Escherichia coli

J Pept Res, 1998 Feb, 51(2), 103 - 9
Interaction of alpha-helical peptides with phospholipid membrane: effects of chain length and hydrophobicity of peptides; Ohmori N et al.; To investigate the interaction of amphiphilic alpha-helical peptides with phospholipid membranes, we synthesized Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4{3}) and three derivatives, in which the chain length and the size of the hydrophobic region of the peptides were different from each other . These peptides formed an alpha-helical structure in the presence of vesicles . In the membrane-perturbation measurement, only 43 showed a strong membrane-perturbation activity below phase-transition temperature (25 degrees C), but above phase-transition temperature (50 degrees C), most peptides showed similar strong activities . On the other hand, in membrane-fusion measurement the long peptides, e.g., Ac-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)3-NHCH3, had strong activities at low peptide concentrations at 25 degrees C . The present study indicated a parallel relationship did not always exist between membrane fusion and perturbation caused by peptides.

J Bacteriol, 1998 Mar, 180(6), 1603 - 6
Trehalose is not relevant for in vivo activity of sigmaS-containing RNA polymerase in Escherichia coli; Germer J et al.; The sigmaS- and sigma70-associated forms of RNA polymerase core enzyme (E) of Escherichia coli have very similar promoter recognition specificities in vitro . Nevertheless, the in vivo expression of many stress response genes is strongly dependent on sigmaS . Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of EsigmaS and thereby contributes to promoter selectivity (S . Kusano and A . Ishihama, J . Bacteriol . 179:3649-3654, 1997) . However, we demonstrate here that a trehalose-free otsA mutant exhibits growth phase-related and osmotic induction of various sigmaS-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced) . We conclude that in vivo trehalose does not play a role in the expression of sigmaS-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.

J Bacteriol, 1998 Mar, 180(6), 1567 - 9
Hybrid Bordetella pertussis-Escherichia coli RNA polymerases: selectivity of promoter activation; Steffen P et al.; We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system . Virulence-associated genes of B . pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E . coli lac promoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E . coli alpha subunit . This suggests that activator contact sites involve a high degree of selectivity.

J Bacteriol, 1998 Mar, 180(6), 1563 - 6
In vivo protein interactions within the Escherichia coli DNA polymerase III core; Jonczyk P et al.; The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies . Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication . It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia coli could be essential for maintaining the optimal fidelity of DNA replication (H . Maki and A . Kornberg, Proc . Natl . Acad . Sci . USA 84:4389-4392, 1987) . We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (alpha subunit {possessing DNA polymerase activity}) and DnaQ (epsilon subunit {possessing 3'-->5' exonucleolytic proofreading activity}) proteins . In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions . In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (theta subunit) proteins . We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype . Our results show that the mutD5 and dnaQ926 mutations do not affect the epsilon subunit-alpha subunit and epsilon subunit-theta subunit interactions . However, the dnaQ49 mutation greatly reduces the strength of interaction of the epsilon subunit with both the alpha and the theta subunits . Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the epsilon protein, which leads to altered interactions within the Pol III core.

J Bacteriol, 1998 Mar, 180(6), 1525 - 32
Activation of Escherichia coli rRNA transcription by FIS during a growth cycle; Appleman JA et al.; rRNA transcription in Escherichia coli is activated by the FIS protein, which binds upstream of rrnp1 promoters and interacts directly with RNA polymerase . Analysis of the contribution of FIS to rrn transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of fis mutations . In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the rrnBp1 FIS sites correlates with cellular levels of FIS . We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made from rrnBp1), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels . FIS activates transcription throughout exponential growth at low culture density, but rrnp1 transcription increases independently of FIS immediately following upshift, before FIS accumulates . These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription from rrnp1 promoters during steady-state growth.

J Bacteriol, 1998 Mar, 180(6), 1425 - 30
A conserved histidine is essential for glycerolipid acyltransferase catalysis; Heath RJ et al.; Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration . We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli . Both the PlsB{H306A} and Aas{H36A} mutants lacked acyltransferase activity . However, the Aas{H36A} mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution . The invariant aspartic acid residue in the HX4D pattern was also important . The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity . Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB{D311A} mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases . These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.

J Bacteriol, 1998 Mar, 180(6), 1411 - 7
The LysR-type transcriptional regulator CbbR controlling autotrophic CO2 fixation by Xanthobacter flavus is an NADPH sensor; van Keulen G et al.; Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy . Maximal induction of the cbb and gap-pgk operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate . The LysR-type transcriptional regulator CbbR regulates the expression of the cbb and gap-pgk operons, but it is unknown to what cellular signal CbbR responds . In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in Escherichia coli and subsequently purified to homogeneity . CbbR of X . flavus is a dimer of 36-kDa subunits . DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located . The addition of 200 microM NADPH, but not NADH, resulted in a threefold increase in DNA binding . The apparent K(dNADPH) of CbbR was determined to be 75 microM . By using circular permutated DNA fragments, it was shown that CbbR introduces a 64 degree bend in the DNA . The presence of NADPH in the DNA-bending assay resulted in a relaxation of the DNA bend by 9 degree . From the results of these in vitro experiments, we conclude that CbbR responds to NADPH . The in vivo regulation of the cbb and gap-pgk operons may therefore be regulated by the intracellular concentration of NADPH.

J Bacteriol, 1998 Mar, 180(6), 1396 - 401
Identification of a sequence motif that confers SecB dependence on a SecB-independent secretory protein in vivo; Kim J et al.; SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in Escherichia coli . However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains . The characteristics which confer SecB dependence on some proteins but not others have not been defined . To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA) . The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo . Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence . We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB . Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif . The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex . Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus.

J Bacteriol, 1998 Mar, 180(6), 1389 - 95
An rne-1 pnp-7 double mutation suppresses the temperature-sensitive defect of lacZ gene expression in a divE mutant; Aiso T et al.; A divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, exhibits differential loss of the synthesis of certain proteins, such as beta-galactosidase and succinate dehydrogenase, at nonpermissive temperatures . In Escherichia coli, the UCA codon is recognized only by tRNA1Ser . Several genes containing UCA codons are normally expressed after a temperature shift to 42 degrees C in the divE mutant . Therefore, it is unlikely that the defect in protein synthesis at 42 degrees C is simply caused by a defect in the decoding function of the mutant tRNA1Ser . In this study, we sought to determine the cause of the defect in lacZ gene expression in the divE mutant . It has also been shown that the defect in lacZ gene expression is accompanied by a decrease in the amount of lacZ mRNA . To examine whether inactivation of mRNA degradation pathways restores the defect in lacZ gene expression, we constructed divE mutants containing rne-1, rnb-500, and pnp-7 mutations in various combinations . We found that the defect was almost completely restored by introducing an rne-1 pnp-7 double mutation into the divE mutant . Northern hybridization analysis showed that the rne-1 mutation stabilized lacZ mRNA, whereas the pnp-7 mutation stabilized mutant tRNA1Ser, at 44 degrees C . We present a mechanism that may explain these results.

J Bacteriol, 1998 Mar, 180(6), 1368 - 74
Contribution of the disulfide bond of the A subunit to the action of Escherichia coli heat-labile enterotoxin; Okamoto K et al.; Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits . These subunits oligomerize into an assembled holotoxin within the periplasm . Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus . This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm . However, it is not known whether other regions of the A subunit contribute to the assembly . The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199 . It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein . We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit . We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes . The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly . A reconstitution experiment in vitro supported the notion . Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs . These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.

FEBS Lett, 1998 Feb 27, 423(3), 347 - 50
Conformational independence of N- and C-domains in ribosomal protein L7/L12 and in the complex with protein L10; Bocharov EV et al.; Isolated N- (1-37) and C-terminal (47-120) fragments of L7 protein, and pentameric (L7)4L10 complex were studied by NMR spectroscopy in solution . The results indicate that the dimer state of the 1-37 fragment with a helical hairpin conformation is identical to the N-terminal structure of the intact L7 dimer . The C-terminal domain of the L7 protein does not participate in (L7)4L10 complex formation . The overall motions of the L7 C-domains are essentially independent both in the L7 dimer and in the (L7)4L10 complex . Conformational motions on a millisecond time scale are detected in the (L7)4L10 complex . The possible relevance of these motions to the biological function of L7/L12 is discussed.

Mol Microbiol, 1998 Feb, 27(4), 787 - 95
Regulation of RssB-dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator-controlled process; Bouche S et al.; Sigma(S) (RpoS) is a highly unstable global regulatory protein in Escherichia coli, whose degradation is inhibited by various stress signals, such as carbon starvation, high osmolarity and heat shock . As a consequence, these stresses result in the induction of sigma(S)-regulated stress-protective proteins . The two-component-type response regulator, RssB, is essential for the rapid proteolysis of sigma(S) and is probably involved in the transduction of some of these stress signals . Acetyl phosphate can be used as a phosphodonor for the phosphorylation of various response regulators in vitro and, in the absence of the cognate sensor kinases, acetyl phosphate can also modulate the activities of several response regulators in vivo . Here, we demonstrate increased in vivo half-lives of sigma(S) and the RpoS742::LacZ hybrid protein (also a substrate for RssB-dependent proteolysis) in acetyl phosphate-free (pta-ackA) deletion mutants, even though no sensor kinase was eliminated . The in vivo data indicate that acetyl phosphate acts through the response regulator, RssB . In vitro, efficient phosphotransfer from radiolabelled acetyl phosphate to the Asp-58 residue of RssB (the expected site of phosphorylation in the RssB receiver domain) was observed . Via such phosphorylation, acetyl phosphate may thus modulate RssB activity even in an otherwise wild-type background . While acetyl phosphate is not essential for the transduction of specific environmental stress signals, it could play the role of a modulator of RssB-dependent proteolysis that responds to the metabolic status of the cells reflected in the highly variable cellular acetyl phosphate concentration.

Mol Microbiol, 1998 Feb, 27(4), 751 - 61
Leucine alters the interaction of the leucine-responsive regulatory protein (Lrp) with the fim switch to stimulate site-specific recombination in Escherichia coli; Roesch PL et al.; The leucine-responsive regulatory protein (Lrp) is a global regulator that controls the expression of numerous operons in Escherichia coli . Lrp can act as a repressor or as an activator of transcription with its effects being potentiated, repressed or unaffected by the presence of exogenous leucine . The phase variation of type 1 fimbria in E . coli provides a unique system in which to investigate the effects of leucine on Lrp, as it is the only known example in which Lrp is a positive regulator and leucine potentiates this effect . Previous studies determined that Lrp binds with high affinity to two sites within the fim switch (fim sites 1 and 2), and binding to these sites stimulates recombination . Here, it is shown that, even though leucine stimulates the fim switch in vivo, it nevertheless causes a slight decrease in Lrp binding to the fim switch in vitro . These contradictory results are explicable by the finding that Lrp binding to a third region adjacent to fim sites 1 and 2 inhibits recombination . According to this model, leucine stimulates recombination by selectively disrupting Lrp binding to this newly characterized region, while having little or no effect on Lrp binding to fim sites 1 and 2.

Mol Microbiol, 1998 Feb, 27(4), 739 - 50
Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli; Britton RA et al.; Era is a low-molecular-weight GTPase essential for Escherichia coli viability . The gene encoding Era is found in the rnc operon, and the synthesis of both RNase III and Era increases with growth rate . Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild-type Era become arrested in the cell cycle at the predivisional two-cell stage . The partially defective Era GTPase mutation (era1) suppresses several temperature-sensitive lethal alleles that affect chromosome replication and chromosome partitioning but not cell division . Our results suggest that Era plays an important role in cell cycle progression at a specific point in the cycle, after chromosome partitioning but before cytokinesis . Possible functions for Era in cell cycle progression and the initiation of cell division are discussed.

Protein Sci, 1998 Jan, 7(1), 211 - 5
Cloning, overexpression, purification, and spectroscopic characterization of human S100P; Gribenko A et al.; The calcium-binding protein S100P has been found to be associated with human prostate cancer . We have overexpressed S100P in Escherichia coli using a T7 expression system . A rapid two-step procedure for the isolation of overexpressed S100P leads to a preparation of >95% pure protein with a yield of approximately 150 mg per liter of culture . The structural integrity of recombinant S100P was analyzed using CD and fluorescence spectroscopic techniques . The far-UV CD shows that secondary structure of recombinant S100P consists predominantly of a-helical structure . Both near-UV CD and tyrosine fluorescence spectra show that aromatic residues are involved in the formation of a specific, well packed structure, indicating that the recombinant S100P protein adopts a compact folded conformation . Ca2+ has a profound effect on S100P structure . Near-UV CD and fluorescence intensity of both internal (tyrosine) and external (ANS) probes suggest significant structural rearrangements in the tertiary structure of the molecule . The similarity of far-UV CD spectrum of S100P in the presence and in the absence of Ca2+ suggests that Ca2+ binding has only minor effects on secondary structure.

Protein Sci, 1998 Jan, 7(1), 193 - 200
Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy; Vohnik S et al.; The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction . In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values . A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity . pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations . The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin . Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions . The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein . Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation . Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site . We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.

J Cell Biochem, 1998 Apr 1, 69(1), 1 - 12
L7 protein is a coregulator of vitamin D receptor-retinoid X receptor-mediated transactivation; Berghofer-Hochheimer Y et al.; The vitamin D receptor (VDR) heterodimerizes with the retinoid X receptor (RXR) and requires additional protein-protein interactions to regulate the expression of target genes . Using the yeast two-hybrid system, we identified the previously described protein L7, that specifically interacted with the VDR in the presence of vitamin D . Deletion analysis indicated, that the N-terminus of L7, which harbours a basic region leucine zipper like domain, mediated interaction with the VDR . Binding assays with purified GST-L7 demonstrated, that L7 specifically pulled down the VDR, that was either expressed in yeast or endogenously contained in the cell line U937 . Interestingly, L7 inhibited ligand-dependent VDR-RXR heterodimerization, when constitutively expressed in yeast . We also demonstrate that L7 repressed binding of VDR-RXR heterodimers to a vitamin D response element . Surprisingly, L7 recruited RXR to the same response element in the presence of 9-cis retinoic acid . Ligand-dependent protein-protein interaction in the yeast two-hybrid system confirmed, that binding of L7 also was targeted at the RXR . Our data suggest, that protein L7 is a coregulator of VDR-RXR mediated transactivation of genes, that modulates transcriptional activity by interfering with binding of the receptors to genomic enhancer elements.

Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 573 - 7
Functional analysis of conserved histidines in ADP-glucose pyrophosphorylase from Escherichia coli; Hill MA et al.; Two absolutely conserved histidines and a third highly conserved histidine are noted in 11 bacterial and plant ADP-glucose pyrophosphorylases . These histidines were individually mutagenized in the E . coli enzyme to glutamine in order to determine their function . Glutamine mutations at residues 143 and 156 produced functional enzymes in cell extracts with slightly lower than wild-type specific catalytic activities and with same heat stability characteristics of the wild-type enzyme . Substitution of residue 83 with glutamine however produced an enzyme having decreased thermal stability . Additional mutageneses at residue 83 with asparagine, arginine, or aspartate gave rise to enzymes having a progressively decreasing trend in thermal stability . These mutants are more susceptible to proteolysis than wild-type enzyme . Kinetic analysis of H83Q and H83N indicates that histidine 83 is not involved in the catalytic mechanism or in substrate binding but possibly in maintenance of the active catalytic structure.

Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 360 - 3
Antibody to a Helicobacter pylori species specific antigen in patients with adenocarcinoma of the stomach; Wang JT et al.; This study attempted to identify a possible antibody response to Helicobacter pylori, which is associated with patients with adeno-carcinoma of the stomach . By using proteins of H . pylori as the antigen, pooled sera from gastric cancer and non-cancer patients were used as the first antibody for Western blot analysis . Antibody responses to a 26 kD secreted protein were observed in pooled cancer sera, but not in pooled sera from non-cancer patients . The protein was purified, while amino acid sequences revealed that it was a H . pylori species specific protein . The gene of this protein was cloned and a recombinant protein was expressed in E . coli . In addition, an antibody to the recombinant protein was tested in each individual patient using Western blot analysis . None of the forty non-gastric cancer patients were positive for the antibody to the recombinantly expressed 26 kD species specific protein . Meanwhile, six of the twenty four cancer patients tested positive (0/40 vs 6/24, p < 0.01) . Results presented herein demonstrate that the species specific protein of H . pylori can be useful in detecting H . pylori associated with adenocarcinoma of the stomach.

Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 131 - 7
Characterization, cloning, and expression of porcine alpha B crystallin; Liao JH et al.; alpha-Crystallin is a major lens protein present in the lenses of all vertebrate species . Recent studies have revealed that bovine alpha-crystallins possess genuine chaperone activity similar to small heat-shock proteins . In order to compare this chaperone-like structural protein from the eye lenses of different mammalian species, we have cloned and expressed one of the main alpha-crystallin subunits, i.e., alpha B crystallin, from the porcine lenses in order to facilitate the structure-function evaluation and comparison of this chaperonin protein . cDNA encoding alpha B subunit chain was obtained using a new "Marathon cDNA amplification" protocol of Polymerase Chain Reaction (PCR) . PCR-amplified product corresponding to alpha B subunit was then ligated into pGEM-T plasmid and prepared for nucleotide sequencing by the dideoxy-nucleotide chain-termination method . Sequencing several positive clones containing DNA inserts coding for alpha B-crystallin subunit constructed only one complete full-length reading frame of 525 base pairs similar to human and bovine alpha B subunits, covering a deduced protein sequence of 175 amino acids including the universal translation-initiating methionine . The porcine alpha B crystallin shows only 3 and 7 residues difference to bovine and human alpha B crystallins respectively, revealing the close relatedness among mammalian eye lens proteins . The sequence differences between porcine and sub-mammalian species such as chicken and bullfrog are much greater, especially at the N- and C-terminal regions of these alpha B crystallins . Expression of alpha B subunit chain in E . coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified alpha B subunit from the native porcine lenses albeit with a much lower activity.

Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 75 - 8
Induction of UCP2 gene expression by LPS: a potential mechanism for increased thermogenesis during infection; Faggioni R et al.; UCP2 has been proposed to regulate thermogenesis and energy expenditure . To identify potential mechanisms underlying the increased energy expenditure and heat production during infection, we investigated whether LPS and cytokines might increase UCP2 mRNA levels in mice . LPS (100 micrograms, i.p.) increased the expression of UCP2 mRNA in liver (28-fold) and muscle and white adipose tissue (5-fold) . In liver, both IL-1 beta (1 microgram, i.p.) and TNF (5 micrograms, i.p.) increased UCP2 mRNA levels, 4- and 3-fold respectively, whereas in muscle and fat tissue, an increase was detectable after TNF, but not IL-1 beta . Indomethacin (10 mg/kg, i.p.) administered immediately before LPS markedly reduced (70%) the ability of LPS to increase UCP2 mRNA in liver, but not in muscle or adipose tissue . These results suggest a role for UCP2 in the heat production and increased energy expenditure that occurs during infection.

Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 79 - 84
Alteration of the fatty acid substrate specificity of lysophosphatidate acyltransferase by site-directed mutagenesis; Morand LZ et al.; The JC201 strain of Eschericia coli contains a temperature-sensitive lesion in lysophosphatidate acyltransferase (LPAT) activity . The LPAT gene from JC201 was isolated by PCR and a single mutant nucleotide, adenine-440, was identified by DNA sequence analysis . Site-directed mutagenesis converted the mutant adenine-440 back to the native guanine-440 nucleotide . The restored LPAT gene rescued JC201 cells at the non-permissive temperature . The fatty acid substrate specificity of LPAT from Eschericia coli was altered by site-directed mutagenesis of a single amino acid in the restored LPAT gene . Threonine-122 of LPAT was changed to alanine or leucine . A change from threonine-122 to alanine increased the substrate specificity in vitro for oleoyl-CoA and linoleoyl-CoA; whereas a change to leucine increased the substrate specificity for lignoceroyl-CoA.

Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 110 - 4
Glyphosate is an inhibitor of plant cytochrome P450: functional expression of Thlaspi arvensae cytochrome P45071B1/reductase fusion protein in Escherichia coli; Lamb DC et al.; Glyphosate (Roundup) is an herbicide used extensively worldwide which acts as an inhibitor of 5'enolpyruvylshikimate-3-phosphate synthase and for which transgenic herbicide resistant plants have been developed . Here we report for the first time that glyphosate is an inhibitor of cytochrome P450 using a functional expression system for Thlaspi arvensae CYP71B1 in Escherichia coli . CYP71B1 was fused to the soluble domain of a plant cytochrome P450 reductase (CPR) from Catharanthus roseus . CYP71B1 could obtain reducing equivalents in this fusion construct and metabolised the polycyclic aromatic hydrocarbon, benzo(a)pyrene . The fusion protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum . Addition of the herbicide resulted in a Type II spectrum indicative of binding via the nitrogen group to haem as a sixth ligand . A Ks of 60 microM was observed and an IC50 of 12 microM was observed for glyphosate inhibition of CYP71B1 activity . The implications of these results are discussed.

Anal Biochem, 1998 Mar 15, 257(2), 203 - 9
A positive selection vector for cloning of long polymerase chain reaction fragments based on a lethal mutant of the crp gene of Escherichia coli; Schlieper D et al.; We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli . The positive selection pressure results from a lethal mutation within the E . coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted . Here, we show that this "suicide" vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the lethal gene.

Plasmid, 1998, 39(2), 114 - 22
New shuttle vectors for the introduction of cloned DNA in Desulfovibrio; Rousset M et al.; The pBG1 replicon from the cryptic plasmid of Desulfovibrio desulfuricans G100A was inserted into pTZ18U derivatives to generate a new family of shuttle vectors . These plasmids are stable both in Escherichia coli and in Desulfovibrio, they present a large number of unique restriction sites, and colonies of recombinant clones can be identified by blue/white screening in E . coli . The pBMC, pBMK, and pBMS series carry the cat, npt, or strAB genes as selectable markers, respectively . The pBMC6, pBMK6, and pBMS6 plasmids can be introduced both in D . desulfuricans and in Desulfovibrio fructosovorans by electrotransformation, and the pBMC7, pBMK7, and pBMS7 plasmids contain additional mobilization functions which makes them suitable for conjugation.

