities were analyzed in various dI+dIII and dI+ecbeta combinations . The abilities of the different dI+dIII combinations to catalyze cyclic transhydrogenation, i.e., the reduction of AcPyAD+ by NADH mediated via tightly bound NADP(H) in dIII, varied in the order: rrI+ecIII approximately rrI+rrIII > rrI+ecbeta >> ecI+ecIII; no measurable activities for ecI+rrIII and ecI+ecbeta were detected . Thus, rrI has a much greater apparent affinity than ecI for ecIII or rrIII or ecbeta . The pH dependences of the cyclic reaction seem to be determined by scalar protonation events on dI, both in rrI+rrIII and ecI+ecIII mixtures as well as in the wild-type R . rubrum and possibly in the E . coli enzyme . Higher reverse activities for rrI+ecbeta than for rrI+ecIII confirmed the regulatory role of dII for the association and dissociation rates of NADP(H). Biochemistry, 1999 Jan 5, 38(1), 303 - 10 N2-hydroxyguanosine 5'-monophosphate is a time-dependent inhibitor of Escherichia coli guanosine monophosphate synthetase; Deras ML et al.; In contrast to several other glutamine amidotransferases including asparagine synthetase, cytidine 5'-triphosphate (CTP) synthetase, carbamoyl phosphate synthetase, and phosphoribosyl pyrophosphate (PRPP) amidotransferase, guanosine monophosphate synthetase (GMPS) will not utilize hydroxylamine as an alternative nitrogen source . Instead, the enzyme is inhibited by an unknown mechanism . One untested hypothesis was that hydroxylamine serves as a substrate and intercepts a xanthosine 5'-monophosphate- (XMP-) adenylate intermediate in the enzyme active site . The nucleotide product of this substitution reaction would be N2-hydroxyguanosine 5'-monophosphate (N2-OH-GMP, 2) . Here we describe the chemoenzymatic preparation of 2, via the nucleotide 2-fluoroinosine 5'-monophosphate (F-IMP, 5), and characterization of both these compounds as inhibitors of Escherichia coli GMPS . F-IMP was conceived as an electronic mimic of a reactive intermediate in the GMPS reaction but was found to bind weakly to the enzyme (IC50 > 2 mM) . In contrast, N2-OH-GMP shows time-dependent inhibition and is competitive with respect to XMP (Ki = 92 nM), representing the first example of a compound that displays these kinetic properties with GMPS . The mechanism of inhibition is proposed to occur via formation of a ternary E.ATP.2 complex, followed by a rate-determining isomerization to a higher affinity complex that has a t1/2 =7.5 min . The contrast in inhibitory activity for 2-substituted purines with GMPS formulates a basis for future inhibitor design . In addition, these results complement recent structural studies of GMPS and implicate the formation of the XMP-adenylate intermediate inducing a probable conformational change that stimulates the hydrolysis of glutamine. Biochemistry, 1999 Jan 5, 38(1), 296 - 302 Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase; Shuttleworth WA et al.; The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported . Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A . Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP) . The K411R and D242A mutants bind S3P only in the presence of GLP . The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported . Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions . These residues, while not essential for catalysis, are most likely important for substrate binding . All of the mutants are shown to be correctly folded by NMR spectroscopy. Biochemistry, 1999 Jan 5, 38(1), 218 - 25 Structural analysis of ternary complexes of Escherichia coli RNA polymerase: ribonuclease footprinting of the nascent RNA in complexes; Milan S et al.; Ternary complexes of RNA polymerase containing the DNA template and nascent RNA are the intermediates in transcript elongation in all cells . We have footprinted the RNA transcript with single-strand-specific ribonucleases in ternary complexes of Escherichia coli RNA polymerase . When complexes are treated with elevated levels of ribonucleases A and T1, the nascent transcript can be cleaved to within 3-4 nucleotides of the 3'-terminus . Ternary complexes containing ribonuclease-cleaved transcripts as short as 3 nucleotides remain stable and active, ensuring that the cleavage occurred within an active ternary complex . However, cleavage by ribonuclease I is restricted, and gives a limited digest product of about 16 nt . At lower concentrations of ribonuclease T1, two regions of partial protection are seen . The first region extends through the