Alcohol Clin Exp Res, 1998 Feb, 22(1), 135 - 41
Acute ethanol intoxication inhibits neutrophil beta2-integrin expression in rats during endotoxemia; Zhang P et al.; The effects of acute ethanol intoxication on neutrophil {polymorphonuclear leukocyte (PMN)} adhesion molecule expression and certain other functional properties during endotoxemia were studied in rats to elucidate the mechanisms underlying the immunosuppressive effects of ethanol . Acute ethanol intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg . Control animals received an intraperitoneal injection of saline . Thirty minutes after intraperitoneal injection, animals were given a 90-min intravenous infusion of Escherichia coli endotoxin (total dose of 112.5 microg/rat in 2.5 ml of saline) or saline . Certain rats received granulocyte colony-stimulating factor (G-CSF; 50 microg/kg in 5% dextrose, subcutaneous injection twice daily) or vehicle pretreatment for 2 days before intravenous endotoxin infusion . Endotoxemia significantly upregulated CD11b/c and CD18 expression on PMNs when compared with those of saline-infused rats . Acute ethanol intoxication inhibited this endotoxin-induced upregulation of CD11b/c and CD18 expression on PMNs . Ethanol intoxication also suppressed the phagocytic activities of PMNs in saline-infused rats, but this suppression failed to reach statistical significance in endotoxin-infused rats . Hydrogen peroxide generation by PMNs in saline- or endotoxin-infused rats was not affected by ethanol intoxication . Histological examination showed extensive PMN sequestration in the liver after endotoxin infusion, and ethanol intoxication significantly attenuated this hepatic sequestration of PMNs . G-CSF pretreatment enhanced neutrophil phagocytosis, CD11b/c and CD18 expression in endotoxin-infused rats, and prevented the ethanol-induced inhibition of neutrophil CD18 expression and phagocytosis . The impairment of beta2-integrin expression on PMNs may be one mechanism underlying ethanol-induced defects of neutrophil delivery into tissue sites of infection . G-CSF may be of benefit to the infected alcoholic host by enhancing leukocyte defense functions.

Protein Sci, 1998 Jan, 7(1), 216 - 9
Fragment of GABA(A) receptor containing key ligand-binding residues overexpressed in Escherichia coli; Xue H et al.; GABA(A) receptor plays a major role in inhibitory synaptic transmission in the central nervous system and is the target of drugs such as the benzodiazepine tranquilizers . The polymeric membrane protein nature of GABA(A) receptor has rendered structural elucidation of the receptor a formidable task, greatly hampering structure-based drug design . We report here the first expression in Escherichia coli of a fragment of GABA(A) receptor . This 131-residue fragment, spanning Cys166 to Leu296 of human GABAA receptor alpha1 subunit, contains residues previously suggested to be involved in benzodiazepine binding . The overexpressed non-fusion recombinant protein was purified to near homogeneity and characterized by circular dichroism (CD), which showed that the recombinant protein has well defined secondary structures where beta-strands are dominant . The stability of the secondary structures was demonstrated by CD spectra at high pH and elevated temperature . Excluding part of the sequences from the carboxyl terminal of the fragment resulted in dramatic changes in the secondary structures comparable to the effects caused by SDS denaturation . Our results therefore suggest that the 131-residue fragment harbors an integral structural domain of the receptor . The overexpression of the recombinant protein fragment thus opens the way to the biochemical and structural studies of a functionally important region of the receptor, and exemplifies an effective approach of expression and characterization that potentially may be extended to other members of the ligand gated channel receptor superfamily, to which the GABA(A) receptor belongs.

J Biomol Struct Dyn, 1998 Feb, 15(4), 689 - 701
Statistical and structural analysis of trp binding sites: comparison of natural and in vitro selected sequences; Haran TE; Two different modes can be used when the trp repressor binds to trp binding sites . In the "full-site mode" each repressor molecule is bound to a DNA target containing at least two conserved five base pair tracts separated by eight base pairs . The binding of the repressor to natural trp operators is of this kind . In the "half-site mode" two repressor molecules are sequence-specifically bound, with infinite cooperativity, to two abutting DNA pentamers . We present evidence suggesting that the sequences obtained by a recent in vitro selection assay (Czernik et al . J . Biol . Chem . 269, 27869-27875, 1994) were selected by the binding of two repressor molecules, and that the repressor is bound to most of these sequences using the half-site mode . Using the results of the selection assay, and the set of natural trp binding sites, we characterize the different sequence requirements of the "full-site" versus the "half-site" binding modes . A statistical analysis of the information content of these binding sites shows that functional information on protein binding modes can be extracted from a set of DNA binding sites by comparing the information content of two different DNA populations, or sub-populations . Furthermore, it shows that the binding of proteins to sequences selected by a functional in vitro assay do not necessarily mimic the binding of the protein to the natural targets, even if the information content is similar in the two DNA target populations, i.e., even if the stringency of the selection assay is adequate for locating natural-like sequences . In addition, we show that the structural requirements for protein-DNA interactions can be achieved by different conformations at the base-pair level . Differences in the structural characteristics of different base-pair steps can be used to determine the binding mode and differential binding affinity, which can be utilized in the regulation of several binding sites by a single specific protein.

Protein Eng, 1997 Nov, 10(11), 1333 - 8
Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser; Mine S et al.; When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)-lysozyme) . The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid . A Met(-2)Pro(-1) or Met(-2)Ser(-1) sequence was introduced at the N-terminus of hen egg-white lysozyme . The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine . From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(-1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N-terminus of hen egg-white lysozyme (Pro(-1)-lysozyme) . Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg-white lysozyme.

Protein Eng, 1997 Nov, 10(11), 1327 - 31
Expression, purification and characterization of the homeodomain of rat ISL-1 protein; Behravan G et al.; Isl-1 is a member of a family of Homeodomains containing proteins that possess an N-terminal pair of zinc binding LIM domains . The Isl-1 gene in rat codes for a protein that binds to the insulin gene enhancer and is also involved in regulation of amylin and proglucagon genes . A DNA sequence coding for 66 amino acid residues containing the C-terminal homeodomain fragment of Isl-1 was expressed as a soluble protein in Escherichia coli . Here, we describe a procedure which allows the rapid native purification of recombinant homeodomain protein fused to an N-terminal tag of six histidines . The purified homeodomain showed DNA-binding activity to its cognate DNA sequence . An enhanced binding activity is observed in the presence of a reducing agent in electrophoretic mobility shift assays . The DNA binding was further characterized by circular dichroism spectroscopy . Addition of DNA to the homeodomain did not change the overall secondary structure content, but the thermal and chemical denaturing profiles were altered . A stabilization of the secondary structure was observed upon DNA binding . The free energy of unfolding at 23 degrees C was 7 kJ mol(-1) in absence of DNA and 29 kJ mol(-1) in the presence of DNA.

Protein Eng, 1997 Nov, 10(11), 1289 - 94
Synthesis and characterization of supramolecular protein aggregates: self-assembled, molecularly-ordered, tubes from electrostatic complementation of glutamine synthetase dodecamers; Chen JP et al.; Dodecameric Escherichia coli glutamine synthetase (GS) is formed from identical subunits arranged in face-to-face hexameric rings . In the presence of Zn2+ and other transition metal ions the individual dodecamers 'stack' to form protein tubes . Previous results have suggested that six binuclear intermolecular metal binding sites are generated at each dodecamer-dodecamer interface by juxtaposition of the N-terminal helices of each subunit adjacent to an analogous helix from a docked dodecamer . In principle, replacement of one of the metal binding sites within each pair of helices with charged amino acids could generate electrostatic interactions that would provide the basis for heterospecific protein-protein interactions . In turn, this would allow for ordered assembly of protein tubes with alternating, chemically distinguishable, components . This hypothesis was tested by replacement of one of the metalligating histidines (His12) with aspartic acid, arginine or cysteine . The H12C mutant was further elaborated by selective thiol modification, with either of the charged reagents 2-iodo-acetic acid or 2-chloro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12 . Light scattering and electron microscopy were used to monitor the 'stacking ability' of these variants in the presence of Zn2+ . No, or few, GS 'tubes' were observed in solutions containing only H12D, H12R, H12C-CA or H12C-IA, in the presence or absence of Zn2+ . In contrast, in mixtures containing H12C-CA and either H12D or H12C-IA, the complementary GS variants stack in the presence of 100 microM Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers . Fluorescence energy transfer experiments with fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA (acceptor) were performed and compared with the energy transfer efficiency with mixtures containing variable ratios of acceptor-labeled and donor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangements of donor and acceptor . The efficiency of metal-dependent energy transfer in mixtures containing the acceptor-labeled H12C-CA and the donor-labeled H12C-IA was 3.2-fold greater than expected for a random distribution of charged variants . Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction between chemically distinguishable dodecamers that align in an ordered one-dimensional array.

Protein Eng, 1997 Nov, 10(11), 1281 - 8
Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments; Villbrandt B et al.; The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity . Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion) . Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase . In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates . Consequently two variants were designed and constructed using site-directed mutagenesis . The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones . Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability . After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank . A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model.

Br J Cancer, 1998 Mar, 77(5), 709 - 19
NAD(P)H:quinone oxidoreductase 1 reduces the mutagenicity of DNA caused by NADPH:P450 reductase-activated metabolites of benzo(a)pyrene quinones; Joseph P et al.; The role of microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase) in the mutagenicity of benzo(a)pyrene-3,6-quinone (BP-3,6-Q) was studied using supF tRNA gene as the mutational target . pUB3 carrying the supF tRNA gene upon transformation into the Escherichia coli ES87 cells exhibited a spontaneous mutation frequency of 0.62 x 10(-6) . Chemical modification of the pUB3 DNA with BP-3,6-Q caused a fourfold increase in the mutation frequency, compared with the spontaneous mutations . P450 reductase catalysed metabolic activation of BP-3,6-Q into reactive products (semiquinone and reactive oxygen species), which caused a further increase in the mutation frequency to eightfold over spontaneous mutations . Oxygen radical scavengers (SOD and catalase) blocked the P450 reductase-activated BP-3,6-Q-induced stimulation of mutations . This indicates that redox cycling of the semiquinone leading to the generation of reactive oxygen species (ROS) was directly responsible for the increased mutation frequency of P450 reductase-activated BP-3,6-Q . Analysis of the mutation spectra revealed that P450 reductase-activated BP-3,6-Q showed a significantly higher preference for frameshift mutations, particularly deletions, compared with the spontaneous mutations and the mutations generated by benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) . The single most frequently observed mutation by P450 reductase-activated quinone (semiquinone + ROS) was deletion of a single guanosine . Among the base substitutions, G:C --> T:A, G:C --> A:T and G:C --> C:G were also noticed . Interestingly, NQO1 competed with P450 reductase and specifically prevented the P450 reductase-activated BP-3,6-Q-induced mutations . However, BP-hydroquinone (BP-3,6-HQ) generated during the metabolic reduction of BP-3,6-Q catalysed by NQO1 caused specific mutations involving the deletion of a single cytosine from the DNA sequence 5'-CCCCC-3' in supF tRNA gene at a significantly high frequency . A similar cytosine deletion was also observed with benzoquinone hydroquinone (HQ), indicating that the deletion of cytosine is associated with a hydroquinone class of compounds . These results suggest that: (1) quinones and P450 reductase-activated products of quinones (semiquinones and ROS) are mutagenic compounds; (2) the mutational spectra of quinones, semiquinones and hydroquinones differ from each other with respect to their mutational frequency and specificity; (3) NQO1 competes with P450 reductase and protects the cells from quinone mutagenicity; and (4) the NQO1 -metabolized quinones (hydroquinones), if not eliminated, cause specific mutations that are not observed with quinones and P450 reductase-activated quinones (semiquinones and ROS).

FEMS Microbiol Lett, 1997 Nov 15, 156(2), 217 - 22
Analysis of the G93E mutant allele of KpsM, the membrane component of an ABC transporter involved in polysialic acid translocation in Escherichia coli K1; Pigeon RP et al.; KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1 . The kpsMG93E allele is a point mutation in the first cytoplasmic loop (Cl) of KpsM which partially disrupts translocation of the capsule . While producing polymer of wild-type length, strains harboring the G93E allele exhibit a decreased production of capsular polymer and a reduced rate of polymer translocation to the cell surface.

J Mol Biol, 1998 Feb 20, 276(2), 505 - 15
Thermodynamic stability and folding of GroEL minichaperones; Golbik R et al.; The apical domain of GroEL (residues 191 to 376) and its C-terminally truncated fragment GroEL(191-345) are expressed with high yield in Escherichia coli to give functional monomeric minichaperones . Owing to the reversible folding behaviour of the minichaperones we can analyse the folding of the polypeptide binding domain of the multidomain GroEL protein, the folding of which is known to be irreversible . The apical domain shows two reversible temperature transitions with transition midpoints at 35 degrees C and at 67 degrees C that can be attributed to the unfolding of the C-terminal helices and the domain core, respectively . The native state of the domain core is stabilized by 5.5 kcal mol-1 relative to the unfolded state . The rate constant of folding of the apical domain core is independent of the minichaperone concentration and the presence of the C-terminal alpha-helices . A folding intermediate on the folding pathway is destabilized relative to the native state by 1.6 kcal mol-1, which is also detected by equilibrium and kinetic binding of the dye bis-ANS . Reversible folding of the polypeptide domain of GroEL guarantees highly efficient chaperonin activity within the GroEL toroid.

J Mol Biol, 1998 Feb 20, 276(2), 405 - 15
Differential cleavage of LexA and UmuD mediated by recA Pro67 mutants: implications for common LexA and UmuD binding sites on RecA; Konola JT et al.; In Escherichia coli, RecA-mediated cleavage of LexA repressor is a key regulatory event required for expression of SOS genes involved in the repair of DNA damage . RecA also mediates the cleavage of UmuD protein to UmuD, a form active in SOS mutagenesis . To determine whether LexA and UmuD have common binding determinants on RecA, we have compared the ability of several recA mutants to function in the cleavage of LexA versus UmuD in vivo . The data reveal that while some recA mutations at Pro67 have a similar effect on LexA and UmuD cleavage, others have striking differential effects . For example, a Pro67-->Trp mutation results in a high level of constitutive cleavage of both proteins . However, Pro67-->Asp and Glu mutations promote constitutive cleavage of LexA and reduce induction of UmuD cleavage to just 5 to 10% of wild-type activity . In contrast, Pro67-->Arg prevents LexA cleavage while allowing nearly 50% of wild-type induction of UmuD cleavage . These results are consistent with the idea that Pro67 is located at a site in the nucleoprotein filament where both LexA and UmuD contact RecA.

J Mol Biol, 1998 Feb 20, 276(2), 355 - 65
FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli; Negre D et al.; The start site of transcription of the ppsA gene, whose expression is controlled by the regulatory protein FruR in Escherichia coli, was determined by primer extension of in vivo transcripts . The interactions of the ppsA promoter with either RNA polymerase or FruR factor were analysed by the base removal method . Our results indicate that: (i) the RNA polymerase binding site has a -10 extended module but lacks its -35 hexamer; (ii) FruR binds to a target DNA region centered around position -45.5 upstream of the ppsA gene . In addition, circular permutation analysis showed that, upon binding to its site, FruR induces a sharp bend of 120 degrees in the DNA helix, which suggests a crucial involvement of FruR-induced bending in ppsA promoter activation . Direct contacts between the upstream activating DNA and RNA polymerase were studied in an in vitro transcription assay by using reconstituted RNA polymerase mutants containing Ala substitutions in C-terminal domain of their alpha subunit . The alpha{L262A}, alpha{R265A} and alpha{N268A} substitutions, which caused the most drastic reduction in the FruR-mediated activation of the ppsA promoter, had previously been shown to inhibit the upstream element-mediated activation at the rrnBP1 promoter.

J Mol Biol, 1998 Feb 20, 276(2), 339 - 53
Molecular analysis of the regulation of csiD, a carbon starvation-inducible gene in Escherichia coli that is exclusively dependent on sigma s and requires activation by cAMP-CRP; Marschall C et al.; The general stress-induced sigma subunit sigma s of Escherichia coli RNA polymerase is closely related to the vegetative sigma factor sigma 70 . In view of their very similar promoter specificity in vitro, it is unclear how sigma factor selectivity in the expression of sigma s-dependent genes is generated in vivo . The csiD gene is such a strongly sigma s-dependent gene . In contrast to sigma s, which is induced in response to many different stresses, csiD, whose expression is driven from a single promoter, is induced by carbon starvation only . To our knowledge, the csiD promoter is the first characterized promoter which is not only exclusively dependent on sigma s-containing RNA polymerase (E sigma s), but also requires an activator, cAMP-CRP . In addition, leucine-responsive regulatory protein (Lrp) acts as a positive modulator of csiD expression . Also in vitro, E sigma s is more efficient than E sigma 70 in csiD promoter binding, open complex formation and run-off transcription, which might be due to the poor match of the csiD -35 region to the sigma 70 consensus and to transcription by E sigma s being less dependent on contacts in this region . By DNase I protection experiments, a cAMP-CRP binding site centered at -68.5 nucleotides upstream of the csiD transcriptional start site was identified . While cAMP-CRP stimulates E sigma 70 binding, it does not promote open complex formation by E sigma 70, but does so in conjunction with E sigma s . With linear templates, cAMP-CRP significantly stimulates E sigma s-mediated in vitro transcription, whereas transcription by E sigma 70 is negligible and hardly stimulated by cAMP-CRP . These findings may reflect different or less stringent positional requirements for an activator site for E sigma s than for E sigma 70, and indicate that cAMP-CRP contributes to sigma factor selectivity at the csiD promoter . In vitro transcription experiments with super-coiled templates, however, revealed significant cAMP-CRP-stimulated transcription also by E sigma 70 . Yet, under these conditions, H-NS was found to restore E sigma s specificity by strongly interfering with cAMP-CRP/E sigma 70-dependent transcription . Lrp strongly and cooperatively binds to multiple sites located between positions -14 and -102 (in a way that suggests DNA wrapping around multiple Lrp molecules) and moderately stimulates in vitro transcription, especially with E sigma s . In summary, we conclude that the csiD promoter has an intrinsic preference for E sigma s, but that also protein factors such as cAMP-CRP, Lrp and probably H-NS as well as DNA conformation contribute to its strong E sigma s selectivity . Furthermore, this strong E sigma s preference in combination with a requirement for high concentrations of the essential activator cAMP-CRP ensures csiD expression under conditions of carbon starvation, but not other stress conditions.

J Mol Biol, 1998 Feb 20, 276(2), 313 - 8
Stepwise selection of TetR variants recognizing tet operator 4C with high affinity and specificity; Helbl V et al.; The TetR PQ39 mutant exhibits a new recognition specificity for the tetO-4C operator, but the affinity is not sufficiently high for use in vivo . A stepwise selection of additional mutations by cassette mutagenesis with randomization of residues in the TetR alpha-helix-turn-alpha-helix motif (HTH) yielded mutant TetR EA37PQ39YM42 showing a similar affinity and increased specificity for tetO-4C as wild-type TetR for tetO . A set of mutants obtained by that approach revealed that the fourth residue of the HTH (Leu41), which points towards the core of the DNA binding domain in TetR, alters the recognition of base-pair 4, e.g . the mutant TetR LV41YM42 exhibits a new recognition specificity for tetO-4G . A small residue at the last position in the turn of the HTH increases the affinity and specificity of DNA binding of TetR mutants containing the PQ39 exchange . Thus, cooperation between residues at positions 37, 39, 41 and 42 in the HTH of TetR is necessary to optimize recognition of base-pair 4 . We conclude that creating a new DNA recognition specificity in the HTH of TetR with high affinity for the tetO-4C operator variant requires exchanges altering flexibility and/or adjustment of the recognition alpha-helix to the target DNA in addition to the contacting residue.

Biochim Biophys Acta, 1998 Feb 11, 1395(3), 309 - 14
Molecular cloning and characterization of the gene encoding glutathione reductase in Brassica campestris; Lee H et al.; We have isolated the Brassica campestris cDNA encoding glutathione reductase of 502 amino acid residues with molecular mass of 54.5 kDa . The deduced amino acid sequences were 92.2%, and 79.5% identical to those of Arabidopsis thaliana, and pea, respectively . As expected, it exhibited a high degree of conservation within the region responsible for the redox reaction and for the binding of GSSG or NADPH . The gene was highly inducible by ozone fumigation or by paraquat treatment.

Biochim Biophys Acta, 1998 Feb 11, 1395(3), 259 - 65
Cloning and expression of the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA; Jehan F et al.; Using a cDNA probe from the rat 24-hydrovitamin D3 24-hydroxylase, the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA has been isolated from a chicken kidney lambda gt11 library . The high degree of similarity with the mammalian 24-hydroxylase cDNAs strongly supports the belief that it is the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA . The deduced amino acid sequences are also very well conserved and 325 of them are identical among the four known 25-hydroxyvitamin D3 24-hydroxylases . This cDNA expressed in E . coli produces 24-hydroxylase activity.

Antisense Nucleic Acid Drug Dev, 1998 Feb, 8(1), 53 - 61
Molecular cloning and expression of cDNA for human RNase H; Wu H et al.; We have cloned, expressed, and purified to electrophoretic homogeneity a human RNase H . The enzyme has a molecular weight of 32 kDa, is Mg2+ dependent, and is inhibited by Mn2+ and N-ethylmaleimide . Its molecular weight and cleavage characteristics are consistent with type 2 human RNase H . The human RNase H we have cloned is highly homologous to Escherichia coli RNase HI (33.6% amino acid identity) and to other RNase H enzymes homologous to E . coli RNase HI . The enzyme is encoded by a single gene that is at least 10 kb in length and is expressed ubiquitously in human cells and tissues.

Acta Biochim Pol, 1997, 44(3), 565 - 78
Expression of Lupinus luteus cDNA coding for PR10 protein in Escherichia coli: purification of the recombinant protein for structural and functional studies; Sikorski MM; The cDNA clones coding for two pathogenesis-related protein homologues of PR10 class, LlPR10.1A and LlPR10.1B, were identified in yellow lupin expression library of uninfected roots . The contribution of PR10 proteins to the overall mechanism of plant defence still remains unknown . In order to elucidate the structure and function of lupin PR10.1A protein, a substantial quantity of the protein was produced in an E . coli expression system using plasmids of pET-series: pET-3a and pET-15b, carrying the T7 promoter . Both plasmids with subcloned Llpr10.1a gene were overexpressed in E . coli, strain BL21(DE3)pLysS . The recombinant LlPR10.1A protein, overproduced in bacterial cells transformed with the pET-3a/Llpr10.1a plasmid, was purified to homogeneity from the insoluble "inclusion bodies" by ammonium sulphate fractionation and two sequential chromatographic steps: ion-exchange chromatography on DE 52 cellulose followed by size exclusion chromatography on Superdex 75 FPLC column . The (His)6 LlPR10.1A protein overproduced in E . coli cells harbouring the pET-15b/Llpr10.1a plasmid was purified by chromatography on Ni2+-charged His.Bind Resin . Western blot analysis with rabbit serum containing anti-LlPR10.1AN antibody revealed identical immunochemical properties of the two recombinant polypeptides and native LlPR10.1A protein . The recombinant protein produced in pET-3a plasmid was renatured from its insoluble form, concentrated up to 22 mg/ml and submitted to crystallisation . However, the LlPR10.1A protein expressed in pET-15b plasmid precipitated from the solution when at a higher concentration (10 mg/ml) . This preparation was used at a lower concentration as an antigen for the preparation of polyclonal antibodies for immunochemical studies.

J Med Microbiol, 1997 Aug, 46(8), 669 - 74
Studies on the lysozyme independence of immune immobilisation of Treponema pallidum and the frequency of lysozyme autoantibodies in syphilitic sera; Zielinski S et al.; The role of lysozyme in the immune immobilisation of Treponema pallidum is not yet fully understood . The T . pallidum immobilisation assay was used to demonstrate that the immobilisation and lysis of T . pallidum in vitro by antibodies (serum, IgG fraction or IgM fraction) and complement proceed in a lysozyme-independent mode . In the presence of lysozyme the rate of immobilisation increased . In contrast with its effect on Escherichia coli, the effect of lysozyme on T . pallidum was governed exclusively by its enzymic activity rather than by the cationic protein nature of the molecule . Lysozyme, released from stimulated phagocytes, induced formation of lysozyme antibodies in 59.6% of syphilis patients as determined by lysozyme antibody ELISA . The highest frequency was found in patients with untreated secondary syphilis, whereas untreated primary syphilis was only rarely accompanied by the presence of lysozyme antibodies . Cross-reactivities between lysozyme and treponemal antigens were excluded by immunoblotting . The autoantibodies did not influence the lysozyme activity . It was concluded that the formation of lysozyme antibodies is only an epiphenomenon in the host defence against treponemal infection.

Biochim Biophys Acta, 1998 Jan 27, 1363(1), 85 - 93
Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis; Schmitz S et al.; The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E . coli . Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies . The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR . Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin . Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR . A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation . In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.

Biochim Biophys Acta, 1998 Jan 27, 1363(1), 35 - 46
Reconstitution of cytochrome b-560 (QPs1) of bovine heart mitochondrial succinate-ubiquinone reductase; Lee GY et al.; The QPs1 subunit of bovine heart mitochondrial succinate-ubiquinone reductase was overexpressed in Escherichia coli DH5 alpha cells as a glutathione S-transferase fusion protein (GST-QPs1) using the expression vector, pGEX/QPs1 . The yield of soluble active recombinant GST-QPs1 fusion protein depends on the IPTG concentration, induction growth time, temperature, and medium . Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth with 0.5 mM IPTG at 27 degrees C in an enriched medium containing betaine and sorbitol . QPs1 is released from the fusion protein by proteolytic cleavage with thrombin . Isolated recombinant QPs1 shows one protein band in SDS-polyacrylamide gel electrophoresis corresponding to subunit III of mitochondrial succinate-ubiquinone reductase . However, partial N-terminal amino acid sequence analysis of recombinant QPs1 shows two extra amino acid residues, glycine and serine, at the N-terminus of mature QPs1, resulting from the recombinant manipulation . When isolated recombinant QPs1 is dispersed in 0.01% dodecyl maltoside, it is in a highly aggregated form with an apparent molecular mass of over 1 million . Recombinant GST-QPs1 contains little cytochrome b-560 heme . However, addition of hemin chloride restores the spectral characteristics of cytochrome b-560 . Cytochrome b-560 restoration varies with the amount of hemin used . Maximum reconstitution is obtained when the molar ratio of heme to fusion protein used in the system is 0.6 . Reconstituted cytochrome b-560 shows a EPR signal at g = 2.91 which corresponds to one of the EPR signals of cytochrome b-560 in a QPs preparation . When GST-QPs1 with reconstituted cytochrome b-560 is treated with thrombin to cleave GST from QPs1, no change in the absorption and EPR characteristics of cytochrome b-560 is observed, indicating that the bis-histidine ligands of reconstituted cytochrome b-560 are provided by QPs1.

Gene, 1998 Jan 30, 207(2), 241 - 9
A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines; Hegedus DD et al.; A series of shuttle vectors have been constructed that allow expression of heterologous proteins in either dipteran or lepidopteran insect cell lines . Constitutive expression in a broad range of host cells is mediated by the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus (OpMNPV) immediate-early 2 (ie2) promoter . Alternatively, if inducible expression is required, for example to express cytotoxic proteins, a vector has been constructed that uses the Drosophila metallothionein (Mtn) promoter for metal-inducible protein expression in dipteran cell lines . A chimeric synthetic bacterial-OpMNPV ie promoter-Zeocin resistance gene cassette has been included to facilitate cloning in E . coli as well as the generation of stably transformed insect cell lines . The utility of the system is demonstrated by the constitutive and inducible expression of the highly processed glycosylphosphatidylinositol-anchored glycoprotein, human melanotransferrin, in transformed insect cell lines.

Gene, 1998 Jan 30, 207(2), 197 - 207
Protein SRP54 of human signal recognition particle: cloning, expression, and comparative analysis of functional sites; Gowda K et al.; Signal recognition particle (SRP) plays a critical role in the targeting of secretory proteins to cellular membranes . An essential component of SRP is the protein SRP54, which interacts not only with the nascent signal peptide, but also with the SRP RNA . To understand better how protein targeting occurs in the human system, the human SRP54 gene was cloned, sequenced, and the protein was expressed in bacteria and insect cells . Recombinant SRP54 was purified from both sources . The protein bound to SRP RNA in the presence of protein SRP19, and associated with the signal peptide of in vitro translated pre-prolactin . Comparative sequence analysis of human SRP54 with homologs from all three phylogenetic domains was combined with high-stringency protein secondary structure prediction . A conserved RNA-binding loop was predicted in the largely helical M-domain of SRP54 . Contrary to general belief, the unusually high number of methionine residues clustered outside the predicted helices, thus indicating a mechanism of signal peptide recognition that may involve methionine-rich loops.

Gene, 1998 Jan 30, 207(2), 187 - 95
Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments; Shingledecker K et al.; Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases . In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected . The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E . coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively . One type of genetic alteration of the RecA intein involved deletion of the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain . The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each . Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region . The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans . The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.

Gene, 1998 Jan 19, 207(1), 53 - 60
Identification of the Dictyostelium discoideum homolog of the N-ethylmaleimide-sensitive fusion protein; Weidenhaupt M et al.; The N-ethylmaleimide-sensitive fusion protein (NSF) is required for vesicular membrane fusion in multiple cellular functions . We have cloned a cDNA encoding the Dictyostelium discoideum homolog of the NSF protein . This cDNA hybridizes with a single fragment in Southern blots suggesting that NSF is encoded by a single gene in the amoeba . It is expressed constitutively during vegetative growth and throughout the differentiation cycle . The encoded gene product comprises 738 aa with a predicted molecular mass of 82 kDa . It shows the characteristic three-domain structure of NSF proteins . A more divergent amino-terminal part is followed by two highly conserved ATP-binding domains featuring Walker A and B signature sequences . The D . discoideum protein presents an overall aa sequence identity of 44% when compared to known NSF homologs . The monoclonal antibody 2E5 directed against Cricetellus griseus NSF recognizes a protein with a molecular weight of approx . 80 000 in a D . discoideum crude extract and the recombinant D . discoideum His6-NSF expressed in Escherichia coli.

J Antimicrob Chemother, 1998 Jan, 41(1), 111 - 4
Association of organic solvent tolerance and fluoroquinolone resistance in clinical isolates of Escherichia coli; Oethinger M et al.; Fluoroquinolone resistance in Escherichia coli is principally caused by two kinds of mutation: those affecting the target proteins of the drugs, i.e . DNA gyrase and topoisomerase IV, and those affecting regulatory genes such as marA, soxS or robA . Recently, overexpression of the latter genes was linked to increased organic solvent tolerance in E . coli . Among 138 clinical fluoroquinolone-resistant and -susceptible clinical isolates of E . coli we found a high association between fluoroquinolone resistance and organic solvent tolerance . This finding suggests that E . coli may undergo an adaptive response to extrinsic substances other than quinolones, while mutating to fluoroquinolone resistance.

Nature, 1998 Mar 5, 392(6671), 101 - 5
Structure of the Sec7 domain of the Arf exchange factor ARNO; Cherfils J et al.; Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state . As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo . Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins . In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic . An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 and GPS1, have been identified . These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain . The Sec7 domain contains the exchange-factor activity . It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Geal and Gea2 . Here we report the crystal structure of the Sec7 domain of human ARNO at 2 A resolution and the identification of the site of interaction of ARNO with Arf.

Tissue Antigens, 1998 Feb, 51(2), 156 - 63
Identification of a human heavy chain antibody fragment directed against human platelet alloantigen 1a by phage display library; Okamoto N et al.; The human platelet alloantigen HPA-1a (PlA1) is responsible for most cases of post-transfusion purpura and neonatal alloimmune thrombocytopenia in the Caucasian population . HPA-1a and HPA-1b are two allelic forms of the platelet membrane glycoprotein IIIa (GPIIIa) gene that differ by a single amino acid . In this report, we describe the development of a recombinant heavy chain antibody fragment capable of distinguishing between the homozygous forms of HPA-1a and HPA-1b . This antibody fragment was isolated from the lymphocytes of an immunized individual through the use of a phage display library system . The recombinant antibody fragment reacted with human platelet lysates from HPA-1a homozygous donors, the HPA-1a form of recombinant N-terminal GPIIIa and intact HPA-1a platelets, but did not react with platelet lysate from HPA-1b homozygous donors, reduced HPA-1a form of platelet GPIIIa or other platelet glycoproteins . This HPA-1a specific human antibody fragment works well in common laboratory assays such as ELISA and flow cytometry, which can assist in identifying HPA-1b homozygous individuals who are known to have a higher risk for developing neonatal alloimmmune thrombocytopenia and post-transfusion purpura . Thus, selection of recombinant antibody fragment using phage display offers a promising alternative to hybridoma technology for the production of human antibodies against human alloantigens and holds potential as a technique in therapeutic applications.

RNA, 1998 Mar, 4(3), 319 - 30
mRNA stabilization by the ompA 5' untranslated region: two protective elements hinder distinct pathways for mRNA degradation; Arnold TE et al.; The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer that can prolong the cytoplasmic lifetimes of a variety of messages to which it is fused . Previous studies have identified two domains of this 5' UTR that together are responsible for its stabilizing effect . One is a 5'-terminal stem-loop . The other is a single-stranded RNA segment (ss2) that contains a ribosome binding site highly complementary to 16S ribosomal RNA . Here we report a detailed investigation of the function of these two stabilizing elements . Our data indicate that mRNA protection by a 5' stem-loop requires no sequence features or thermodynamic stability beyond the minimum necessary for stem-loop formation . Stabilization by ss2 appears to result not from a high frequency of translation initiation, but rather from a high degree of occupancy of this 5' UTR segment by bound ribosomes . Although close spacing of translating ribosomes is not critical for message stabilization by the ompA 5' UTR, mRNA longevity does require the periodic passage of ribosomes through the protein-coding region . Unlike bound ribosomes, which hinder mRNA cleavage by RNase E, the 5' stem-loop appears to impede degradation of ompA mRNA via a distinct pathway that is RNase E-independent . These findings imply that the ompA 5' UTR prolongs mRNA longevity by impeding multiple pathways for mRNA degradation.

Zhonghua Yi Xue Za Zhi (Taipei), 1998 Jan, 61(1), 39 - 43
Bilateral idiopathic infantile pyoceles: a case report; Hsieh DS et al.; A 15-day-old infant was admitted to the 803 Military Hospital with swelling, bilateral erythematous changes, a tender firm mass in the scrotum and fever of six hours' duration . Scrotal sonography showed accumulated fluid with internal echoes in both hydrocele sacs . Aspirated fluid was found to contain Escherichia coli and bilateral scrotal pyoceles was diagnosed . The most common etiologic factor for this is meconium peritonitis, with surgical drainage being the necessary treatment . The case is reported, with a review of the literature.

Mol Cells, 1997 Dec 31, 7(6), 807 - 15
Expression of a cDNA encoding Phytolacca insularis antiviral protein confers virus resistance on transgenic potato plants; Moon YH et al.; To develop an antiviral agent and virus-resistant plants, a cDNA clone encoding Phytolacca insularis antiviral protein (PIP) was isolated from a cDNA library constructed with poly(A)+ RNA purified from leaves of P . insularis . The PIP cDNA contains an open reading frame encoding 307 amino acids . The deduced amino acid sequence includes a putative signal sequence of 22 amino acids at the N-terminus . The amino acid sequence of PIP shares 84% homology with that of the pokeweed antiviral protein (PAP) . In addition, the mature PIP exhibits the conserved putative active site found in other ribosome-inactivating proteins (RIPs) . Recombinant PIP (rPIP) synthesized in Escherichia coli inhibits protein synthesis in vitro in rabbit reticulocyte lysate through the N-glycosidase activity in a similar manner with other RIPs . Local lesion assays with purified rPIP revealed that it inhibits infection of various viruses to plants . Transgenic potato plants expressing the PIP cDNA under the control of the cauliflower mosaic virus 35S promoter are resistant to viruses, such as potato virus X, potato virus Y, and potato leafroll virus . These results suggest that the PIP cDNA could be used for the development of an antiviral agent and transgenic plants resistant against a broad spectrum of plant viruses infecting through both mechanical and aphid transmission.

Mol Cells, 1997 Dec 31, 7(6), 769 - 76
Cloning and analysis of the DNA polymerase-encoding gene from Thermus filiformis; Jung SE et al.; The gene encoding Thermus filiformis (Tfi) DNA polymerase was cloned and its nucleotide sequence was determined . The primary structure of Tfi DNA polymerase was deduced from its nucleotide sequence . Tfi DNA polymerase is comprised of 833 amino acid residues and its molecular mass was determined to be 93,890 Da . The deduced amino acid sequence of Tfi DNA polymerase showed a high sequence homology to E . coli DNA polymerase I-like DNA polymerases: 78.5% homology to Taq DNA polymerase, 78.4% to Tca DNA polymerase, and 41.8% to E . coli DNA polymerase I . An extremely high sequence identity was observed in the region containing polymerase activity . The G + C content of the coding region for the Tfi DNA polymerase gene was 68.5%, which was higher than that of the chromosomal DNA (65%) . The G + C contents in the first, second, and third positions of the codons used were 71.8%, 40.9%, and 92.7% respectively . Codon usage in Tfi DNA polymerase was heavily biased towards the use of G + C in the third position . Rare codons with U or A as the third base were sometimes used to avoid using GA(A/T) TC and TCGA sequences, as they are recognition sites for the restriction endonucleases TfiI and TaqI.

Jpn J Ophthalmol, 1997 Nov-Dec, 41(6), 355 - 61
Synthetic lipid A-induced uveitis and endotoxin-induced uveitis--a comparative study; Hanashiro RK et al.; Endotoxin-induced uveitis (EIU) is an animal model of ocular inflammation induced by lipopolysaccharide (LPS) . The lipid A (LA) region of the LPS chemical structure is believed to be responsible for virtually all the biological activities induced by LPS . The aim of this study was to perform a more detailed investigation of the potency of LA in reproducing EIU . Various doses of either LPS or LA were injected into the footpad of an inbred strain of Lewis rat and the inflammation patterns were compared by assessing the protein concentration, by cytological study, and by determining the inflammatory cell content in samples of aqueous humor obtained during 96-hour follow-up . Evaluation of the cell number and protein concentration ratio of both groups showed the LA-stimulated group presented a higher ratio than the LPS group (Welch's t-test, (P < 0.00001) . It was noteworthy that even the injection of high doses of LPS could not reproduce the level of cellular infiltration induced by LA . Histological study confirmed the enhanced cellularity in the LA group, neutrophils being predominant in both the LPS- and the LA-stimulated groups . The divergent findings in these two models of uveitis may be valuable to further investigations of the process of inflammatory cell migration into the anterior chamber of the eye.

Mol Reprod Dev, 1998 Apr, 49(4), 426 - 34
Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins; Crosby JA et al.; During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida . To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs) . The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody . The recombinant truncated beta-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation . Three of the five recombinant forms (truncated beta-acrosin, Ser/Ala222-truncated beta-acrosin, and truncated beta-acrosin "heavy chain") had the ability to bind ZPGPs . The two shorter forms (the amino and carboxy termini of truncated beta-acrosin) failed to bind . The catalytic site mutant (Ser/Ala222) of truncated beta-acrosin does not differ from the recombinant truncated beta-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process . Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site.

Neuroendocrinology, 1998 Feb, 67(2), 109 - 16
Endotoxin induces interleukin-1beta and nitric oxide synthase mRNA in rat hypothalamus and pituitary; Satta MA et al.; The gases nitric oxide (NO) and carbon monoxide (CO) may be involved in hypothalamo-pituitary-adrenal axis (HPA) modulation . In the brain, NO is synthesized by two forms of NO synthase (NOS), a constitutive neuronal form (nNOS) and an inducible form (iNOS) . There are also a constitutive heme oxygenase (HO2) and an inducible form (HO1) which generate CO . We have therefore investigated the effect of peripheral lipopolysaccharide (LPS) administration on the gene expression of these enzymes along with interleukin-1beta (IL-1beta) gene expression in the hypothalamus, pituitary and liver . Male Wistar rats (200-250 g body weight) were injected intraperitoneally with endotoxin (Escherichia coli, 055 B5) dissolved in sterile normal saline {250 microg/kg first group, 2.5 mg/kg (second group) and 6.25 mg/kg (third group)} in a final volume of 0.5 ml, or saline alone in the control group . The first and the second groups were studied 1, 3, 8 and 24 h after LPS (n = 4 per group); the third group was studied at 3 h . Total RNA was extracted from the hypothalamus, pituitary and liver, and cDNA was made using standard reverse transcriptase methods . Duplex polymerase chain reaction (PCR) was standardised in order to quantify the expression of a specific gene in relation to the 'house-keeping' gene beta-actin . The specific genes studied were iNOS, nNOS, HO1, HO2 and IL-1beta . The PCR products were separated on agarose gel and densitometric analysis of the bands allowed semi-quantification . In the second group, iNOS and IL-1beta were induced in hypothalamus, pituitary and liver, showing a peak at 3 h (p < 0.001), returning to baseline levels at 24 h . Neuronal NOS was not expressed in the liver under basal conditions or after LPS; in the hypothalamus and pituitary, nNOS was expressed basally but there was no change after LPS . In the first group, iNOS and IL-1beta were again induced in all three tissues studied, but with a delayed time course compared to the second and third groups; the peak change for IL-1beta occurred at 8 h (p < 0.05), again returning to baseline levels at 24 h . The peak for iNOS occurred at 24 h . HO1 and HO2 were expressed in all three tissues under basal conditions; HO1 was increased at 1 h in the liver in the second group, and at 3 h in the pituitary in the third group . There was no change in either HO1 or HO2 in the hypothalamus at any dose at any time point . We conclude that IL-1beta and iNOS are induced in rat hypothalamus and pituitary following various doses of endotoxin . We speculate that while IL-1beta may mediate stimulation of the HPA by endotoxin, NO generation may be involved in the counter-regulation of this response.

Arch Biochem Biophys, 1998 Feb 15, 350(2), 283 - 90
Truncation of human squalene synthase yields active, crystallizable protein; Thompson JF et al.; Squalene synthase catalyzes the first committed step in cholesterol biosynthesis and thus is important as a potential target for therapeutic intervention . In order to determine the important functional domains of the protein, the amino and carboxyl terminal regions thought to be involved in membrane association of the enzyme were removed genetically . The 30 N-terminal amino acids were deleted with no apparent effect on activity . Additional deletion of 81 or 97 amino acids from the C-terminus completely ablated activity . However, a protein with a C-terminal deletion of 47 amino acids retained full activity . The latter enzyme was readily overexpressed in Escherichia coli and purified to homogeneity . The pure, doubly truncated enzyme exhibited a specific activity similar to that reported for the protease-solubilized rat liver enzyme, had a KM for farnesyl diphosphate similar to that observed for native enzyme, and was inhibited by anionic compounds to the same degree as native enzyme . Using the vapor diffusion method, the protein was crystallized as an enzyme-inhibitor complex, yielding orthorhombic crystals which diffracted to 2.2 A .

Mutat Res, 1998 Jan 13, 412(1), 63 - 7
Bone marrow and liver mutagenesis in lacZ transgenic mice treated with hexavalent chromium; Itoh S et al.; The mutagenic effects of the hexavalent chromium compound K2CrO4 in lacZ transgenic mice (Muta Mouse) were investigated at two sampling times . K2CrO4 was administered intraperitoneally to five male mice per treatment group at a single dose of 40 mg/kg . The animals were sacrificed on days 1 and 7 after the treatment . Mutant frequencies in the bone marrow and liver were analyzed by the positive selection method using Escherichia coli C (galE-) strain and phenyl beta-D-galactoside . K2CrO4 induced a significant increase in mutant frequency in the bone marrow on day 1, but not on day 7 after the treatment . In the liver, on the other hand, a significant induction in the mutant frequency was seen on day 7, whereas no induction was observed on day 1 . The reason for the different responses to the mutagenic activity of K2CrO4 between these organs may be related to their cell turnover rates . The mutations induced by K2CrO4 in the bone marrow may have occurred in more differentiated cells than stem cells, and the rapid proliferative activity may have caused a rapid decrease in mutated cells by day 7 . These results suggest that experiments on mutagenesis should be done with more than one sampling point, a short expression time in addition to a longer one, so as to detect mutations induced in organ with high cell proliferation.

J Clin Microbiol, 1998 Mar, 36(3), 840 - 2
Enteroaggregative, Shiga toxin-producing Escherichia coli O111:H2 associated with an outbreak of hemolytic-uremic syndrome; Morabito S et al.; Shiga toxin-producing Escherichia coli O111:H2 strains from an outbreak of hemolytic-uremic syndrome showed aggregative adhesion to HEp-2 cells and harbored large plasmids which hybridized with the enteroaggregative E . coli probe PCVD432 . These strains present a novel combination of virulence factors and might be as pathogenic to humans as the classic enterohemorrhagic E . coli.