first 15-16 nucleotides from the 3'-OH terminus; the second region extends from position 30 out to position 45 . We interpret these regions of partial protection as defining two RNA product binding sites on the RNA polymerase that bind the product to the enzyme during elongation . Our results rule out the existence of a stable RNA-DNA hybrid in these ternary complexes of greater than 3 base pairs in length. Biochemistry, 1999 Jan 5, 38(1), 207 - 17 Modified nucleosides in the first positions of the anticodons of tRNA(Leu)4 and tRNA(Leu)5 from Escherichia coli; Horie N et al.; Minor leucine tRNA species, tRNA(Leu)4 and tRNA(Leu)5, from Escherichia coli B have been reported to recognize leucine codons UUA and UUG {Goldman, E., Holmes, W . M., and Hatfield, G . W . (1979) J . Mol . Biol . 129, 567-585} . In the present study, these two tRNA(Leu) species were purified from E . coli A19, and the nucleotide sequences were determined by a post-labeling method . tRNA(Leu)5 was found to correspond to the tRNA gene reported as su degrees6 tRNA {Yoshimura, M., Inokuchi, H., and Ozeki, H . (1984) J . Mol . Biol . 177, 627-644} . The first letter of the anticodon was identified to be 2'-O-methylcytidine (Cm) . tRNA(Leu)4 was identified as the minor leucine tRNA that has been sequenced previously (tRNA(Leu)UUR) {Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J . A . (1980) J . Biol . Chem . 255, 2220-2225} . There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA(Leu)4 . Nucleoside N* was isolated to homogeneity (1 A260 unit) . By 1H NMR spectroscopy, nucleoside N was found to be a 2'-O-methyluridine derivative with a substituent having a -CH2NH2+CH2COO- moiety in position 5 of the uracil ring . On the basis of these NMR analyses together with mass spectrometry, the chemical structure of nucleoside N* was determined as 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um) . Nucleoside N* was thus found to be a novel type of naturally occurring modified uridine . Because of the conformational rigidity of Cm and cmnm5Um in the first position of the anticodon, these tRNA(Leu) species recognize the leucine codons UUA++ and UUG correctly, but never recognize the phenylalanine codons UUU and UUC. Hybridoma, 1998 Dec, 17(6), 535 - 40 Generation of human Fab monoclonal antibodies against preS1 of hepatitis B virus using repertoire cloning; Choi IH et al.; Human monoclonal antibodies (MAbs) have considerable potential in the prevention and treatment of many viral diseases . A combinatorial antibody library of heavy (Fd)- and light-chain genes derived from peripheral blood lymphocytes of a volunteer with high antibody titer to preS1 of HBV was constructed and expressed on the surface of filamentous phages . The library contained 7 x 10(9) independent clones . A phage antibody population from the third panning against preS1 was converted to one expressing soluble Fabs by removal of the g3 sequences from the pComb3 phagemid vector and subsequent transformation into E . coli TG1 cells . Screening of the library led to the identification of two clones, 3DW and 8GW, showing high reactivity toward preS1 . The authenticity of the Fabs was confirmed by immunoblot analysis which yielded approximately 60 and approximately 30 kDa bands under nonreducing and reducing conditions, respectively . The soluble Fabs of 3DW and 8GW exhibited relative affinities of 6 x 10(5) and 8 x 10(6) M(-1), respectively . The sequencing results demonstrate that all Fd sequences belong to subgroup II and all light chain sequences belong to subgroup I . There are differences in CDR length and composition, especially in the FW3 and CDR3 regions of the heavy- and light-chain genes . These human Fab MAbs specific to preS1, generated from a combinatorial library, represent prototypes of passive immunotherapy candidates for viral hepatitis B. Dev Biol Stand, 1998, 96, 97 - 104 Betaseron; Lin L; Betaseron, an analogue of human beta-interferon where serine was genetically engineered to substitute for cysteine at position 17, is produced in E . coli . The molecule is a small polypeptide of 165 amino acids with a single disulphide bond, and is non-glycosylated . The site-specific substitution was made to obtain a product that is more stable upon storage . Similar to native IFN-beta, Betaseron is hydrophobic in nature and has been shown to have the same panel of biological activities which includes antiviral activity against a variety of viruses, inhibition of cell growth, activation of natural