J Clin Microbiol, 1998 Mar, 36(3), 652 - 6
Epidemiological study of a food-borne outbreak of enterotoxigenic Escherichia coli O25:NM by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis; Mitsuda T et al.; This study investigated the applicability of molecular epidemiological techniques to the identification of the causal agent of an outbreak of diarrhea caused by ingestion of food contaminated with enterotoxigenic Escherichia coli (ETEC) . The outbreak occurred at four elementary schools in July 1996 and affected more than 800 people . Illness was most strongly associated with eating tuna paste (relative risk, 1.79; 95% confidence interval = 1.16 to 2.79; P = 0.0001) . To evaluate the epidemiological characteristics of the pathogen, the DNAs from numerous isolated ETEC strains were subjected to randomly amplified polymorphic DNA analysis, pulsed-field gel electrophoresis of nuclease S1-treated plasmid DNA, and analysis of genomic DNA restriction fragment length polymorphisms . All ETEC isolates were of the O25:NM (nonmotile) serotype, which carries a heat-stable enterotoxin Ib gene . Genotypic analysis demonstrated that the strains isolated from the patients at all four schools were identical . The isolates of ETEC O25:NM obtained from the tuna paste that had been served for lunch at these schools were genetically indistinguishable from those isolated from the patients . Results suggest that this outbreak was food borne . The molecular biology-based epidemiological techniques used in this study were useful in characterizing the causal agent in this food-borne epidemic.

Oncol Res, 1997, 9(10), 553 - 63
Expression of the verotoxin receptor glycolipid, globotriaosylceramide, in ovarian hyperplasias; Arab S et al.; The presence of cell surface receptor glycolipid, globotriaosylceramide (Gb3), is essential to confer susceptibility to the E . coli-derived verotoxin (VT) . Our earlier studies showed that Gb3 is expressed in ovarian carcinoma cell lines . The Gb3 content of normal ovary, benign and malignant primary ovarian tumors, and their metastases have now been compared by verotoxin thin-layer chromatogram (TLC) overlay of the glycolipid tissue extracts . FITC-labeled VT1 B subunit binding to frozen tumor sections was also monitored histochemically . Low to undetectable levels of Gb3 were found in "normal" ovarian tissue . Gb3 was markedly increased in both benign and malignant tumors, suggesting that increased Gb3 may be related to proliferation, rather than malignancy per se . Mucinous tumors showed the least Gb3 elevation; serous tumors were variable, showing higher levels of Gb3 in less differentiated malignant tumors . By far the highest Gb3 content was observed for secondary ovarian metastases and tumors refractory to chemotherapy . Frozen sections of neoplastic ovarian tissue overlaid with fluorescein-conjugated VT1 B subunit show extensive binding to tumor cells, particularly in poorly differentiated samples and blood vessels adjacent to, and within, the tumor mass . Tumor foci were stained but stromal tissue was consistently negative both in primary tumors and metastases . VT staining of well-differentiated primary ovarian tumor sections was weak, corresponding to their low Gb3 content, but strong staining was observed in sections from a highly differentiated primary tumor from a patient who was unexpectedly refractory to clinical chemotherapy . These studies suggest that verotoxin/Gb3 targeting may provide the basis for new treatments for ovarian cancer.

Biochim Biophys Acta, 1998 Jan 15, 1382(1), 47 - 54
Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica; Saavedra-Lira E et al.; The parasite Entamoeba histolytica is an organism whose main energetic source comes from glycolysis . It has the singularity that several of its glycolytic enzymes use pyrophosphate as an alternative phosphate donor . Thus, pyruvate phosphate dikinase (PPDK), an inorganic pyrophosphate (PPi)-dependent enzyme, substitutes pyruvate kinase present in humans . We previously cloned and sequenced the gene that codifies for PPDK in E . histolytica . We now report its expression in a bacterial system and its purification to 98% homogeneity . We determined its K(m) for phosphoenolpyruvate, AMP and PPi (21, < 5 and 100 microM, respectively) . Unlike PPDK from maize and bacteria and pyruvate kinase from other cells, EhPPDk is dependent on divalent cations but does not require monovalent cations for activity . The enzyme has an optimum pH of 6.0, it is labile to low temperatures and has a tetrameric structure . Since EhPPDK is a PPi-dependent enzyme, we also tested the effect of some pyrophosphate analogs as inhibitors of activity . Studies on the function and structure of this enzyme may be important for therapeutic research in several parasitic diseases, since it has no counterpart in humans.

Electrophoresis, 1997 Dec, 18(15), 2852 - 6
Variability in the pattern of random amplified polymorphic DNA; Khandka DK et al.; The random amplified polymorphic DNA (RAPD) technique is a simple method to detect DNA polymorphism . It is sensitive to reaction conditions . Small changes in the reactants' concentration cause variations in amplification products . Using DNA from Asparagus officinalis, Dactylis glomerata, Mercurialis annua and Escherichia coli, we examined variability in the amplification pattern associated with reaction constituents . An increase in the ratio of Taq DNA polymerase to DNA in the reaction increased the number of amplified fragments . Increasing the concentration of primer resulted in the amplification of low molecular weight DNA fragments, while lowering the concentration resulted in high molecular weight fragments . Subsets of amplified fragments required different concentrations of magnesium for their highest intensity . Mechanical shearing of DNA obtained by sonication led to reduction in amplification of a subset of products . Enzymatic fragmentation of DNA by restriction enzymes led to loss or gain of specific fragments, depending on the DNA, primer, and restriction enzyme . RAPD markers of pooled DNA of anonymous pedigree should be critically evaluated for frequent 'false positive' markers.

Electrophoresis, 1997 Dec, 18(15), 2703 - 8
Unique identification of proteins from small genome organisms: theoretical feasibility of high throughput proteome analysis; Cavalcoli JD et al.; We evaluate current levels of accuracy for estimation of molecular weight (Mr) and isoelectric point (pI) to proteins on two-dimensional (2-D) gels as well as the distribution and clustering of proteins in the predicted proteome of E . coli . We also examine the ability to find single candidates within the predicted proteome for matching to a protein seen on 2-D gels, based on the current level of accuracy . We discuss the levels of accuracy needed to match predicted proteins to observed proteins based solely on Mr and pI criteria obtained from genomic information, and propose methodology to achieve this level of accuracy . In addition, we will address the future goals of this work since the small genomes of bacteria provide a foundation and stepping stone to similar studies in higher organisms.

J Virol Methods, 1997 Dec, 69(1-2), 147 - 58
Expression of Coxsackievirus B4 proteins VP0 and 2C in Escherichia coli and generation of virus protein recognizing antisera; Harkonen T et al.; Coxsackievirus B4 (CBV-4) capsid protein VP0 and non-structural 2C protein were expressed and purified using a glutathione-S-transferase (GST) fusion protein expression system . We used a full-size CBV-4 cDNA as a template to amplify the genes by polymerase chain reaction (PCR) . The genes were cloned into expression vector pGEX-2T and expressed as a fusion protein with GST . The GST-fusion proteins (GST-2C and GST-VP0) were purified in denatured and native forms and used to generate antibodies in rabbits . The antisera raised against GST-VP0 fusion protein recognized the corresponding structural proteins (VP0, VP2 and VP4) from purified CBV-4 preparations and infected cell lysates . In addition, cross-reactivity with CAV-9 and CBV-5 capsid proteins was observed . Anti-GST-2C antisera precipitated viral 2C protein in CBV-4-infected GMK cells, showing that the antibodies recognize the corresponding natural antigen.

Biol Chem, 1998 Jan, 379(1), 33 - 8
RNase E, the major player in mRNA degradation, is down-regulated in Escherichia coli during a transient growth retardation (diauxic lag); Barlow T et al.; The endoribonuclease RNase E plays a major part in mRNA degradation in Escherichia coli in addition to its role in processing rRNA . RNase E is encoded by an essential gene, rne, also known as ams and hmp, which is autoregulated post-transcriptionally . Here we report a transient decrease in the steady state level of the full-length rne transcript and a corresponding decline in the amount of the protein and enzymatic activity . During this period an mRNA fragment, lacking an intact 5' end, accumulates . This down-regulation of RNase E occurs under aerobic growth conditions in rich medium during a short diauxic lag in mid-exponential phase; it most likely reflects an exhaustion of a not yet identified medium compound which is followed by switching on a new metabolic pathway . During this lag, the levels of bulk protein are maintained . Our results suggest that a transient drop in the intracellular RNase E level is a means of cells to retard mRNA turnover in a period of adjustment to medium utilization . Furthermore, the here described regulation of the rne transcript and its cognate gene product seems to occur by an RNase E-independent mechanism responsive to changes in growth conditions.

J Biochem (Tokyo), 1998 Jan, 123(1), 128 - 35
Mechanism of mitochondrial import of adenylate kinase isozymes; Nobumoto M et al.; Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of the cellular adenine nucleotide composition . Three isozymes, AK1, AK2, and AK3, have so far been characterized in vertebrates . They are located in different tissues, while their primary and tertiary structures are similar . Among them, AK2 and AK3 are located in mitochondria, but unlike most mitochondrial proteins, both proteins lack a cleavable presequence . In this study, we first confirmed that AK2 is distributed in liver cells in both the cytosol and the intermembrane space of mitochondria, while AK3 is localized exclusively in the mitochondrial matrix . Next, we analyzed the process of import of AK2 and AK3 by incubating isolated rat mitochondria with proteins that were synthesized in a reticulocyte lysate translation system . The results indicated that both AK2 (an intermembrane-space-targeting protein) and AK3 (a matrix-targeting protein) require an inner membrane electrochemical potential for their import . This finding for AK2 is in contrast with those of other noncleavable intermembrane-space-targeting proteins such as cytochrome c and cytochrome c heme lyase, which do not require the membrane potential for their import . In the transport process, AK2 and AK3 competed with the adrenodoxin precursor, which is imported into the matrix through a mechanism common to other mitochondrial matrix proteins . Thus, AK2 and AK3 were thought to be translocated into mitochondria through the same pathway as that for most mitochondrial protein precursors . Neither AK2, that was previously synthesized in reticulocyte lysates, nor AK2, that was purified from an Escherichia coli overexpression system, was imported into mitochondria in a post-translational import manner . In contrast, AK3 was imported into mitochondria after completion of protein synthesis . Thus, the import of AK2 is likely to be co-translational, and the co-translational import mechanism might contribute to the bi-topological distribution of AK2 in both the cytosol and mitochondria.

J Biochem (Tokyo), 1998 Jan, 123(1), 47 - 54
Cloning and expression of human adenylate kinase 2 isozymes: differential expression of adenylate kinase 1 and 2 in human muscle tissues; Lee Y et al.; A cDNA clone coding for adenylate kinase 2B was isolated from fetal liver, and the expression of AK2 was investigated in human tissues . The ORF in the cDNA clone for human AK2B predicted a protein comprising 232 amino acids (25.6 kDa) . The features of AK2A and AK2B sequences in human were the same as those in the bovine system . Each of the recombinant proteins, AK2A and AK2B, was expressed in Escherichia coli cells, and the purified recombinant proteins were enzymatically active . The distribution of AK2 transcripts in various human tissues was examined by Northern analysis . Unlike in the bovine system, it was found that the AK2A transcript was the major form of AK2 mRNA species in all human tissues . The transcripts of AK2 isozymes were relatively abundant in heart, liver, and also in skeletal muscle, where the expression level of AK2 was known to be low . Western blot analysis of AK isozymes in human heart and skeletal muscle revealed that AK2 protein was found only in heart, whereas AK1 was detected in both tissues . These tissue-specific expressions of the AK isozymes in human might suggest the presence of organ-specific regulation of the AK2 gene including a post-transcriptional control in skeletal muscle.

J Biochem (Tokyo), 1998 Jan, 123(1), 33 - 41
Nonadditive effects of double mutations at the flexible loops, glycine-67 and glycine-121, of Escherichia coli dihydrofolate reductase on its stability and function; Ohmae E et al.; The structure, stability, and enzymatic function of dihydrofolate reductase (DHFR) from Escherichia coli are influenced by point mutations at sites 67 and 121 in two flexible loops {Gekko et al . (1994) J . Biochem . 116, 34-41; Ohmae et al . (1996) J . Biochem . 119, 703-710} . In the present study, eight double mutants at sites 67 and 121 (G67V/G121S, G67V/G121A, G67V/G121C, G67V/G121D, G67V/G121V, G67V/G121H, G67V/G121L, and G67V/G121Y) were constructed in order to identify interactions between the two sites of DHFR . The far-ultraviolet circular dichroism spectra of double mutants were clearly different from those of the respective single mutants, with significant changes being observed for three mutants, G67V/G121A, G67V/G121L, and G67V/G121S . The Gibbs free energy change of urea unfolding of double mutants could not be expressed by the sum of those of the respective single mutants except for G67V/G121H . The steady-state kinetic experiments showed that the effect of double mutations manifests itself not in Km but in k(cat), and the transition-state stabilization energy for G67V/G121A, G67V/G121C, and G67V/G121L is not equal to the sum of those for the single mutants . These results indicate that the additivity rule essentially does not hold for these double mutants, and that long-range interactions occur between sites 67 and 121, even though they are separated by 27.7 A . This is evidence that the flexible loops play important roles in the stability and function of this enzyme through structural perturbations, in which a small alteration in local atomic packing due to amino acid substitution is cooperatively magnified over almost the whole molecule.

Gac Med Mex, 1997 Nov-Dec, 133(6), 511 - 25
{Continuous and common epitopes present in fimbriae of enterotoxigenic Escherichia coli (ETEC)}; Lopez-Vidal Y; Colonization factor antigens (CFAs and PCFs) are important virulence factors of Escherichia coli (ETEC) diarrhea . Antibodies to CFAs produced after ETEC infection are protective; however, the CFA epitopes which induce protective antibodies have not yet been characterized . This study is the characterization of the immune response to CFAs at molecular level and identification of the epitopes associated with inhibition of cell-adherence and protection that will lead to the development of methods to prevent ETEC infection and disease . The aim of this study was the characterization of the linear epitopes of CFA/I that react with sera from acute and convalescent phase of ETEC-in-fected children, with adult sera from endemic and non-endemic areas, with monoclonal antibodies (Mabs) and with hyperimmune antiserum to CFAs and PCFs different from CFA/I . Three linear and common epitopes were recognized among the CFA/I in child sera and adult sera from endemic areas and with hyperimmune sera against other known CFAs and putative ETEC colonization factors.

FEMS Microbiol Lett, 1998 Feb 15, 159(2), 343 - 7
Modification of topA in Synechococcus sp . PCC 7942 resulted in mutants capable of growing under low but not high concentration of CO2; Gabay C et al.; Insertion of a cartridge encoding kanamycin resistance within an open reading frame, ORF839, in the cyanobacterium Synechococcus sp . PCC 7942 resulted in merodiploids bearing both the normal and the modified ORF839, suggesting that its gene product is essential for growth . In the absence of kanamycin the mutants were able to grow like the wild type, but in its presence the mutants grew under 0.015% CO2 in air but not under 5% CO2 in air . ORF839, identified in this study, is highly homologous to topA encoding topoisomerase I in several organisms, but it does not contain the zinc-binding motif identified in the C-terminal region of the enzyme from Escherichia coli.

FEMS Microbiol Lett, 1998 Feb 15, 159(2), 201 - 7
Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei; Schupp T et al.; The 54-kbp Type I polyketide synthase gene cluster, most probably involved in rifamycin biosynthesis by Amycolatopsis mediterranei, was cloned in E . coli and completely sequenced . The DNA encodes five closely packed, very large open reading frames reading in one direction . As expected from the chemical structure of rifamycins, ten polyketide synthase modules and a CoA ligase domain were identified in the five open reading frames which contain one to three polyketide synthase modules each . The order of the functional domains on the DNA probably reflects the order in which they are used because each of the modules contains the predicted acetate or propionate transferase, dehydratase, and beta-ketoacyl-ACP reductase functions, required for the respective step in rifamycin biosynthesis.

Vopr Med Khim, 1997 Nov-Dec, 43(6), 440 - 56
{Isolation and characterization of an evolutionary precursor of human monoamine oxidases A and B}; Singer TP et al.; An interesting flavoprotein-type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned by Schilling and Lerch (1995a,b) The properties of this MAO, as well as a substantial part of its amino acid sequence resemble those of both MAO A and B from higher animals, raising the possibility that it may be an evolutionary precursor of these mitochondrial enzymes . It differs from MAO A and B in several respect, however, including the fact that it is soluble and of peroxisomal localization and that the FAD is non-covalently attached . We have overexpressed the fungal enzyme (MAO-N) in Escherichia coli, isolated it for the first time in pure form, and, in collaboration with Dr . Elena Sablin, crystallized it . Since several of the observations of previous workers on MAO-N could not be reproduced and seem to be erroneous, we have reexamined its, substrate specificity, interaction with reversible and irreversible inhibitors and other catalytic and molecular properties . MAO-N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme . Some aspects of the substrate specificity resemble those of MAO B, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots . Contrary to the report of Schilling and Lerch (1995a), however, the fungal enzyme does not oxidize serotonin, norepinephrine, dopamine or other biogenic amines . MAO-N is irreversibly inhibited by stoichiometric amounts of both (-)deprenyl and clorgyline in a mechanism-based reaction, forming flavocyanine adducts with N(5) of the FAD, like the mammalian enzymes, but inactivation is much faster with clorgyline than deprenyl, suggesting again a closer resemblance to MAO A than B . The dissociation constants for a large number of reversible competitive inhibitors have been determined for MAO-N and comparison with similar values for MAO A and B again pointed to a much greater similarity to the former than the latter . Experiments designed to change the linkage of the FAD to covalent form by site-directed mutagenesis and to dissociate.

Nippon Yakurigaku Zasshi, 1997 Oct, 110 Suppl 1, 1P - 6P
{Transgenic plants as medicine production systems}; Okada Y; Transgenic plants are emerging as an important system for the expression of many recombinant proteins, especially those intended for therapeutic purpose . The production of foreign proteins in plants has several advantages . In terms of required equipment and cost, mass production in plants is far easier to achieve than techniques involving animal cells . Successful production of several proteins in plants, including human serum albumin, haemoglobin, monoclonal antibodies, viral antigens (vaccines), enkephalin, and trichosanthin, has been reported . Particularly, the demonstration that vaccine antigens can be produced in plants in their native, immunogenic forms opens exciting possibilities for the "bio-farming" of vaccines . If the antigens are orally active, food-based "edible vaccines" could allow economical production . In this review, I will discuss the progress that has been made by several groups in what is now an expanding area of medicine research that utilizes transgenic plants.

Biochem Mol Biol Int, 1998 Jan, 44(1), 157 - 63
Additive effects of bovine serum albumin, dithiothreitol, and glycerol on PCR; Nagai M et al.; The effects of bovine serum albumin, dithiothreitol, and glycerol on PCR were studied . PCR under standard conditions failed the amplification of an enterohemorrhagic E . coli DNA fragment when the boiled bacterial cell lysate was used as the template . The addition of either one of bovine serum albumin, dithiothreitol, or glycerol in the reaction mixture allowed the specific fragment amplification; and the optimum concentrations were as follows: bovine serum albumin, 1 mg/ml; dithiothreitol, 10 mM; and glycerol, 5% . In addition, when all of the three agents were included at the above concentrations, the PCR yield was further increased . The effect of the three-agent mixture was not the template specific . Furthermore, the mixture enabled long PCR over 20 kb when Taq DNA polymerase with 3'-5' exonuclease activity was used for the amplification . Our simple PCR method allows robust PCR independent of template purity or amplification length.

Biochem Mol Biol Int, 1998 Jan, 44(1), 69 - 77
Expression of the cDNA and purification of P0 ribosomal protein from Lupinus luteus; Mikolajczyk K et al.; Eukaryotic ribosomal protein P0 is localized on the large ribosomal subunit at the base of the specific "stalk" structure, close to the central protuberance . Recently we have obtained a clone coding for P0 ribosomal protein from L . luteus (yellow lupin) cDNA library . Here we present its expression in E . coli cells and purification of the target protein.

Am J Trop Med Hyg, 1998 Feb, 58(2), 144 - 51
Evaluation of recombinant dengue viral envelope B domain protein antigens for the detection of dengue complex-specific antibodies; Simmons M et al.; To increase the specificity of dengue (DEN) diagnosis based on antibody detection, we have evaluated recombinant proteins as antigens that incorporate most of the B domain of the DEN virus envelope protein fused to the trpE protein of Escherichia coli (trpE-DEN) . A pooled antigen consisting of trpE-DEN proteins representing all four serotypes of DEN virus was used in an indirect ELISA for the detection of IgG or IgM antibody . This assay was compared with a standard IgG indirect ELISA and an IgM-capture ELISA using DEN virus-infected cell culture pooled antigens . The results indicated that the trpE-DEN antigens and the cell culture antigens were equally sensitive for detecting IgM and IgG antibodies in convalescent sera from Peru and Indonesia representing virus isolation-confirmed primary and secondary DEN infections, respectively . Fourteen day postinfection IgG antibody-positive sera obtained from individuals infected with DEN-1 virus who had been vaccinated with other flaviviruses were more strongly reactive with the cell culture antigen than with the recombinant antigen, but by day 21 postinfection, a strong antibody response to the trpE-DEN antigens was present . These results suggested that the early antibody response was directed predominantly towards shared flavivirus group antigens that were not detected with the trpE-DEN antigens . Comparison of the trpE-DEN-1 recombinant antigen with a DEN-1 virus-infected cell lysate antigen for the detection of IgG antibody in sera from a cohort of 55 individuals from Peru who seroconverted over a one-year period indicated greater specificity for the recombinant antigens . Also, sera from individuals with no known DEN infections that had been sequentially vaccinated with yellow fever and Japanese encephalitis reacted with the DEN virus cell culture antigen in the IgG ELISA, but did not react with the trpE-DEN pooled antigens . Similarly, YF IgM antibody positive samples that showed cross-reactivity with the DEN virus cell culture antigens, did not react with the trpE-DEN pooled antigens . These results indicated that the trpE-DEN pooled antigen provided a more specific diagnosis of dengue infections than DEN virus-infected cell culture antigen and avoided the biohazards associated with handling live virus during the preparation of diagnostic reagents . The trpE-DEN pooled antigen should permit a better approach to distinguish between past DEN and other flavivirus infections in epidemiologic surveys, and also increase the specificity of serologic diagnosis of acute DEN infections.

DNA Cell Biol, 1998 Feb, 17(2), 169 - 75
Polyribonucleotide phosphorylase is a double-stranded DNA-binding protein; Zhang P et al.; Polyribonucleotide phosphorylase (PNPase) is one of the critical components of the E . coli RNA degradosome, which consists of both PNPase and endoribonuclease RNase E . The function of this complex is to control the rate of mRNA degradation . The PNPase possesses two enzymatic activities, namely 3'-5' processive exoribonuclease activity and 5'-3' RNA polymerase activity . In the present study, we used conventional chromatography to purify an E . coli protein that binds to a specific double-stranded DNA sequence . Microsequencing of the purified protein showed that this DNA-binding protein was PNPase . Our data further demonstrate that PNPase binds to DNA in a sequence-specific manner . These data suggest that PNPase may have previously unappreciated DNA-related functions in addition to its known role in mRNA degradation.

Neuroscience, 1998 Apr, 83(4), 1245 - 50
The sympathetic nervous system tonically inhibits peripheral interleukin-1beta and interleukin-6 induction by central lipopolysaccharide; De Luigi A et al.; To study the role of the sympathetic nervous system in the induction of inflammatory cytokines elicited by central lipopolysaccharide, sympathetic chemical denervation was performed by intraperitoneal injection of 6-hydroxydopamine . Rats received the neurotoxin according to the following schedule: 50 mg/kg on days 1 and 2, 100 mg/kg on days 3, 4 and 7 . On day 8, lipopolysaccharide (2.5 microg/6 microl/rat) was injected intracerebroventricularly and rats were killed 2 h later . 6-Hydroxydopamine reduced noradrenaline and dopamine content in the spleen by 88.7% and 88.8% respectively, without affecting striatal contents indicating that the chemical sympathectomy had been effective and selective . In sympathectomized rats, lipopolysaccharide raised interleukin-1beta and interleukin-6 serum levels more than in control rats given the vehicle . Tumour necrosis factor-alpha serum levels in sympathectomized rats were no different from those in vehicle-treated rats . Interleukin-1beta and interleukin-6 messenger RNA expression, measured by northern blot analysis, was clearly detectable in adrenals and spleen of rats given lipopolysaccharide . Sympathectomy increased lipopolysaccharide-induced interleukin-1beta and interleukin-6 messenger RNA in adrenals and spleen . Corticosterone basal levels were raised by central lipopolysaccharide and not further changed by sympathectomy . The present study shows that sympathetic nervous system denervation enhances the synthesis and production of peripheral interleukin-1beta and interleukin-6 in rats given central lipopolysaccharide and suggests a tonic inhibitory control of the sympathetic nervous system on these inflammatory cytokines.

J Pharm Biomed Anal, 1997 Dec, 16(4), 561 - 72
A non-isotopic probe-hybridization assay for residual DNA in biopharmaceuticals; Riggin A et al.; Although most biopharmaceuticals are highly purified, there is a theoretical concern that such recombinant products could be contaminated with oncogenic or bacterial DNA . A crucial part of the control of such biologicals is to ensure they do not contain more residual DNA than a safety limit suggested by the regulatory agency . Currently, the FDA has suggested a 100 pg per dose limit for residual DNA . DNA probes labeled with a radioisotope such as 32P have been commonly used in hybridization tests . Because of the radiation safety concern, we chose to develop a procedure for assessing DNA levels by either a dot or slot blot hybridization technique using a nonisotopic DNA probe and immuno-enzymatic detection . A minimum detectable limit (MDL) of < 10 pg DNA mg(-1) protein can be achieved . Method validation data demonstrated that the precision, reproducibility, and robustness of this approach are appropriate for quality control.

Invest Ophthalmol Vis Sci, 1998 Mar, 39(3), 658 - 61
Mice deficient in tumor necrosis factor receptors p55 and p75, interleukin-4, or inducible nitric oxide synthase are susceptible to endotoxin-induced uveitis; Smith JR et al.; PURPOSE: To investigate the roles of tumor necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4), and inducible nitric oxide synthase (iNOS) in endotoxin-induced uveitis (EIU) using gene knock-out mice . METHODS: Mice (C57BL/6 x 129) either of normal phenotype or deficient in the genes encoding one or both tumor necrosis factor receptors (TNFR p55 and TNFR p75), IL-4, or iNOS were given footpad injections of 400 micrograms Escherichia coli lipopolysaccharide . Animals were killed 24 hours later, and infiltrating cells were counted on 5-micron ocular cross-sections through the optic nerve . RESULTS: All abnormal mouse phenotypes were susceptible to EIU . Yet, TNFR p55 and IL-4 gene knock-out mice experienced less ocular inflammation than control animals (P = 0.021 and 0.007, respectively), whereas disease was not reduced for iNOS-deficient mice . Mice deficient in TNFR p55 and TNFR p75 experienced milder EIU than mice lacking TNFR p75 alone (P = 0.046) . CONCLUSIONS: Mice deficient in TNFR p55 and TNFR p75, IL-4, or iNOS retain the susceptibility to EIU, but TNF-alpha and IL-4 influence the influx of inflammatory cells to the eye during this disease.

Exp Parasitol, 1998 Jan, 88(1), 43 - 50
Dirofilaria immitis: molecular cloning and expression of a cDNA encoding a selenium-independent secreted glutathione peroxidase; Tripp C et al.; A cDNA clone, Di29, encoding a homolog of glutathione peroxidase, was isolated from a Dirofilaria immitis adult female cDNA expression library by a combination of polymerase chain reaction amplification with primers designed from the Brugia pahangi glutathione peroxidase gene sequence and hybridization screening of D . immitis cDNA libraries . The Di29 nucleotide and deduced amino acid sequences were very similar to those described for lymphatic filariae and predicted a secreted form of glutathione peroxidase with a cysteine residue substituted for selenocysteine in the active site . The cDNA clone was expressed in Escherichia coli and Spodoptera frugiperda Sf9 insect cells, and the resulting recombinant proteins were purified for antibody production and assessment of enzymatic properties, respectively . An antiserum generated against the E . coli-expressed protein detected a protein of 29 kDa in D . immitis via immunoblotting . This protein is expressed in adult worms (both sexes) and fourth stage larvae generated via 6 days of in vitro culture, but was undetectable in microfilariae, and third stage larvae obtained either directly from mosquitoes or following 2 days of culture . The Di29-encoded recombinant protein was secreted from Sf9 insect cells and displayed low-level glutathione peroxidase activity against a range of hydroperoxide substrates, including hydrogen peroxide.

Biosci Biotechnol Biochem, 1998 Jan, 62(1), 193 - 5
Compensation for D-glutamate auxotrophy of Escherichia coli WM335 by D-amino acid aminotransferase gene and regulation of murI expression; Liu L et al.; D-glutamate, an indispensable component of peptidoglycans of bacteria, is provided by glutamate racemase in E . coli cells . Compensation for D-glutamate auxotrophy of E . coli WM335 cells lacking the glutamate racemase gene, murI, with the D-amino acid aminotransferase gene suggests that presence of a threshold concentration for the D-glutamate required by E . coli cells, as well as a regulation system for murI expression.

Biosci Biotechnol Biochem, 1998 Jan, 62(1), 136 - 41
Expression of biologically active human calpastatin in baculovirus-infected insect cells and in Escherichia coli; Hitomi K et al.; Calpastatin, an endogeneous inhibitor protein acting on calpain (Ca(2+)-dependent cysteine proteinase), is widely distributed in animal tissues and cells . Two different expression systems, baculovirus-infected Spodoptera frugiperda (Sf9) insect cells and Escherichia coli, were used for overexpression of the human calpastatin tagged with N-terminal hexahistidine peptide . Recombinant calpastatin was purified to homogeneity by nickel ion affinity chromatography and gel filtration separation . Purified recombinant proteins from both systems have similar inhibitory activity for calpain.

Biosci Biotechnol Biochem, 1998 Jan, 62(1), 78 - 82
Molecular interaction between proteins involved in EvgAS signal transduction of Escherichia coli; Tanabe H et al.; EvgA and EvgS constitute one two-component signal transduction system in Escherichia coli . Although probable signaling domains of these proteins have been estimated, the molecular mechanism of their interaction remains to be elucidated . Here, we investigated protein to protein interactions between EvgA and EvgS and also between the EvgAS system and other related signaling pathways by means of surface plasmon resonance . EvgA and EvgS interacted directly and inhibition of phosphorylation of their functional domains abolished formation of the EvgAS complex . No interaction was observed either between EvgA and Bordetella BvgS or BvgA and EvgS . OmpR, a response regulator for the osmoregulative gene expression of E . coli, had similar but not identical behavior towards EvgS to that of EvgA . These results indicate that interaction between the signaling proteins is closely related to phosphorylation of the functional domain of the proteins.

Biosci Biotechnol Biochem, 1998 Jan, 62(1), 60 - 5
Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth; Takaya N et al.; We cloned a chitinase-encoding gene from Aspergillus nidulans by polymerase chain reaction using degenerated oligonucleotide primers designed from the conserved amino acid sequences among chitinases from yeasts and Rhizopus spp . The cloned gene, named chiA, encoded a polypeptide consisting of 660 amino acids . Disruption of chiA had no effect on hyphal or conidiophore morphology, but germination frequency and hyphal growth rate decreased substantially . Expression of chiA was investigated using Escherichia coli beta-galactosidase as a reporter enzyme . The beta-galactosidase activity was present during hyphal growth and increased twice as the conidiophores developed . In situ staining of beta-galactosidase activity found high expression in metulae, phialides, and conidia during conidiophore development, indicating that the expression of chiA is developmentally regulated . This is the first report to isolate a chitinase gene from A . nidulans and investigate its functions using the gene disruption technique and gene fusion methods in filamentous fungi.

Appl Environ Microbiol, 1998 Mar, 64(3), 1163 - 5
Expression of psychrophilic genes in mesophilic hosts: assessment of the folding state of a recombinant alpha-amylase; Feller G et al.; Alpha-Amylase from the antarctic psychrophile Altermonas haloplanktis is synthesized at 0 +/- 2 degrees C by the wild strain . This heat-labile alpha-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation . In the described expression system, a compromise between enzyme stability and E . coli growth rate is reached at 18 degrees C.

Rev Sci Tech, 1997 Aug, 16(2), 472 - 81
Risks and prevention of contamination of dairy products; Cullor JS; Consumers and regulatory officials are becoming increasingly aware of the human health risk of the presence of micro-organisms or chemicals in the agricultural environment . Providing 'on-farm food safety' programmes which address the daily management of the production unit with regard to animal health and well-being, public health and environmental health must be a top priority for agriculturalists and veterinarians . Developing critical control point management (CCPM) procedures for animal and human health concerns is a viable approach to aid in alleviating public concerns about dairy products and the food supply in general . Such CCPM programmes may be created for individual production units based upon risk analysis, total quality management and hazard analysis and critical control point principles . Implementation of these programmes will be essential both in addressing food safety concerns for the resident population of a nation and in developing or maintaining international markets for the export of animal products.

Transplantation, 1998 Feb 27, 65(4), 583 - 5
Serologic association of human herpesvirus eight with posttransplant Kaposi's sarcoma in Saudi Arabia; Qunibi W et al.; BACKGROUND: This study investigates the association between human herpesvirus eight (HHV8) and Kaposi's sarcoma (KS), the most common cancer occurring in renal transplant recipients in Saudi Arabia . METHODS: A cross-sectional study of seroreactivity to HHV8 antigens in posttransplant KS patients from a tertiary care hospital in Riyadh, Saudi Arabia, and in control subjects without KS was conducted . Seroreactivity rates were determined using immunoblotting assays to detect antibodies to two lytic cycle HHV8 antigens: p40, an antigen found in infected cells, and sVCA, an HHV8-encoded small viral capsid antigen expressed in Escherichia coli . RESULTS: Antibodies to HHV8 p40 and sVCA were present in a significantly higher proportion of renal transplant patients with KS (13 of 14 patients) compared to renal transplant patients without KS (5 of 18; P<0.001) and compared to other control individuals (6 of 44; P<0.001) . HHV8 seroreactivity was more common among patients with renal failure (28%) than among other control groups (7%) . CONCLUSIONS: The serologic results provide evidence of a strong association between HHV8 and posttransplant KS in Saudi ArabiaPIP: In Saudi Arabia, Kaposi's sarcoma occurs in 4.1% of renal transplant recipients and accounts for 70% of malignancies in this group . Human herpes virus 8 (HHV8) has been identified in the DNA of many of these patients . The association between HHV8 and Kaposi's sarcoma was investigated further in post-renal transplant Kaposi's sarcoma patients from a tertiary care hospital (King Faisal Specialist Hospital and Research Center) in Riyadh, Saudi Arabia (n = 14), and non-Kaposi's sarcoma controls with renal transplant (n = 18), chronic renal failure (n = 14), other cancers that did not affect renal function (n = 15), and healthy volunteers (n = 15) . The median time from transplant to Kaposi's sarcoma was 13 months . A serum sample was assumed to have antibodies to HHV8 if antibody to either p40 or sVCA was detected . The prevalence of HHV8 seroreactivity was 13/14 (93%) in cases, 5/18 (28%) in renal transplants without Kaposi's sarcoma, and 11/62 (18%) in the aggregate control group . HHV8 seroreactivity was significantly more common (p 0.001) among transplant patients with Kaposi's sarcoma than those without this cancer (odds ratio, 33.80; 95% confidence interval, 2.96-904) . These findings suggest an etiologic link between HHV8 and Kaposi's sarcoma presumably due to immunologic or cellular factors that influence host-virus interactions .

Cancer Res, 1998 Mar 1, 58(5), 1013 - 20
Engineering human DNA alkyltransferases for gene therapy using random sequence mutagenesis; Encell LP et al.; O6-Alkylguanine-DNA alkyltransferase (AGT) is a suicide enzyme that repairs alkylation damage at the O6 position of guanine in DNA . The essentiality of a limited number of amino acid residues at the active site has been determined by site-directed mutagenesis . We used random mutagenesis techniques to create a plasmid library of > 10(6) human AGT mutants with substitutions at residues 150-172 and selected for clones with mutations rendering Escherichia coli resistant to both the alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the AGT inhibitor, O6-benzylguanine (BG) . On sequencing surviving clones resistant to MNNG in the presence and absence of BG, we found that a majority of the clones contained multiple substitutions at mostly nonconserved positions . We selected nine mutants resistant to a combination of MNNG and BG, and the survival of these mutants was as much as 341-fold higher than that of cells harboring wild-type AGT under the same conditions . Each of the mutants contained at least three amino acid substitutions and as many as eight, suggesting that maximum resistance to MNNG in the presence of BG requires even more substitutions than resistance to MNNG alone . BG is being tested clinically as a way to sensitize tumors to chemotherapeutic alkylating agents . Therefore, our BG-resistant mutants hold strong potential as gene therapy candidates for protecting normal tissue in patients receiving BG in combination with alkylating agents for the treatment of cancer.

Adv Exp Med Biol, 1997, 428, 333 - 41
Skeletal muscle function, oxygenation and biochemistry in an endotoxemic model of SIRS; Iannoli ED et al.; We have developed a reproducible low-dose endotoxin model which is useful for the investigation of early SIRS . The data confirm that organ function cannot be inferred from whole animal data (e.g . SVR vs . MVR) . Thus, the study of SIRS at the organ and cellular level is essential . Decreased skeletal muscle oxygen consumption with 4 Hz exercise in early SIRS may be related to depletion of physiologic reserves, especially microcirculatory reserves, as suggested by decreased myoglobin saturation and decreased energy charge . Using this model, we will investigate whether organ dysfunction in SIRS is due to oxygen-limited cellular ATP production or impaired cellular metabolism.

Nurs Res, 1998 Jan-Feb, 47(1), 19 - 24
Meperidine attenuates the secretion but not the transcription of interleukin 1 beta in human mononuclear leukocytes; McCarthy DO et al.; BACKGROUND: The infusion of amphotericin-B (AmB) often produces clinically distressing rigors and chills, which promptly abate with intravenous injection of meperidine, although its mechanism of action is unknown . OBJECTIVE: To examine the effects of meperidine on the transcription or secretion of Interleukin 1 beta (IL-1 beta) in human mononuclear leukocytes (MNL) exposed in vitro to the lipopolysaccharide (LPS) contained in Escherichia coli endotoxin or to AmB . METHODS: Blood was drawn from eight healthy adult volunteers . The blood was centrifuged, and the layer containing MNL was separated; incubated with various combinations of medium, meperidine, and AmB; then tested for IL-1 content to determine the effect of meperidine on MNL secretion of IL-1 beta . To determine the effect on MNL transcription of IL-1 beta, the RNA was extracted from cells and the IL-1 beta was measured using one of two different methods . RESULTS: Incubation of human MNL in the presence of LPS or AmB significantly increased transcription of IL-1 beta mRNA and secretion of IL-1 beta . Addition of meperidine to these cultures significantly reduced LPS-induced, but not AmB-induced, secretion of IL-1 beta in vitro . Meperidine did not alter IL-1 beta mRNA levels in MNL exposed to LPS or AmB . CONCLUSIONS: These data suggest that meperidine decreases rigors and chills in part by decreasing MNL secretion of IL-1 beta through a posttranscriptional mechanism.

Biotechnol Appl Biochem, 1998 Feb, 27 ( Pt 1), 55 - 61
Structure and expression of the D-amino-acid oxidase gene from the yeast Rhodosporidium toruloides; Liao GJ et al.; D-Amino-acid oxidase (DAO2; EC 1.4.3.3) catalyses the oxidative deamination of D-amino acids to alpha-keto acids and ammonia . The purified DAO protein from Rhodosporidium toruloides was used to determine its amino acid sequence . Three internal peptide sequences, YCQYLARELQ, IAGIDDQAAEPIR and RCTMDSSDP, were obtained and used to synthesize four fully degenerated oligonucleotides for cloning of the DAO gene . Both cDNA and genomic DNA encoding R . toruloides DAO were cloned and sequenced . Comparison of these two DNA sequences revealed that the DAO gene contains six exons and five introns . The gene encodes a polypeptide of 368 amino acids with a calculated molecular mass of 40,079 Da . Using an Escherichia coli protein expression system, the DAO protein of R . toruloides can easily be produced in an active form and purified in a large quantity.

Arch Biochem Biophys, 1998 Feb 15, 350(2), 333 - 9
Structural determinants of progesterone hydroxylation by cytochrome P450 2B5: the role of nonsubstrate recognition site residues; He YQ et al.; The highly related rabbit cytochromes P450 2B4 and 2B5 differ in only 12 amino acid positions, but only 2B5 has activity toward progesterone . Previously, simultaneous site-directed mutagenesis of four key substrate recognition site (SRS) residues (114, 294, 363, and 367) was shown to result in interconversion of the androstenedione hydroxylase specificities of cytochrome P450 2B4 and 2B5 . However, the progesterone metabolite profiles of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue were not identical to that of P450 2B5 . Therefore, single mutants of P450 2B5 at the remaining seven positions were constructed, expressed in Escherichia coli, and studied with progesterone as the substrate . The single mutants at positions 120 and 221, which are outside any known SRS, exhibited a significant alteration in progesterone hydroxylation . Based on these results, Ile-114, Arg-120, Ser-221, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 were replaced simultaneously with Phe, His, Pro, Thr, Val, and Ala, respectively, from 2B5 . This yielded a mutant with a very similar progesterone metabolite profile to that of 2B5, although the total activity was lower . An additional substitution at residue 370 produced a multiple mutant P450 2B4 I114F-R120H-S221P-S294T-I363V-V367A- T370M with very similar or identical substrate specificity, regio- and stereospecificity and kinetic properties to that of P450 2B5 wild type .

Arch Biochem Biophys, 1998 Feb 15, 350(2), 324 - 32
Role of the alanine at position 363 of cytochrome P450 2B2 in influencing the NADPH- and hydroperoxide-supported activities; Hanna IH et al.; Escherichia coli was used to express the two closely related cytochromes P450 2B1 and 2B2 and two mutants of 2B2 in which residues Gly-303 and Ala-363 were replaced by Ser and Val, respectively . The expressed proteins were partially purified and assayed for benzphetamine and n-octylamine (NOA) binding and 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (EOD), benzphetamine N-demethylation (BND) and 7,12-dimethylbenz{a}anthracene (DMBA) hydroxylation activities in the presence and absence of cytochrome b5 . The Kd values for benzphetamine and NOA obtained for the wild-type enzymes were similar to reported values . The Ala-363 --> Val mutant (A363V) of 2B2 exhibited Kd values for both ligands that were more similar to 2B1 than to 2B2 . The EOD and BND activities of the A363V mutant were 10- and 3.8-fold those exhibited by 2B2, respectively . With DMBA, the A363V mutation led to a 6-fold increase in the hydroxylation activity at the 7-methyl substituent while the hydroxylation activity at the 12-methyl substituent was slightly suppressed . The 7-hydroxymethyl:12-hydroxymethyl product ratio obtained with the A363V mutant (1.3) was much closer to the ratio obtained with 2B1 (1 . 9) than to that obtained with 2B2 (0.17) . Conversely, the Gly-303 --> Ser substitution did not influence the characteristics of the 2B2-catalyzed metabolism of DMBA to the same magnitude . When cumene hydroperoxide (CHP) was used to support the EOD activities of the proteins, 2B2 exhibited a 2- to 20-fold greater activity than 2B1 or either of the mutants . Examination of the CHP-derived products of the EOD reactions revealed the formation of mainly 2-phenyl-2-propanol due to the heterolytic cleavage of CHP . However, only the 2B1 EOD-reaction mixture also contained the P450-mediated CHP-isomerization products 2-phenyl-1,2-propanediol and 2-(p-hydroxyphenyl)-2-propanol . The formation of these products with 2B1 but not 2B2 may explain why 2B1 is not as efficient as 2B2 or 2B2-G303S in carrying out the CHP-supported reactions .

Arch Biochem Biophys, 1998 Feb 15, 350(2), 298 - 306
Cloning, expression, and characterization of a type II 3-dehydroquinate dehydratase gene from Streptomyces hygroscopicus; Florova G et al.; A gene encoding dehydroquinate dehydratase (DHQase) was cloned from Streptomyces hygroscopicus var . ascomyceticus . The 528-bp open reading frame specified a primary translation product of 175 amino acids with a calculated Mr of 18,789 . The predicted amino acid sequence of the DHQase showed similarities to bacterial and fungal type II DHQases . Overexpression of the dhq gene was accomplished in Escherichia coli using a gene fusion technique in which a malE, the gene encoding the maltose binding protein (MBP), was fused via a short oligonucleotide region to the beginning of dhq . The recombinant MBP-DHQase fusion protein was purified by affinity chromatography and cleaved using thrombin . The resulting DHQase, separated from the MBP, demonstrated typical properties of a type II DHQase: a relatively high Km for the dehydroquinate substrate (650 microM) and extreme thermal stability . The subunit Mr estimated by SDS-PAGE was 19,000, and the native Mr estimated by gel-exclusion chromatography and sucrose-density centrifugation was 130,000, suggesting that the enzyme is a homoheptamer (type II DHQases are typically homododecamers) . The MBP-DHQase complex also adopted a heptameric structure and was a thermostable, fully active DHQase, indicating that the N-terminus is not involved in formation of protomer-protomer complexes . Previous analyses have supported positioning the N-terminus of type II DHQases close to the active site and a conformational change in this region coincident with ligand binding . Nonetheless, the Km and relative kcat obtained for MBP-DHQase were indistinguishable from those observed for DHQase . Inactivation data of the DHQase from S . hygroscopicus with the arginine-specific reagent phenylglyoxal showed that a modified Arg residue(s) is likely close to the N-terminus and active site of DHQase, but does not play an essential role in catalysis .