killer cells, and binding to interferon receptors on the cell surface . Betaseron has been tested in a wide variety of clinical settings since 1983 . The pivotal trial for the treatment of relapsing-remitting multiple sclerosis began in 1988 . A PLA was filed for this indication in 1992 by Berlex and Chiron, and FDA approval was received in 1993 . Betaseron is synthesized in E . coli and deposited as inclusion bodies . The manufacturing process involves solubilization and reduction of the insoluble protein, followed by purification by organic extraction, cystine oxidation and size exclusion chromatographic steps . The purified Betaseron is formulated with human serum albumin to maintain solubility at neutral pH . The complete primary sequence of Betaseron was verified by amino and carboxy-terminal sequence analysis, peptide mapping, amino acid analysis and fragment analysis after chemical cleavages . Overlapping amino acid sequence information confirmed that the amino acid sequence is the same as predicted by the DNA sequence . The amino-terminal methionine of Betaseron is removed after synthesis in E . coli . An intramolecular disulphide bond between Cys 31 and Cys 141 formed during the manufacturing process is routinely confirmed by peptide map analysis . The purity of Betaseron is assessed using a panel of analytical methods including non-reducing and reducing SDS-PAGE and reversed phase HPLC analysis where minor product-related components can be identified . These minor species were characterized with respect to their biological and biochemical properties, and identified using a variety of approaches including construction of additional, beta-interferon analogs . There is significant redundancy in the release testing of Betaseron . The amount of characterization information available on this relatively simple molecule along with the extensive manufacturing experience would suggest that some redundant testing could be eliminated for this well-characterized protein. Adv Exp Med Biol, 1998, 453, 229 - 34 Structure and function of smooth muscle myosin light chain kinase; Kishi H et al.; Myosin light chain kinase (MLCK) plays a central role in regulating the actin-myosin interaction of smooth muscle . MLCK phosphorylates the light chain of myosin in the presence of Ca2+ and calmodulin (CaM) thereby activating myosin so that it can interact with actin . Besides this kinase activity, MLCK shows i) actin-binding activity that can assemble actin filaments into their bundles and ii) myosin-binding activity that can form myosin filaments . To localize the actin- and myosin-binding activities in the MLCK molecule and to examine their possible role in regulating the actin-myosin interaction, we expressed various fragments of cDNA encoding MLCK in Escherichia coli as recombinant proteins . We found that MLCK consists of an N-terminal actin-binding domain, a central kinase domain, and a C-terminal myosin-binding domain . The Met1-Pro41 sequence is responsible for Ca2+/CaM-sensitive binding to actin . This binding site exerts an inhibitory effect on the actin-myosin interaction only when myosin is phosphorylated . MLCK binds to myosin at the C-terminal domain, the sequence of which is identical to telokin, an abundant myosin-binding protein in smooth muscle cells . This domain itself has no regulatory role in the interaction . However, the interaction was stimulated when this domain was extended to include the sequence known to regulate the activity of the kinase domain . The stimulation was observed only when myosin was unphosphorylated. Nucleic Acids Res, 1999 Feb 1, 27(3), 910 - 1 A novel method for increasing the transformation efficiency of Escherichia coli-application forbacterial artificial chromosome library construction; Zhu H et al.; Bacterial artificial chromosome (BAC) libraries play a pivotal role in genomics studies . A crucial step in BAC library construction is the transformation of Escherichia coli by electroporation . Absolute efficiency (cfu/microgram DNA) is affected by a number of factors including the topological form and treatment of DNA samples . Here we report a simple new protocol using tRNA assisted precipitation that increased transformation efficiency by 70-fold for BAC ligations and up to 400-fold for plasmid ligations . The mechanism may involve altering or stabilizing the topographical form of the DNA molecules. Nucleic Acids Res, 1999 Feb 1, 27(3), 895 - 902 In vitro analysis of processing at the 3'-end of precursors