Arch Biochem Biophys, 1998 Feb 15, 350(2), 266 - 72
Salt-stable complexes of the Escherichia coli RecBCD enzyme bound to double-stranded DNA; Gabbidon MR et al.; We have examined binding of the RecBCD enzyme to linear double-stranded DNA under two types of conditions . Binding in the absence of ATP can be measured by a nitrocellulose filter binding assay or by gel retardation on polyacrylamide gels . The binding is tightest to ends with four-nucleotide single-stranded 3'-termini (3'-overhang > 5'-overhang > blunt ends) . The Kd for blunt ends is 3 . 0 (+/-0.5) nM, in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 . The binding is weakened in the presence of NaCl, with none detected at 0.5 M NaCl . Binding in the presence of ATP and low MgCl2 concentrations ("unwinding conditions") can be measured by the filter assay and on agarose gels . Enzyme-DNA complexes allowed to form under unwinding conditions are not affected by 0.5 M NaCl . ATP hydrolysis continues and the complexes dissociate at a rate similar to those to which no salt is added . These enzyme-DNA complexes can be trapped with EDTA, and they are unaffected for at least 1 h by 0.5 M NaCl or heparin . The results show that the enzyme-DNA interactions are different when the enzyme is bound to partially unwound DNA compared to when it is bound to the ends of fully duplex DNA .

Arch Biochem Biophys, 1998 Feb 15, 350(2), 223 - 32
Analysis of four residues within substrate recognition site 4 of human cytochrome P450 3A4: role in steroid hydroxylase activity and alpha-naphthoflavone stimulation; Domanski TL et al.; Sequence alignment of human cytochrome P450 3A4 with bacterial enzymes of known structure has provided a basis from which to predict residues involved in substrate oxidation . Substitutions were made at four residues (I301, F304, A305, and T309) predicted to be located within the highly conserved substrate recognition site 4 . Site-directed mutants engineered to contain carboxy-terminal histidine tags were expressed in Escherichia coli and purified on a metal affinity column . The integrity of each protein was assessed by SDS-polyacrylamide gel electrophoresis and immunoblotting . Functional analysis was performed using progesterone and testosterone as substrates and alpha-naphthoflavone as an activator . In testosterone hydroxylase assays, all of the mutants displayed rates of total product formation similar to wild-type 3A4, with several mutants showing small differences in specific products formed . However, with progesterone as the substrate, mutants F304A, A305V, and T309A exhibited altered product ratios and/or changes in the rates of product formation . F304A and A305V also displayed altered flavonoid stimulation that resulted in product ratios dramatically different from wild-type 3A4 . Therefore, the kinetics of progesterone hydroxylation of these mutants and the wild-type enzyme were further assessed, and the data were analyzed with the Hill equation . Results with wild-type 3A4 and F304A indicated that at high progesterone concentrations, hydroxylation rates and product ratios are independent of the presence of alpha-NF . This suggests that progesterone may be equivalent to alpha-NF as an activator . In contrast, A305V exhibited autoactivation by progesterone but inhibition by alpha-NF .

Anal Biochem, 1998 Feb 15, 256(2), 185 - 91
One-day enzymatic synthesis and purification of UDP-N- {1-14C}acetyl-glucosamine; Leiting B et al.; UDP-GlcN{1-14C}Ac was synthesized in a single enzymatic reaction from {1-14C}acetate and commercially available precursors on both a microcurie (micromole) and a millicurie (millimole) scale . The reaction was catalyzed by the action of acetyl coenzyme A synthetase, inorganic pyrophosphatase, and the bifunctional Escherichia coli GlmU protein . Within 2 h 86 to 94% reaction is attained, and it approaches 99% completion overnight . GlmU protein was prepared in the form of a fusion suitable for nickel chelate affinity chromatography . Several methods were developed for rapid purification of UDP-GlcN{1-14C}Ac: an HPLC method handled micromole (microcurie) loads . Alternatively, ion exchange chromatography over DOWEX AG1 X-2 using a batch elution procedure was compatible with millimole (millicurie) amounts of radiolabel and yielded both chemically and radiochemically homogeneous UDP-GlcN{1-14C}Ac . These methods allow laboratories to quickly produce and purify microcurie to millicurie quantities of N-acetyl-labeled UDP-GlcNAc by a choice of methods from relatively inexpensive precursors .

Blood, 1998 Mar 1, 91(5), 1609 - 15
Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD100 Escherichia coli; Taylor FB et al.; Antitissue factor antibody attenuated the coagulopathic and lethal responses to LD100 Escherichia coli, whereas active site inhibited factor Xa inhibited only the coagulopathic response . In this study, we wished to determine: (1) whether active site inhibited factor VIIa blocks the coagulopathic and/or attenuates the lethal effects of LD100 E coli and (2) whether these effects are accompanied by attenuation of the inflammatory cytokine response to LD100 E coli . Eight baboons infused for 2 hours with LD100 E coli also were given five bolus infusions of DEGR VIIa of 280 microg/kg at T = -10 minutes, +2, 4, 6, and 8 hours and observed for changes in vital signs, and the concentrations of hemostatic components (fibrinogen, platelets, fibrin degradation products) and inflammatory mediators (tumor necrosis factor {TNF}, interleukin-6 {IL-6}, IL-8) at T = 0, 1, 2, 4, 6, and 8 hours . Eight control baboons were also infused with LD100 E coli alone and followed as described above . Four of the eight baboons treated with DEGR VIIa were permanent 7-day survivors versus none in the control group . The mean survival times for the treated and control groups were 116 +/- 22 and 26 +/- 8 hours, respectively . These values differed significantly from each other, (P = .0008) . The decrease in platelet and fibrinogen concentrations and the increase in fibrin degradation products observed in the control group were significantly attenuated in the treated group, as was thrombosis of renal glomerular capillaries . Treatment with DEGR VIIa showed no effect on the peak TNF response to LD100 E coli at T = 2 hours (170 +/- 32 v 120 +/- 35 ng/mL) . DEGR VIIa, however, did attenuate the IL-6 and IL-8 responses at T = 8 hours (ie, the IL-6 concentrations were 81 +/- 10 for treated and 1,256 +/- 236 for the control groups and the IL-8 concentrations were 28 +/- 3.9 for the treated and 60 +/- 8.2 for the control group) . These values for IL-6 and IL-8 differed significantly from each other between the treated and control groups (P = .0001 and .0074, respectively) . It should be noted that the initial responses of IL-6 and IL-8 up to T = 4 hours were not attenuated . We concluded that DEGR VIIa treatment attenuates inflammatory, as well as hemostatic system responses to LD100 E coli . We hypothesize that this occurs through interference with the assembly and/or interactions of tissue factor/VIIa complexes.

Genes Dev, 1998 Feb 15, 12(4), 547 - 56
Different core promoters possess distinct regulatory activities in the Drosophila embryo; Ohtsuki S et al.; There are numerous examples of shared enhancers interacting with just a subset of target promoters . In some cases, specific enhancer-promoter interactions depend on promoter competition, whereby the activation of a preferred target promoter precludes expression of linked genes . Here, we employ a transgenic embryo assay to obtain evidence that promoter selection is influenced by the TATA element . Both the AE1 enhancer from the Drosophila Antennapedia gene complex (ANT-C) and the IAB5 enhancer from the Bithorax complex (BX-C) preferentially activate TATA-containing promoters when challenged with linked TATA-less promoters . In contrast, the rho neuroectoderm enhancer (NEE) does not discriminate between these two classes of promoters . Thus, certain upstream activators, such as Ftz, prefer TATA-containing promoters, whereas other activators, including Dorsal, work equally well on both classes of promoters . These results provide in vivo evidence that different core promoters possess distinct regulatory activities . We discuss the possibility that an invariant TFIID complex can adopt different conformations on the core promoter.

Genes Dev, 1998 Feb 15, 12(4), 527 - 37
The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase; Lafontaine DL et al.; Many or all of the sites of pseudouridine (Psi) formation in eukaryotic rRNA are selected by site-specific base-pairing with members of the box H + ACA class of small nucleolar RNAs (snoRNAs) . Database searches previously identified strong homology between the rat nucleolar protein Nap57p, its yeast homolog Cbf5p, and the Escherichia coli Psi synthase truB/P35 . We therefore tested whether Cbf5p is required for synthesis of Psi in the yeast rRNA . After genetic depletion of Cbf5p, formation of Psi in the pre-rRNA is dramatically inhibited, resulting in accumulation of the unmodified rRNA . Protein A-tagged Cbf5p coprecipitates all tested members of the box H + ACA snoRNAs but not box C + D snoRNAs or other RNA species . Genetic depletion of Cbf5p leads to depletion of all box H + ACA snoRNAs . These include snR30, which is required for pre-rRNA processing . Depletion of Cbf5p also results in a pre-rRNA processing defect similar to that seen on depletion of snR30 . We conclude that Cbf5p is likely to be the rRNA Psi synthase and is an integral component of the box H + ACA class of snoRNPs, which function to target the enzyme to its site of action.

J Biol Chem, 1998 Feb 20, 273(8), 4556 - 62
Mutational analysis of the roles of residues in Escherichia coli elongation factor Ts in the interaction with elongation factor Tu; Zhang Y et al.; The crystal structure of the Escherichia coli elongation factor (EF)-Tu.Ts complex indicates that there are extensive contacts between EF-Tu and EF-Ts . To determine the importance of these contacts in the interaction between E . coli EF-Tu and EF-Ts, residues in EF-Ts at the interface of these two proteins were mutated . The binding constants governing the interaction of the resulting EF-Ts variants with E . coli EF-Tu were determined . The effects of these mutations on the ability of EF-Ts to stimulate GDP exchange with EF-Tu.GDP and on its ability to stimulate the activity of EF-Tu in polymerization were tested . The results indicate that Arg-12, Met-19, and Met-20 in the N-terminal domain of EF-Ts and His-147 and Lys-166 and/or His-167 in subdomain C of EF-Ts are crucial in the interaction between EF-Tu and EF-Ts . Lys-23, Val-234, Met-235, and the C-terminal helix h13 are less important . The binding constants of the EF-Ts variants governing their interactions with EF-Tu correlate well with their activities in stimulating GDP exchange with EF-Tu . Mutations prepared in EF-Tu indicate that His-19 and Gln-114 but not Glu-348 in EF-Tu are moderately important for its interaction with EF-Ts.

J Biol Chem, 1998 Feb 20, 273(8), 4387 - 91
Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach; Laurberg M et al.; The function of the N-terminal region of elongation factor Tu is still unexplained . Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly . In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate . The resulting four point mutants were expressed and purified using the pGEX system . The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA) . The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA . Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding . Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region.

J Biol Chem, 1998 Feb 20, 273(8), 4317 - 22
Identification of histidine 45 as the axial heme iron ligand of heme oxygenase-2; Ishikawa K et al.; A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (DeltaHHO2) was expressed in Escherichia coli . To identify the axial heme ligand of HO-2, His-45 to Ala (DeltaH45A) and His-152 to Ala (DeltaH152A) mutants have been prepared using this expression system . DeltaH45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity . A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-DeltaH45A complex . The DeltaH152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis . The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction . The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume . The gel filtration-purified DeltaH152A exhibited spectroscopic and enzymatic properties identical to those of wild-type . We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch . Biochem . Biophys . 302, 402-408; McCoubrey, W . K., Jr., Huang, T . J., and Maines, M . (1997) J . Biol . Chem . 272, 12568-12574), that His-45, but not His-152, in heme oxygenase isoform-2 is the proximal ligand of the heme and is essential for the heme degradation activity of the enzyme . His-152 appears to play a structural role in stabilization of the heme oxygenase protein.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1944 - 9
Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone; Schuurink RC et al.; We have used Arabidopsis calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins . The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K+ channels . This cDNA was designated HvCBT1 (Hordeum vulgare CaM binding transporter) . Hydropathy plots of HvCBT1 showed the presence of six putative transmembrane domains, but sequence alignment indicated a pore domain that was unlike the consensus domains in K+ and olfactory channels . Expression of a subclone of amino acids 482-702 in Escherichia coli generated a peptide that bound CaM . When a fusion protein of HvCBT1 and green fluorescent protein was expressed in barley aleurone protoplasts, fluorescence accumulated in the plasma membrane . Expression of HvCBT1 in the K+ transport deficient Saccharomyces cerevisiae mutant CY162 showed no rescue of the mutant phenotype . However, growth of CY162 expressing HvCBT1 with its pore mutated to GYGD, the consensus sequence of K+ channels, was compromised . We interpret these data as indicating that HvCBT1 acts to interfere with ion transport.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1546 - 51
Escherichia coli NusA is required for efficient RNA binding by phage HK022 nun protein; Watnick RS et al.; The Nun protein of phage HK022 is an RNA binding protein of the arginine-rich motif family . Nun binds the phage lambda boxB RNA sequence (BOXB) on nascent lambda transcripts and arrests transcription elongation . Binding to BOXB is inhibited by Zn2+ and stimulated by the Escherichia coli NusA protein . Deletion of the Nun C-terminal region enhances BOXB binding and makes it independent of Zn2+ and NusA . The C terminus of Nun thus appears to interfere with the N-terminal RNA binding motif . NusA relieves this interference by binding to the Nun C terminus and forming a complex with Nun and BOXB . However, NusA also inhibits transcription arrest in vitro, in the absence of the other Nus factors . Nun deleted for its C terminus fails to bind RNA polymerase (RNAP) (RNAP) or NusA in vitro or to arrest transcription in vivo or in vitro . Our findings are consistent with the idea that NusA inhibits transcription arrest by binding to the Nun C terminus, thus blocking the interaction between Nun and RNAP . NusG, NusB, and NusE factors restore transcription arrest, presumably by promoting transfer of Nun from NusA to RNAP.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1528 - 33
A previously unidentified host protein protects retroviral DNA from autointegration; Lee MS et al.; Integration of a DNA copy of the viral genome into a host chromosome is an essential step in the retrovirus life cycle . The machinery that carries out the integration reaction is a nucleoprotein complex derived from the core of the infecting virion . To successfully integrate into host DNA, the viral DNA within this complex must avoid self-destructive integration into itself, a reaction termed autointegration . We have previously shown {Lee, M . S . and Craigie, R . (1994) Proc . Natl . Acad . Sci . USA 91, 9823-9827} that viral nucleoprotein complexes isolated from Moloney murine leukemia virus-infected cells exhibit a barrier to autointegration . This autointegration barrier could be destroyed by stripping factors from the complexes and subsequently restored by incubation with a host cell extract, but not by incubation with an extract of disrupted virions . We have now used this autointegration barrier reconstitution assay to purify the host factor from uninfected NIH 3T3 fibroblasts . It is a single polypeptide of 89 aa that does not match any previously identified protein . The identity of the protein was confirmed by expressing it in Escherichia coli and demonstrating the activity of the heterologously expressed protein in the reconstitution assay.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1523 - 7
Localization of F plasmid SopB protein to positions near the poles of Escherichia coli cells; Kim SK et al.; The subcellular localization of the SopB protein, which is encoded by the Escherichia coli F plasmid and is involved in the partition of the single-copy plasmid, was directly visualized through the expression of the protein fused to the jellyfish green fluorescent protein (GFP) . The fusion protein, but not GFP itself, was found to localize to positions close but not at the poles of exponentially growing cells . Neither the presence of other F-encoded proteins nor the binding of SopB to its recognition sites within the sopC locus of F is required for this localization . Examination of derivatives of the fusion protein lacking various regions of SopB suggests that the signal for the cellular localization of SopB resides in a region close to its N terminus . It is plausible that the near polar localization of SopB may serve the function of keeping a segregated pair of F plasmids apart while the cell septum is being formed . The plausible relation between the specific location of SopB and its suppression of sopC-linked genes when overexpressed is also discussed.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1375 - 80
Amber suppression in Escherichia coli by unusual mitochondria-like transfer RNAs; Bourdeau V et al.; The "cloverleaf" base-pairing pattern was established as the structural paradigm of active tRNA species some 30 years ago . Nevertheless, this pattern does not accommodate the folding of certain mitochondrial tRNAs . For these recalcitrant tRNAs, we have proposed structures having from 5 to 10 base pairs in the anticodon stem rather than the canonical 6 . The absence of these types of tRNAs in cytoplasmic translation systems, however, raises the possibility that they may not be bona fide alternate folding patterns for active tRNA molecules . For this reason, we have designed new tRNA genes based on our model of unusual mitochondrial tRNAs, having 7, 8, 9, and 10 base pairs in the anticodon stem with other modifications to the D-stem and connector regions . We show here that these synthetic genes produce tRNAs that actively suppress amber codons in vivo.

Nucleic Acids Res, 1998 Feb 15, 26(4), 1128 - 9
UGUS, a reporter for use with destabilizing N-termini; Zaccaria Roberta Greco D et al.; Due to constraints in vector construction, reporter polypeptides often carry N-terminal sequences of extraneous origin . Since protein half-life can be influenced by small determinants in the N-terminus, such foreign sequences can destabilize proteins and compromise results of reporter-based studies . We provide a real-life example of this problem (destabilizing sequences derived from a ribosomal protein) and show that it can be solved with the ubiquitin fusion technique, in which ubiquitin sequences are placed upstream of the reporter, in our case beta-glucuronidase . Post-translational processing by characterized pathways removes the ubiquitin together with destabilizing sequences, generating a stable reporter whose N-terminus is constant.

Nucleic Acids Res, 1998 Feb 15, 26(4), 1107 - 15
GeneMark.hmm: new solutions for gene finding; Lukashin AV et al.; The number of completely sequenced bacterial genomes has been growing fast . There are computer methods available for finding genes but yet there is a need for more accurate algorithms . The GeneMark . hmm algorithm presented here was designed to improve the gene prediction quality in terms of finding exact gene boundaries . The idea was to embed the GeneMark models into naturally derived hidden Markov model framework with gene boundaries modeled as transitions between hidden states . We also used the specially derived ribosome binding site pattern to refine predictions of translation initiation codons . The algorithm was evaluated on several test sets including 10 complete bacterial genomes . It was shown that the new algorithm is significantly more accurate than GeneMark in exact gene prediction . Interestingly, the high gene finding accuracy was observed even in the case when Markov models of order zero, one and two were used . We present the analysis of false positive and false negative predictions with the caution that these categories are not precisely defined if the public database annotation is used as a control.

Nucleic Acids Res, 1998 Feb 15, 26(4), 954 - 60
Translational termination in Escherichia coli: three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing signals; Poole ES et al.; The observations that the Escherichia coli release factor 2 (RF2) crosslinks with the base following the stop codon (+4 N), and that the identity of this base strongly influences the decoding efficiency of stop signals, stimulated us to determine whether there was a more extended termination signal for RF2 recognition . Analysis of the 3' contexts of the 1248 genes in the E.coli genome terminating with UGA showed a strong bias for U in the +4 position and a general bias for A and against C in most positions to +10, consistent with the concept of an extended sequence element . Site-directed crosslinking occurred to RF2 from a thio-U sited at the +4, +5 and +6 bases following the UGA stop codon but not beyond (+7 to +10) . Varying the +4 to +6 bases modulated the strength of the crosslink from the +1 invariant U to RF2 . A strong selection bias for particular bases in the +4 to +6 positions of certain E . coli UGANNN termination sites correlated in some cases with crosslinking efficiency to RF2 and in vivo termination signal strength . These data suggest that RF2 may recognise at least a hexanucleotide UGA-containing sequence and that particular base combinations within this sequence influence termination signal decoding efficiency.

Nucleic Acids Res, 1998 Feb 15, 26(4), 948 - 53
The Escherichia coli MutL protein stimulates binding of Vsr and MutS to heteroduplex DNA; Drotschmann K et al.; Vsr DNA mismatch endonuclease is the key enzyme of very short patch (VSP) DNA mismatch repair and nicks the T-containing strand at the site of a T-G mismatch in a sequence-dependent manner . MutS is part of the mutHLS repair system and binds to diverse mismatches in DNA . The function of the mutL gene product is currently unclear but mutations in the gene abolish mutHLS -dependent repair . The absence of MutL severely reduces VSP repair but does not abolish it . Purified MutL appears to act catalytically to bind Vsr to its substrate; one-hundredth of an equivalent of MutL is sufficient to bring about a significant effect . MutL enhances binding of MutS to its substrate 6-fold but does so in a stoichiometric manner . Mutational studies indicate that the MutL interaction region lies within the N-terminal 330 amino acids and that the MutL multimerization region is at the C-terminal end . MutL mutant monomeric forms can stimulate MutS binding.

Nucleic Acids Res, 1998 Feb 15, 26(4), 932 - 41
Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII; Harrison L et al.; Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS) . How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study . Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break . The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion . Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3' . Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand . Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break . With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion . Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII . If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed . These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.

Nucleic Acids Res, 1998 Feb 15, 26(4), 925 - 31
Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus; Akbar Behjatnia SA et al.; The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication . Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form . The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV . Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region . Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined) . Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element . This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.

Nucleic Acids Res, 1998 Feb 15, 26(4), 896 - 902
The nature of the minimal 'selenocysteine insertion sequence' (SECIS) in Escherichia coli; Liu Z et al.; The UGA codon, usually a stop codon, can also direct the incorporation into a protein of the modified amino acid selenocysteine . This UGA decoding process requires a cis -acting mRNA element called 'selenocysteine insertion sequence' (SECIS) that can form a stem-loop structure . In Escherichia coli the SECIS of the selenoprotein formate dehydrogenase (FdhH) mRNA has been previously described to consist of at least 40 nucleotides following the UGA codon . Here we determined the nature of the minimal SECIS required for the in vivo UGA-directed selenocysteine incorporation in E.coli . Our study is based on extensive mutational analysis of the fdhF SECIS DNA located in a lac' Z fusion . We found that the whole stem-loop RNA structure of the E.coli fdhF SECIS previously described is not required for the UGA-directed selenocysteine incorporation in vivo . Rather, only its upper stem-loop structure of 17 nucleotides is necessary on the condition that it is located in a proper distance (11 nucleotides) from the UGA codon . Based on these observations, we present a new model for the minimal E.coli SECIS.

Hum Mol Genet, 1997 Dec, 6(13), 2223 - 31
Activation of the human transglutaminase 1 promoter in transgenic mice: terminal differentiation-specific expression of the TGM1-lacZ transgene in keratinized stratified squamous epithelia; Yamada K et al.; Transglutaminase 1 (TGase 1) is a tissue-specific enzyme which is expressed in the keratinized stratified squamous epithelia and which catalyzes straightepsilon-(gamma-glutamyl) lysine cross-links of proteins to form the cell envelope at the periphery of cornified cells . A transient expression assay using a luciferase reporter gene linked to the 2.5 kb 5' upstream region of the human TGase 1 gene (TGM1) showed phorbol ester-responsive promoter activity in cultured normal human keratinocytes . To assess its promoter activity in vivo, we generated transgenic mice expressing the Escherichia coli beta-galactosidase gene (lacZ) directed by the 5' upstream region . beta-Galactosidase histochemistry revealed that the TGM1-lacZ transgene was expressed in terminally differentiating keratinocytes in upper layers of stratified squamous epithelia in embryonic, neonatal and adult transgenic mice . The expression pattern was similar to that of endogenous TGase 1 mRNA detected by in situ hybridization . Furthermore, topical application of a phorbol ester to adult tail skin enhanced expression of the transgene as well as TGase 1 mRNA in the epidermis . Thus, the 2.5 kb 5' upstream sequence of TGM1 includes elements regulating tissue- and terminal differentiation-specific gene expression in stratified squamous epithelia.

Mutat Res, 1997 Dec, 385(3), 259 - 67
Frameshifts, base substitutions and minute deletions constitute X-ray-induced mutations in the endogenous tonB gene of Escherichia coli K12; Kanbashi K et al.; We have analyzed the DNA sequence changes in a total of 127 X-ray-induced mutations in the endogenous tonB gene of Escherichia coli cells . Frameshifts accounted for 61 mutations among which 51 were a - 1 frameshift . The second most commonly found mutations were base substitutions (20 transversions and 8 transitions) . Twelve of the 16 deletion mutations were the minute-size deletion of 3-25 base pairs, three were the medium-size deletion of 294-643 base pairs and the remaining one was the deletion of 8375 base pairs . Half of the frameshifts and deletions had a run of several identical bases or short direct repeats at the sites of mutation . The spectrum was not in good agreement with the spectrum of spontaneous endogenous tonB mutation nor with the spectra obtained from a mutated gene on a plasmid which had been irradiated in vitro and used to transfect cells for the assay . We discuss the possibility that an X-ray-induced DNA strand break produces local alteration of DNA structure which increases aberrant DNA replication leading to frameshift and minute-size deletion mutations.

Mutat Res, 1997 Dec, 385(3), 213 - 22
The pre-UV nutritional stresses increase UV resistance, decrease UV mutagenesis and inhibit excision repair; Slezarikova V et al.; Nutritional stresses applied to E . coli prior to UV irradiation increase UV resistance and decrease UV mutagenesis . This effect is uvrA-dependent and might reflect a more efficient excision of pyrimidine dimers {1} . The data presented here, however, indicate that after prestarvation for glucose or amino acids pyrimidine dimer excision (PDE) was partly inhibited . It appears that the stress conditions stimulate a mode of uvr-dependent tolerance of lesions, efficient and precise . Possible modes of PDE inhibition and lesion tolerance are discussed.

Mutat Res, 1997 Dec, 385(3), 195 - 203
Function of the homologous regions of the Escherichia coli DNA excision repair proteins UvrB and UvrC in stabilization of the UvrBC-DNA complex and in 3'-incision; Moolenaar GF et al.; The nicking of damaged DNA during the nucleotide excision repair reaction in E . coli, is the result of a multi-step process involving three enzymes, UvrA, UvrB and UvrC . The UvrB protein is loaded on the site of the damage by UvrA, forming a stable UvrB-DNA complex . This complex is recognized by UvrC and in the resulting UvrBC-DNA complex dual incision takes place, first on the 3'-side and next on the 5'-side of the damaged nucleotide . A domain in the C-terminal part of UvrB has been identified to be essential for formation of the specific UvrBC-DNA complex that induces the 3'-incision {1} . The N-terminal half of UvrC contains a region that is homologous to this C-terminal domain of UvrB . Using site-directed mutagenesis of a conserved phenylalanine in the homologous regions of UvrB and UvrC two mutants were constructed, UvrB(F652L) and UvrC(F223L) . Both proteins were tested in vitro using a DNA substrate with a defined cisplatin lesion . The protein-DNA and protein-protein interactions were studied using bandshift assays and DNAse I footprinting . We show that both domains are important for the binding of UvrC to the UvrB-DNA complex.

FEBS Lett, 1998 Feb 13, 423(1), 31 - 4
Recombinant protein kinase C-gamma phorbol binding domain upon microinjection blocked insulin-induced maturation of Xenopus laevis oocytes; Pawelczyk T et al.; The second cysteine-rich (Cys-2) domain of rat brain PKC-gamma regulatory region C1 (92-173) was expressed in Escherichia coli cells and purified . NMR studies of Cys-2 protein identified the phorbol and other phospholipid binding sites within this molecule (Xu, R.X., Pawelczyk, T., Xia, T-H . and Brown, S.T . (1997) Biochemistry 37, 10709-10717) . Here, we tested the ability of this domain to bind other proteins . Using an overlay assay we show that the Cys-2 domain binds other proteins in Xenopus oocyte soluble fraction . Unlike the kinase activity, binding of Cys-2 to other proteins was detected in the absence of added phospholipids . Microinjection of Cys-2 protein into Xenopus leavis oocytes inhibited insulin-induced but not progesterone-induced maturation . The smallest dose that enhanced insulin-induced maturation was 0.45 x 10(-12) mol injected Cys-2 . These results demonstrate that the PKC-gamma Cys-2 domain beside being the binding site for phorbol ester/DAG and phosphatidylserine binds also other proteins . The proteins that interact with Cys-2 domain of PKC are essential for insulin-induced maturation program in oocytes.

FEBS Lett, 1998 Feb 13, 423(1), 1 - 4
Secondary structure and limited proteolysis give experimental evidence that the precursor of pulmonary surfactant protein B contains three saposin-like domains; Zaltash S et al.; The 42 kDa precursor of surfactant protein B generates the 79 residue mature SP-B polypeptide, which belongs to the family of saposin-like proteins and has unique functional roles in pulmonary surfactant . From sequence comparisons it has been suggested that proSP-B, in addition to SP-B, contains two saposin-like domains, but their existence has until now not been experimentally verified . The 381 residue human proSP-B was now fused to an N-terminal poly-His tag, expressed in Escherichia coli, and purified from inclusion bodies by resolubilisation with 2.5% (w/v) SDS and, after removal of SDS, subsequent metal affinity chromatography . Recombinant proSP-B thus obtained exhibits about 35% alpha-helical structure in sodium phosphate buffer and is proteolytically cleaved preferentially between the three saposin-like domains . These results experimentally support that proSP contains, in addition to SP-B, two further saposin-like domains.

J Virol Methods, 1998 Jan, 70(1), 37 - 44
Recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus; Ndifuna A et al.; The nucleocapsid protein of the Gray strain of infectious bronchitis virus (IBV) is highly immunogenic and cross-reactive among various distinct serotypes . Recombinant nucleocapsid polypeptide expressed in bacteria with a histidine tag at the amino terminus has been used as antigen for developing an assay to detect IBV-specific antibody . This fusion protein was produced readily in bacteria and easily purified with a nickel column which bound to the histidine tag . Conditions were optimized for using these preparations for an IBV-specific ELISA . Although differences in optical densities could be detected between pre-immune and positive sera for the Ark, Mass, and Gray strains with antigen concentrations between 50 and 0.1 microg per well, the greatest differences could be detected with 3 and 1.5 microg of protein per well . Maximum differences in optical densities between pre-immune and positive sera were obtained using 2.4 microg per well of protein and sera diluted between 1:80 and 1:160 . In addition, as little as 30 ng/dot of recombinant nucleocapsid consistently detected IBV-specific sera in immunoblot assays which have convenient field applications.

Arch Virol, 1998, 143(1), 181 - 9
Detection of bovine immunodeficiency-like virus infection in experimentally infected calves; Baron T et al.; Detection of BIV virus infection by serological means, PCR and virus isolation in experimentally infected calves is described . Viral sequences were specifically detected by PCR in peripheral blood mononuclear cells (PBMCs), with primer systems located in the gag, pol and tat regions of the viral genome . An enzyme-linked oligosorbent assay (ELOSA) in microtiter plates is described, for the detection of PCR products, the sensitivity of which was shown to be comparable to that of membrane hybridization detection . Serological response of the animals against the BIV p26 protein was shown, using a recombinant fusion protein ((His)6p26) expressed in E . coli and purified by metal affinity chromatography, in ELISA and Western blot studies . The presence of infectious virus was demonstrated by its rescue, by virus isolation in cell cultures, from PBMCs during a one year follow-up.

Genetics, 1998 Feb, 148(2), 545 - 57
Chi-dependent intramolecular recombination in Escherichia coli; Friedman-Ohana R et al.; Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5'-GCTGGTGG-3') that interacts with RecBCD . To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage . Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers . Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes . Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site . A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic . These data suggest a role for Chi at both ends of the linear substrate . Chi was lost in all recombinational exchanges stimulated by a single Chi site . Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval . These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.

Cytokine, 1998 Jan, 10(1), 1 - 12
IL-8 derivatives with a reduced potential to form homodimers are fully active in vitro and in vivo; Horcher M et al.; Interleukin 8 (IL-8) is a member of the CXC subfamily of chemokines which attracts and activates preferentially neutrophilic granulocytes . At nanomolar concentrations monomeric and dimeric forms of the molecule are in equilibrium, with the monomer being the prevalent form . Five amino acids from position 23 to 29 of the 72-amino acid IL-8 sequence form the dimer interface, with Leu25 and Val27 being highly conserved among the CXC chemokines . To investigate the contribution of these amino acids to the dimerization of IL-8, we produced in escherichia coli IL-8 derivatives with phenylalanine substitutions at position 25 or 27, or both 25 and 27 . All three recombinant proteins were characterized by a significantly impaired potential to form dimers in solution as seen in chemical crosslinking experiments . IL-8 Val27 also could not be crosslinked as a dimer on its receptors . Receptor affinities and in vitro chemotactic activities, however, were not significantly different between wild-type and IL-8 with single mutations . The dimerization deficient IL-8 analogue had also full inflammatory activity in vivo . Thus, the monomer is the biologically active form of IL-8.

Virology, 1998 Mar 1, 242(1), 118 - 27
DNA requirements in vivo for phage T4 packaging; Lin H et al.; Phage T4 terminase, comprising the products of genes 16 and 17, packages headfuls of DNA from a concatemer but its mechanism of DNA recognition remains to be determined . Phage T4 terminase gene sequences were introduced into prophage lambda imm434 and plasmids in order to assess their effect on packaging as measured by transduction frequency and DNA content of T4-transducing particles . Multiple copy prophage lambda imm434 genes were transduced at 100-fold higher frequency, and high copy plasmids were transduced at 1000-fold higher frequency than single copy prophage or chromosomal genes T4 16 gene inserts enhanced both prophage and plasmid packaging; terminase gene-containing plasmid DNA in T4 transducing particles could exceed 10% of the total . Deletion or base change of the 24-bp gene 16 3' region which is required for sequence specific amplification of terminase gene 17 (Hp 17 mutations) depressed these elevated plasmid transduction frequencies, suggesting that this is a preferred T4 pac sequence . Moreover, a specific gene 16-containing pac fragment could be detected in mature, packaged phage T4 DNA following restriction endonuclease digestion . We conclude that both the copy number of homologous sequences and the DNA pac sequence(s) themselves are important for packaging, consistent with a synapsis model for regulation of terminase cutting and packaging in phage T4.

Virology, 1998 Mar 1, 242(1), 6 - 13
Distinct regions of EBV DNase are required for nuclease and DNA binding activities; Liu MT et al.; Epstein-Barr virus (EBV) DNase possesses both endonuclease and exonuclease activities and accepts both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as substrates . To map regions of EBV DNase responsible for nuclease and DNA binding activities, a series of mutant DNase polypeptides was expressed using a bacterial system for the nuclease assay and in an in vitro transcription/translation system to assay binding activity to dsDNA or ssDNA cellulose . The results indicated that the C-terminus of EBV DNase, residues 450-460, is essential for nuclease activity but dispensable for DNA binding . However, deletion of residues 441-470 resulted in the loss of both nuclease and DNA binding activities . Substitution of Phe452 and Val458 led to inactive enzymes . In the N-terminus, deletion of residues 23-28 and residues 7-61 resulted in the loss of nuclease activity but the DNA binding activities of the deleted enzymes were intermediate and low, respectively . Mutation of Leu23 to Gly showed drastically reduced nuclease activity but its DNA binding ability was not affected . Based on the amino acid sequence alignment of various herpesvirus DNases, we chose four highly conserved and two less well conserved regions as controls for mutagenesis studies . These six internal deletion (ID) mutants were prepared using a recombinant PCR method . Each of the polypeptides was expressed in a bacterial system for the nuclease assay and using an in vitro transcription/translation system for the DNA binding assay . DNA binding and nuclease activities of all six internal deletion mutants were abolished, except that mutant ID2, with deletion of residues 138-152, retained an intermediate ability to bind DNA . These data indicate that since mutations at distinct regions within EBV DNase resulted in the loss of nuclease and/or DNA binding activities, it is suggested that these distinct regions are required for maintenance of an intact and highly ordered structure(s) for both activities.

Biochem Biophys Res Commun, 1998 Feb 24, 243(3), 700 - 5
Src homology-2 domains protect phosphotyrosyl residues against enzymatic dephosphorylation; Brunati AM et al.; The SH2 domain of c-Fgr (class 1A) has been expressed in E . coli as GST fusion protein and tested for its ability to prevent the dephosphorylation of a variety of phosphotyrosyl (poly)peptides by three distinct protein tyrosine phosphatases (TC-PTPase, YOP, and Low Mr PTPase) . Dephosphorylation of HS1 protein and of a derived phosphopeptide, HS1 (388-402), exhibiting the motif selected by class 1A SH2 domains is inhibited in a dose dependent manner with full inhibition promoted by a 2- to 3-molar excess of GST/SH2 domain irrespective of either the nature or the amount of phosphatase used . The IC50 values for inhibition of these and other phosphotyrosyl substrates roughly correlates with their expected affinity for class 1A SH2 domain . Inhibition is partially reversed by the addition of D-myo-inositol 1,4,5-triphosphate, which competes for the binding to the SH2 domains . Our data on one side show that additional mechanism(s) besides mere competition must assist PTPases to dissociate SH2-PTyr complexes and on the other suggest a role for SH2 domains in protecting phosphotyrosyl residues from premature dephosphorylation.

J Magn Reson, 1998 Feb, 130(2), 335 - 40
HSQC-based methyl group selection via gradients in mutidimensional NMR spectroscopy of proteins; Diercks T et al.; A quadruple-quantum filtered HSQC (QQF-HSQC) for the selection of methyl groups which minimizes the line-broadening effects of proton homonuclear couplings is presented . The scheme uses gradients for n-quantum coherence selection and solvent suppression . In contrast to the heteronuclear quadruple-quantum coherence (HQQC) approach, the QQF-HSQC allows for long constant-time (CT) evolution, making use of the generally favorable relaxation properties of methyl groups . The increase in resolution and concomitant gain in sensitivity is discussed in theory and demonstrated in practice on the 14-kDa human nonpancreatic synovial phospholipase A2 (hnps-PLA2) . The constant-time version is particularly useful for obtaining high-resolution spectra as demonstrated on hnps-PLA2 . The applicability of the CT-QQF-HSQC module in multidimensional experiments is demonstrated using a 3D CT NOESY-QQF-HSQC spectrum of the 31-kDa homodimeric IIAMan domain of the mannose transporter of E . coli .

Arch Biochem Biophys, 1998 Mar 1, 351(1), 8 - 16
Site-directed mutagenesis of histidine-90 in Escherichia coli L-threonine dehydrogenase alters its substrate specificity; Johnson AR et al.; Escherichia coli L-threonine dehydrogenase is a member of the Zn(2+)-containing alcohol/polyol dehydrogenase family . Methylation of His-90 of L-threonine dehydrogenase was recently found to cause total inactivation (J . P . Marcus and E . E . Dekker, 1995 Arch . Biochem . Biophys . 316, 413-420) . Since His-90 is not conserved among the related dehydrogenases, this residue was changed to arginine, asparagine, and alanine by site-directed mutagenesis in order to probe its role . All three purified, homogeneous mutants, like wild-type enzyme, contained one Zn2+ atom/subunit and exhibited a sequential catalytic mechanism; the kcat value for each, however, was reduced approximately 10-fold . The K(m) value for threonine was elevated from 3 mM for wild-type enzyme to 31, 328, and 417 mM, respectively, for mutants H90R, H90N, and H90A . The activation energy of catalysis for mutant H90A was increased by 6.6 kcal/mol, suggesting that in the wild-type enzyme His-90 forms at least one crucial hydrogen bond in the transition state . Whereas wild-type enzyme catalyzed the oxidation of threonine amide (0.75 M) about twice as fast as this same concentration of threonine or 0.375 M L-2-amino-3-hydroxypentanoate, the reaction rate of mutant H90A with 0.75 M threonine amide or threonine methyl ester was 33- to 35-fold higher than with this level of threonine . Similarly, mutant H90N used 0.75 M threonine methyl ester or threonine amide as substrate 9- to 13-fold better than it used this concentration of threonine . Mutants H90A and H90N were more reactive with 0.225 M L-threonine hydroxamate than with 0.75 M threonine, but mutant H90A did not oxidize L-2-amino-3-hydroxypentanoate (0.375 M) and mutant H90N used this substrate poorly . The best substrates for mutant H90R were threonine methyl ester, threonine, and threonine amide (all tested at 0.75 M); 0.375 M L-2-amino-3-hydroxypentanoate was a poor substrate . The isolation and characterization of these first His-90 mutants of E . coli L-threonine dehydrogenase confirm the importance of this residue in catalysis and suggest that His-90 is an active-site residue which modulates the substrate specificity of L-threonine dehydrogenase.

Mol Cell Probes, 1997 Dec, 11(6), 407 - 26
Rapid and sensitive detection of Chlamydia trachomatis using a ligatable binary RNA probe and Q beta replicase; Stefano JE et al.; A simple assay format was developed for the direct detection of C . trachomatis rRNA utilizing ligation of recombinant MDV-1 probe RNA fragments hybridized to 23S rRNA after capture and release from a solid support . Assay background (equivalent to 10(4) targets) was suppressed by blocking sequences in the 5' MDV reporter probe fragment complementary to the 3' fragment by prehybridization of a DNA oligonucleotide . A pair of reporter fragments bearing a deletion within the region, obtained by a hydrid-selection-amplification protocol, yielded a low level of assay background which was reduced to < 2% with a blocker directed against the remaining pairing sequence . This probe set showed a sensitivity of 10(3) molecules of 23S rRNA (> 95% responding) and could detect a single elementary body (EB) of Chlamydia trachomatis or 1-10 EB added to a clinical matrix of pooled negative human cervical swab samples . The time of first appearance of amplification products by real-time fluorescence detection showed a linear response to log increases in the target level over a 10(5)-fold range, permitting the determination of target level within an order of magnitude . The assay showed approximately 10(9)-fold discrimination over Chlamydia pneumonae (TWAR) rRNA . High levels of cultured C . albicans, E . coli, S . aureus, or N . gonorrhoeae had no detectable effect on assay background or the ability to detect a single elementary body.

FEBS Lett, 1998 Feb 6, 422(3), 311 - 4
Recombinant Plasmodium falciparum glutathione reductase is inhibited by the antimalarial dye methylene blue; Farber PM et al.; Plasmodium falciparum glutathione reductase (PfGR) has emerged as a drug target against tropical malaria . Here we report the expression of PfGR in Escherichia coli SG5(DE3) and isolation procedures for this protein . Recombinant PfGR does not differ from the authentic enzyme in its enzymic properties, the turnover number being 9900 min(-1) . The dimeric flavoenzyme exhibits redox-dependent absorption spectra; the single tryptophan residue (per 57.2 kDa subunit) is strongly fluorescent . PfGR can be inhibited by the antimalarial drug methylene blue at therapeutic concentrations; the Ki for non-competitive inhibition is 6.4 microM . The sensitivity to methylene blue is observed also at high ionic strength so that, by analogy to human GR, analysis of crystalline enzyme-drug complexes can be envisaged.

Bioorg Khim, 1997 Dec, 23(12), 949 - 52
{Recombinant proteins containing amino acid sequences of two ectatomin chains}; Esipov RS et al.; Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors . Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB) . Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained.

J Virol, 1998 Mar, 72(3), 2297 - 304
Molecular characterization of hemagglutination domains on the fibers of subgenus D adenoviruses; Pring-Akerblom P et al.; The adenovirus fiber mediates the agglutination of erythrocytes . Based on differential hemagglutinating properties, subgenus D adenoviruses can be subdivided into clusters DI, DII, and DIII . While subgenus DI adenoviruses agglutinate rat and human erythrocytes, DII adenoviruses simply agglutinate rat erythrocytes and DIII adenoviruses display no or only weak rat erythrocyte agglutination . Amino acid sequence comparisons revealed distinct domains on the fiber knob which could be involved in hemagglutination . In order to localize and characterize the domains responsible for the interaction with rat and human erythrocytes, potential hemagglutination domains of the adenovirus type 9 (Ad9) (subgenus DI) fiber knob were introduced into Ad17 (subgenus DII) and Ad28 (subgenus DIII) fiber knobs by primer-directed mutagenesis . Furthermore, rat erythrocyte hemagglutination domains were also introduced into the Ad3 (subgenus B) fiber knob, which only agglutinated monkey erythrocytes . Altogether, 27 chimeric and mutated fiber proteins were expressed in Escherichia coli and subsequently tested for hemagglutination activity . The hemagglutination tests revealed that at least two domains can mediate the agglutination of rat erythrocytes . While one domain is located on the GH loop, the other domain extends from the C beta strand to the CD loop . The domain on the GH loop was partially conserved in all adenoviruses showing an incomplete hemagglutination pattern with rat erythrocytes . The domains involved in the agglutination of human erythrocytes are located on the CD and HI loops of the subgenus DI fiber knob.