of M1 RNA, the catalytic subunit of Escherichia coli RNase P: multiple pathways and steps for the processing; Kim S et al.; M1 RNA of 377 nucleotides, the catalytic subunit of Escherichia coli RNase P, is produced by a 3' processing reaction from precursor M1 RNA, a major transcript from the rnpB gene . We analyzed products and intermediates generated by the in vitro processing reaction using a 40% ammonium sulfate precipitate of the S30 fraction (ASP-40) and determined their involvement in the processing . From the results we proposed a model of two pathways for 3' processing of M1 RNA . In this model, one pathway (pathway I) involves +385/+386 intermediates and the other pathway (pathway II) does not . The position of the 3'-end of the precursor molecule determined the choice of the pathways . The precursor having the 3'-end of +413 was processed by both pathways while that having the +415 end was processed only by pathway II . The ASP-40 fraction generated processing products (termed +378/+379 RNA) containing one or two more nucleotides at the 3'-end than M1 RNA, regardless of which pathway was used . Therefore, both pathways require the final 3' trimming for complete processing . The endonucleolytic generation of +378/+379 RNA by pathway II was blocked by the rne-3071 mutation, suggesting that this step is carried out by RNase E. Nucleic Acids Res, 1999 Feb 1, 27(3), 848 - 53 A signal encoded in vertebrate DNA that influences nucleosome positioning and alignment; Stein A et al.; Evidence is provided that the nucleotide triplet con-sensus non-T(A/T)G (abbreviated to VWG) influences nucleosome positioning and nucleosome alignment into regular arrays . This triplet consensus has been recently found to exhibit a fairly strong 10 bp periodicity in human DNA, implicating it in anisotropic DNA bendability . It is demonstrated that the experimentally determined preferences for nucleosome positioning in native SV40 chromatin can, to a large extent, be pre-dicted simply by counting the occurrences of the period-10 VWG consensus . Nucleosomes tend to form in regions of the SV40 genome that contain high counts of period-10 VWG and/or avoid regions with low counts . In contrast, periodic occurrences of the dinucleotides AA/TT, implicated in the rotational positioning of DNA in nucleosomes, did not correlate with the preferred nucleosome locations in SV40 chromatin . Periodic occurrences of AA did correlate with preferred nucleosome locations in a region of SV40 DNA where VWG occurrences are low . Regular oscillations in period-10 VWG counts with a dinucleosome period were found in vertebrate DNA regions that aligned nucleosomes into regular arrays in vitro in the presence of linker histone . Escherichia coli and plasmid DNA, which fail to align nucleosomes in vitro, lacked these regular VWG oscillations. Nucleic Acids Res, 1999 Feb 1, 27(3), 817 - 21 Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway; Costa M et al.; Human DNA helicase VIII (HDH VIII) was isolated in the course of a systematic study of the DNA unwinding enzymes present in human cells . From a HeLa cell nuclear extract a protein with an Mrof 68 kDa in SDS-PAGE was isolated, characterised and micro-sequenced . The enzyme shows ATP- and Mg2+-dependent activity is not stimulated by RPA, prefers partially unwound 3'-tailed substrates and moves along the bound strand in the 5' to 3' direction . HDH VIII can also unwind partial RNA/DNA and RNA/RNA duplexes . Microsequencing of the polypeptide showed that this enzyme corresponds to G3BP, an element of the Ras pathway which binds specifically to the GTPase-activating protein . HDH VIII/G3BP is analogous to the heterogeneous nuclear ribonucleoproteins and contains a sequence rich in RGG boxes similar to the C-terminal domain of HDH IV/nucleolin, another DNA and RNA helicase. Nucleic Acids Res, 1999 Feb 1, 27(3), 771 - 7 Apurinic/apyrimidinic endonuclease genes from the trypanosomatidae leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli; Perez J et al.; Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity . We have isolated cDNAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capable of complementing the deficiency of exonuclease III and dUTPase in the Escherichia coli mutant BW286 . This double mutant is non-viable at 37 degreesC due to an accumulation of non-repaired sites following excision of uracil from DNA . The genes were expressed as beta-galactosidase-AP endonuclease fusion proteins and as such are active in repair of AP sites in E . coli . The Trypanosoma and Leishmania sequences have unique N-termini containing sequences that correspond to probable nuclear transport signals, while the C-terminal domains exhibit pronounced similarity to exonuclease III . The L.major gene was overexpressed as a histidine-tagged protein and recombinant enzyme exhibited endonuclease activity on AP DNA in vitro . Furthermore, expression of the enzymes in AP endonuclease-deficient E.coli mutants conferred significant resistance to killing by methylmethane sulphonate and peroxides . This study constitutes one of the first descriptions of DNA repair enzymes in these pathogenic organisms where oxidative stress is an important mechanism of both drug-mediated and intracellular killing. EMBO J, 1999 Jan 15, 18(2), 470 - 9 Structure-function analysis of the Z-DNA-binding domain Zalpha of dsRNA adenosine deaminase type I reveals similarity to the (alpha + beta) family of helix-turn-helix proteins; Schade M et al.; RNA editing alters pre-mRNA through site-selective adenosine deamination, which results in codon changes that lead to the production of novel proteins . An enzyme that catalyzes this reaction, double-stranded RNA adenosine deaminase (ADAR1), contains two N-terminal Z-DNA-binding motifs, Zalpha and Zbeta, the function of which is as yet unknown . In this study, multidimensional NMR spectroscopy was used to show that the topology of Zalpha is alpha1beta1alpha2alpha3beta2beta3 . Long-range NOEs indicate that beta1 and beta3 interact with each other . Site-directed mutagenesis was used to identify residues in alpha3, beta3 and the loop connecting beta2 to beta3 that affect Z-DNA binding . Also identified were 11 hydrophobic residues that are essential for protein stability . Comparison with known structures reveals some similarity between Zalpha and (alpha + beta) helix-turn-helix proteins, such as histone 5 and the family of hepatocyte nuclear factor-3 winged-helix-turn-helix transcription factors . Taken together, the structural and functional data suggest that recognition of Z-DNA by Zalpha involves residues in both the alpha3 helix and the C-terminal beta-sheet. EMBO J, 1999 Jan 15, 18(2), 363 - 74 A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases; Meyer M et al.; The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival . In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E . VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV) . They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family . VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer . VEGF-E and VEGF-A were found to possess similar bioactivities, i.e . both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo . Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1) . VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2 . These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis. EMBO J, 1999 Jan 15, 18(2), 306 - 12 Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase; Myllyharju J et al.; Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens . The {alpha(I)}2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the {alpha(II)}2beta2 type II enzyme is not . We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids . Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer . A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10 . This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module . Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase . Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase EMBO J, 1999 Jan 15, 18(2), 297 - 305 The structure of a PKD domain from polycystin-1: implications for polycystic kidney disease; Bycroft M et al.; Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene . The PKD1 gene codes for a large cell-surface glycoprotein, polycystin-1, of unknown function, which, based on its predicted domain structure, may be involved in protein-protein and protein-carbohydrate interactions . Approximately 30% of polycystin-1 consists of 16 copies of a novel protein module called the PKD domain . Here we show that this domain has a beta-sandwich fold . Although this fold is common to a number of cell-surface modules, the PKD domain represents a distinct protein family . The tenth PKD domain of human and Fugu polycystin-1 show extensive conservation of surface residues suggesting that this region could be a ligand-binding site . This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.
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