J Virol, 1998 Mar, 72(3), 2160 - 7
Intercapsomeric disulfide bonds in papillomavirus assembly and disassembly; Li M et al.; In order to analyze bonding contacts that stabilize the virion or promote capsid assembly, bovine papillomavirus (BPV) virions were subjected to buffer conditions known to disrupt polyomavirus virions . At physiologic ionic strength, incubation with dithiothreitol (DTT), EGTA, or DTT plus EGTA did not disrupt BPV virions as determined by electron microscopy . However, incubation of virions with DTT rendered the BPV L1 protein susceptible to trypsin cleavage at its carboxy terminus and rendered the genome susceptible to digestion with DNase I . When DTT-treated BPV virions were analyzed by analytical ultracentrifugation, they sedimented at 230S compared with 273S for untreated virions, suggesting a capsid shell expansion . Incubation with EGTA had no effect on trypsin or DNase I sensitivity and only a small effect upon the virion S value . A single cysteine residue conserved among BPV and human papillomavirus (HPV) L1 proteins resides within the trypsin-sensitive carboxy terminus of L1, which is required for capsid assembly . A recombinant HPV type 11 L1 protein, which was purified after expression in Escherichia coli and which has a Cys-to-Gly change at this position (Cys424), formed pentamers; however, unlike the wild-type protein, these mutant pentamers could no longer assemble in vitro into capsid-like structures . These results indicate an important role for interpentamer disulfide bonds in papillomavirus capsid assembly and disassembly and suggest a mechanism of virus uncoating in the reducing environment of the cytoplasm.

J Virol, 1998 Mar, 72(3), 1949 - 58
Pseudorabies virus glycoprotein gK is a virion structural component involved in virus release but is not required for entry; Klupp BG et al.; The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J . Baumeister, B . G . Klupp, and T . C . Mettenleiter, J . Virol . 69:5560-5567, 1995) . To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase-gK fusion protein expressed in Escherichia coli . In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions . After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized . This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type . For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKbeta) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB- genome (PrV-gK(gB)) . Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle . After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible . In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation . Interestingly, the plating efficiency of PrV-gKbeta was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry . Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus . However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells . Thus, we postulate that presence of gK is important to inhibit immediate reinfection.

FEBS Lett, 1998 Feb 6, 422(3), 285 - 90
Crystal structure of a murine alpha-class glutathione S-transferase involved in cellular defense against oxidative stress; Krengel U et al.; Glutathione S-transferases (GSTs) are ubiquitous multifunctional enzymes which play a key role in cellular detoxification . The enzymes protect the cells against toxicants by conjugating them to glutathione . Recently, a novel subgroup of alpha-class GSTs has been identified with altered substrate specificity which is particularly important for cellular defense against oxidative stress . Here, we report the crystal structure of murine GSTA4-4, which is the first structure of a prototypical member of this subgroup . The structure was solved by molecular replacement and refined to 2.9 A resolution . It resembles the structure of other members of the GST superfamily, but reveals a distinct substrate binding site.

Virus Res, 1997 Oct, 51(2), 231 - 7
Molecular dissection of the reovirus lambda1 protein nucleic acids binding site; Bisaillon M et al.; A recent study has shown that the reovirus lambda1 protein can unwind double-stranded nucleic acid molecules, a process energetically coupled to the hydrolysis of nucleoside 5'-triphosphates . In the present study, it was demonstrated that lambda1, expressed as a fusion protein with the Escherichia coli maltose-binding protein (MBP), possesses a non-specific affinity for various nucleic acids . The study also showed that the first ten amino acids of the protein are sufficient for binding while a deletion of the first five amino acids prevented the binding to nucleic acids . These data indicate that the basic charges located at the extreme amino-terminal portion of the protein constitute the major determinant responsible for nucleic acids binding . Finally, a quantitative gel retardation assay using purified solubilized MBP-lambda1 fusion protein confirmed the importance of the amino-terminal region and also revealed that the lambda1 protein possesses a higher affinity for single-stranded RNA than for double-stranded nucleic acids, a property shared by many helicases.

Virus Res, 1997 Oct, 51(2), 197 - 201
Identification of bovine viral diarrhea virus nonstructural polypeptide NS4B/P38; van Olphen AL et al.; The bovine pestivirus polyprotein is processed into at least 11 mature polypeptides . Previous studies with polyprotein region-specific antiserum raised against beta-galactosidase fusion proteins or synthetic peptides allowed the assignment of viral non-structural proteins to specific segments of the BVDV genome . However, the gene product from the NS4B/P38 region of the viral genome remained to be demonstrated directly . BVDV cDNA fragments predicted to encode part of NS4B/P38 (from codon 2521 to 2838 of the BVDV open reading frame) and a portion of NS5A/P58 (from codon 3008 to 3340) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli . Polyclonal rabbit antibodies prepared against each of the purified fusion proteins, GST-2521-2838 and GST-3008-3340, were used to immunoprecipitate viral polypeptides present in BVDV-infected cell lysates . Rabbit antiserum to GST-2521-2838 bound a polypeptide of 38 kDa identified as the mature NS4B/P38 polypeptide; while anti GST-3008-3340 lacked this specificity and bound NS5A/P58 . Moreover, both antisera recognized a 96 kDa polypeptide, previously identified as a NS4B-NS5A/PP96 precursor . The function of the newly identified and highly conserved NS4B/P38 protein in viral replication remains to be determined.

J Biochem (Tokyo), 1997 Dec, 122(6), 1241 - 5
Isolation and characterization of an Escherichia coli mutant lacking the major serine transporter, and cloning of a serine transporter gene; Ogawa W et al.; L-Serine as well as L-valine inhibits the growth of Escherichia coli cells, and L-isoleucine releases this growth inhibition . We isolated an E . coli mutant (designated as WAT9) that was able to grow on lactate (or glucose) as a carbon source even in the presence of L-serine, the parent not being able to . Cells of WAT9 were not able to grow on L-serine as a carbon source even if L-isoleucine was present in the culture medium, while the parental cells grew . This mutant was shown to lack the principal L-serine transporter in E . coli, the Na+/ serine symporter . This mutant is useful for analysis of the role(s) of the Na+/serine symporter in cell physiology and as a host for the cloning of L-serine transporter gene(s) . In fact, we cloned a gene encoding a serine transporter from chromosomal DNA of E . coli using WAT9 as the host . The gene enabled the mutant cells to grow on L-serine . Transport activity for L-serine was restored in the mutant cells harboring a plasmid carrying the gene . We partially sequenced the gene and found that it was the tdcC gene . We showed that TdcC is an H+/serine symporter.

J Biochem (Tokyo), 1997 Dec, 122(6), 1133 - 8
Inhibition by anti-RCC1 monoclonal antibodies of RCC1-stimulated guanine nucleotide exchange on Ran GTPase; Azuma Y et al.; Nine monoclonal antibodies to RCC1, the guanine nucleotide exchange factor on Ran GTPase, were obtained using recombinant RCC1 as the antigen . Epitopes of three monoclonal antibodies, which did not inhibit RCC1 function, were localized in the N-terminus outside the RCC1 repeat, while epitopes of the other 6 monoclonal antibodies were localized within the RCC1 repeat . Three of the latter 6 monoclonal antibodies, 2B6, 6C3, and 8D9, inhibited RCC1-stimulated nucleotide release . Two of them, 2B6 and 6C3, recognized the same amino acid residues in the N-terminus of the second RCC1 repeat, Tyr89, Ser90, Phe91, and Gly92, of which one, Gly92, is conserved in Saccharomyces cerevisiae and mutated in an rcc1(-) strain, mtr1-2 . The monoclonal antibody 8D9 recognized two amino acid residues, Arg320 and Ala321, downstream of Gly319 in the N-terminus of the 6th RCC1 repeat, which corresponds to Gly92 in the second RCC1 repeat . The monoclonal antibodies which inhibited RCC1 function bound to RCC1 in homogeneous solution and stained cellular RCC1 . We propose that the N-terminus of the RCC1 repeat is exposed at the surface of RCC1 on the coated plate or in fixed cells, and is involved in the RCC1-stimulated nucleotide exchange on the Ran GTPase.

J Natl Cancer Inst, 1998 Mar 4, 90(5), 370 - 80
Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase gene therapies; Aghi M et al.; BACKGROUND: A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer . The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects . METHODS: Rat 9L gliosarcoma cells were transfected with plasmids containing the E . coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells) . The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured . The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored . The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies . To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied . RESULTS AND CONCLUSIONS: The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.

J Infect Dis, 1998 Mar, 177(3), 793 - 5
Parenteral nutrition facilitates activation of coagulation but not of fibrinolysis during human endotoxemia; van der Poll T et al.; Venous thrombosis and bacterial infections are common complications of parenteral nutrition . To test the hypothesis that infection facilitates activation of coagulation during parenteral nutrition, healthy subjects were intravenously injected with endotoxin (2 ng/kg) after they had received either 1 week of standard parenteral nutrition (n = 7) or normal enteral feeding (n = 8) . Compared with enteral feeding, parenteral nutrition was associated with a selectively enhanced activation of the coagulation system (plasma levels of thrombin-antithrombin III complexes) during endotoxemia . Activation of the fibrinolytic system (plasminogen activator activity, tissue-type plasminogen activator, plasminogen activator inhibitor type 1) proceeded similarly in both study groups . In patients receiving parenteral nutrition, one common complication (bacterial infection) may facilitate the occurrence of another common complication (venous thrombosis) by synergistic stimulation of the coagulation system.

J Infect Dis, 1998 Mar, 177(3), 662 - 7
Milk immunoglobulin with specific activity against purified colonization factor antigens can protect against oral challenge with enterotoxigenic Escherichia coli; Freedman DJ et al.; Enterotoxigenic Escherichia coli (ETEC) is the most commonly isolated pathogen responsible for travelers' diarrhea and the cause of up to 650 million cases of pediatric diarrhea per year in the developing world . As a safe alternative to the prophylactic use of antibiotics, a hyperimmune bovine milk antibody product with specific activity against purified colonization factor antigens (CFAs) was developed and evaluated in a human challenge study . Twenty-five healthy adult volunteers were challenged orally with 10(9) cfu of a virulent CFA/I-bearing ETEC . In the randomized double-blind trial, 7 of 10 volunteers receiving a lactose-free placebo developed clinical diarrhea after challenge, compared with only 1 of 15 cases in volunteers receiving active product (Fisher's exact test, P < .0017) . It is concluded that antibodies against CFAs alone are sufficient for protection and that prophylaxis with milk-derived immunoglobulin is a feasible alternative to existing drug interventions.

J Infect Dis, 1998 Mar, 177(3), 642 - 50
Phylogenetic analysis of Escherichia coli strains causing neonatal meningitis suggests horizontal gene transfer from a predominant pool of highly virulent B2 group strains; Bingen E et al.; Phylogenetic relationships of 69 neonatal meningitis Escherichia coli strains isolated worldwide were studied . Restriction fragment length polymorphism of rrn operons (rrn RFLP) in these isolates was compared with that of the 72 strains of the ECOR reference collection . Distributions of K1 antigen, of polymerase chain reaction-detected ibe10 gene, pap, afa, sfa/foc, hly, and aer operons, and of a 14.9-kb rrn-containing HindIII fragment previously associated with neonatal meningitis were compared . Oligoclonality was observed for the meningitis strains . Factorial analysis of correspondence on the rrn RFLP data showed a frequency gradient of meningitis strains from the phylogenetic B2 group (68%) to the A group (6%), via the D and B1 groups (26%) . The distribution of the virulence determinants argues for their horizontal transfer during the evolution of E . coli . Analysis of the status of some neonates further suggests that neonatal meningitis results from a balance between bacterial genes of virulence and host factors.

Carcinogenesis, 1998 Feb, 19(2), 305 - 10
Mutagenic specificity of the food mutagen 2-amino-3-methylimidazo{4,5-f}quinoline in Escherichia coli using the yeast URA3 gene as a target; Broschard TH et al.; 2-Amino-3-methylimidazo{4,5-f}quinoline (IQ), a strong mutagen/carcinogen, belongs to a group of heterocyclic amines that are formed (ng/g amounts) during the cooking of protein containing food . The mutational specificity of IQ in Escherichia coli was determined in a forward mutation assay using the yeast URA3 gene as a target . The plasmid pTU-AC, containing the target URA3, was randomly modified in vitro using N-hydroxy-IQ, and subsequently transformed into an E . coli pyrF strain (DB6656) . Mutant clones were directly selected by their ability to grow on medium containing 5-fluoro-orotic acid which is toxic to URA3+ clones and thereby selects for URA3- mutants . Single Strand Conformation Polymorphism (SSCP) was used to map the mutation-containing regions of URA3, so that it was necessary to sequence only the relevant, mutation-containing fragment and not the entire gene . At a modification level of 7 IQ-lesions/URA3 gene, the predominant mutations were base substitutions (approximately 70%), followed by complex gene rearrangements (approximately 20%) and frameshifts (approximately 10%) . More than 96% of the base substitutions occurred at G:C base pairs and were predominantly G:C-->A:T transitions, followed by G:C-->T:A and G:C-->C:G transversions . Next neighbour analysis revealed that deoxyguanosines situated within the sequence 5'-TGC were more susceptible to mutations induced by IQ . With one exception, all frameshift mutations were -1 deletions at runs of three consecutive dGs . At higher IQ-modification levels, predominantly complex sequence rearrangements were observed.

Graefes Arch Clin Exp Ophthalmol, 1998 Feb, 236(2), 146 - 50
Effects of controlled mutations on the N- and C-terminal extensions of chick lens beta B1 crystallin; Coop A et al.; BACKGROUND: While gamma-crystallin exists as a monomer, beta-crystallin, which unlike gamma-crystallin contains N- and C-terminal arms, associates to form homodimers . METHODS: In order to answer the question of whether the extensions are involved in dimerisation of chick lens beta B1 crystallin, we have developed a heterologous expression system for chicken beta B1 crystallin in Escherichia coli, and produced three mutations by site-directed mutagenesis . We have substituted residues in the PAPA segment of the N-terminal extension, curtailed the N-terminal extension by five residues, and deleted 16 residues from the C-terminal extension . RESULTS: High-resolution gel filtration chromatography and non-denaturing gel electrophoresis show that the mutations did not influence dimerisation of the beta B1 crystallin, while circular dichroism and tryptophan fluorescence indicated that the mutations did not have a major influence on beta B1 crystallin structure or its heat stability . CONCLUSIONS: Our experiments show that as with rat lens beta B2 crystallin, dimerisation of beta B1 crystallin is not affected by alterations to the conserved PAPA region and that the peptide linker region rather than the N- and C-terminal extensions must be important in dimerisation.

Protein Expr Purif, 1998 Feb, 12(1), 138 - 43
Affinity chromatographic purification of antibodies to a biotinylated fusion protein expressed in Escherichia coli; de Dios Alche J et al.; HOP1, a protein component of the synaptonemal complex in Saccharomyces cerevisiae which is believed to play an important role in meiotic synapsis, was expressed in Escherichia coli as a fusion protein incorporating a "tag" polypeptide which is biotinylated naturally in the bacteria . The HOP1 fusion protein was produced in an insoluble form within the bacteria; once solubilized using a denaturing agent, the protein was purified by avidin monomer affinity chromatography . The recombinant protein was used to immunize rabbits and produce polyclonal antibodies . Procedures for affinity purification of antibodies using the recombinant protein attached to the avidin column and a magnetic method for concentration of antibodies are described . Antibody elution conditions in these procedures do not affect the affinity of the column for the recombinant protein, which can be recovered afterward . Affinity-purified antibodies show high binding capacity to HOP1 recombinant protein in immunoblotting experiments, but reduced background compared with crude antiserum or purified IgG fraction . The affinity-purified antibodies recognize a major band around 70 kDa in Western blots of yeast protein extracts following meiotic induction .

Protein Expr Purif, 1998 Feb, 12(1), 122 - 32
Solubility of proteins isolated from inclusion bodies is enhanced by fusion to maltose-binding protein or thioredoxin; Sachdev D et al.; When the mammalian aspartic proteinases, procathepsin D or pepsinogen, are expressed in Escherichia coli both accumulate in inclusion bodies . While pepsinogen is efficiently refolded in vitro, recovery of procathepsin D is limited by insolubility . We expressed procathepsin D and pepsinogen in E . coli, with E . coli maltose-binding protein (MBP) or thioredoxin (trx) fused to their C-termini (aspartic proteinase-MBP or aspartic proteinase-trx) . The fusion proteins were still found in inclusion bodies . However, the recovery of soluble procathepsin D-MBP and procathepsin D-trx after refolding was facilitated by the bacterial fusion partners . Maltose-binding protein was more efficient than thioredoxin in increasing the recovery of soluble protein . The vector, pET23bMBPH6, can be used for general expression of heterologous proteins in E . coli . The vector includes a histidine tag at the C-terminus of MBP to allow one-step purification of the fusion proteins under denaturing conditions . After purification, the protein of interest can be cleaved from MBP with factor Xa protease and separated from the MBP partner . Refolded pepsinogen-MBP and pepsinogen-trx were enzymatically active, but procathepsin D-MBP and procathepsin D-trx were soluble but largely inactive . The results show that the limited recovery of activity upon refolding of procathepsin D is not the consequence of competing aggregation . Thus, the fusions do not necessarily facilitate native refolding, but they do enhance the recovery of soluble protein . Such fusions could provide a system to study, in soluble form, folding states which are otherwise inaccessible because of aggregation and precipitation .

Protein Expr Purif, 1998 Feb, 12(1), 38 - 44
Expression of rat histone H1d in Escherichia coli and its purification; Bharath MM et al.; Histone H1 is involved in the folding of linear polynucleosomal filament into a 30-nm fiber . In an effort to understand the role of different domains of histone H1 in chromatin folding, we have now expressed rat histone H1d in Escherichia coli using pTrc99A expression vector by providing a 6-His tag at the C-terminus to facilitate its purification . The expressed protein histone H1d was purified from the soluble extract of E . coli by employing Ni2+ NTA-agarose and heparin-agarose chromatography . The recombinant histone H1d was shown to be authentic by its N-terminal amino acid analysis, its secondary structural characteristics, and its ability to (a) condense DNA and (b) bind specifically to synthetic four-way junction DNA .

Protein Expr Purif, 1998 Feb, 12(1), 25 - 8
An expression system of rat calmodulin using T7 phage promoter in Escherichia coli; Hayashi N et al.; An efficient expression system of rat calmodulin in Escherichia coli is presented . To express rat calmodulin cDNA, we employed a pET expression vector which contains the T7 phage promoter and terminator . After transformation of E . coli BL21(DE3) strain which carries T7 phage RNA polymerase inducible with isopropyl-beta-D-thiogalactopyranoside, induction of the expression, and chromatography of soluble proteins on a phenyl-Sepharose column, about 250 mg of recombinant rat calmodulin was obtained from 1 liter of E . coli culture . The recombinant calmodulin lacked the N-terminal methionine, and posttranslational modifications such as Nalpha-acetylation and methylation . This system facilitates the large amount preparation of calmodulin and the mutant proteins required for the structural analysis by NMR spectrometry and/or X-ray crystallography .

Protein Expr Purif, 1998 Feb, 12(1), 17 - 24
The human trifunctional enzyme of de novo purine biosynthesis: heterologous expression, purification, and preliminary characterization; Poch MT et al.; The cDNA for the human trifunctional enzyme of de novo purine biosynthesis, which encodes glycinamide ribonucleotide synthetase, aminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide trans-formylase, has been overexpressed in Escherichia coli and its protein product has been purified to homogeneity . The glycinamide ribonucleotide transformylase activity, which constitutes the C-terminal domain of the trifunctional enzyme, has been characterized with respect to its kinetic constants, Vmax = 3.03 +/- 0.15 micromol/min-mg and Km values for beta-glycinamide ribonucleotide and 10-formyl-5,8-dideazafolate of 0.94 +/- 0.21 and 1.58 +/- 0.25 microM, respectively, and its kinetic mechanism, which is ordered-sequential with the folate substrate binding first . The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional .

Protein Expr Purif, 1998 Feb, 12(1), 7 - 16
Isolation and expression of murine carbonic anhydrase IV; Hurt JD et al.; A 1193-bp cDNA containing the complete murine carbonic anhydrase IV coding sequence was isolated from a Balb/c kidney cDNA library . The entire coding sequence plus shorter segments was used in an Escherichia coli T7 expression vector system to produce four forms of murine CA IV, including (1) a protein representing the full-length coding sequence, (2) an amino-truncated protein lacking the 18 N-terminal amino acid plasma membrane targeting sequence, (3) a protein which lacked the plasma membrane targeting sequence and 26 C-terminal amino acids, and (4) a protein which lacked both 36 N-terminal residues (the plasma membrane targeting sequence plus 18 additional amino acids which included the first two cysteines) and 26 C-terminal residues . All four proteins were expressed as catalytically inactive inclusion bodies . After rapid dilution of washed, guanidine hydrochloride-denatured inclusion bodies into a glutathione-, l-arginine-containing renaturation buffer, an active carbonic anhydrase IV at yields of 3-4 mg/liter was easily purified from cultures expressing the form lacking the N-terminal targeting sequence and 26 C-terminal residues . The longest and shortest forms of carbonic anhydrase IV failed to refold into active enzyme under these conditions . The activity of purified recombinant carbonic anhydrase IV was highly resistant to sodium dodecyl sulfate, as is the native enzyme . This resistance presumably results from intramolecular disulfide bonds maintaining a functional active site configuration even in the presence of denaturing agents .

Protein Expr Purif, 1998 Feb, 12(1), 1 - 6
Expression in Escherichia coli of the elongation factor 1beta gene and its nucleotide T160C mutant from the archaeon Sulfolobus solfataricus; Ianniciello G et al.; The guanine nucleotide exchange factor EF-1beta gene from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1beta) was amplified by PCR and cloned into the pT7-7 expression vector . One of four selected clones harbored the T160C nucleotide substitution leading to the Y54H amino acid change in a hydrophobic region of SsEF-1beta, caused by a nucleotide misincorporation of the Taq DNA polymerase during PCR . The resulting plasmids were used to transform the Escherichia coli BL21(DE3)pLysE strain . Upon induction with isopropyl beta-d-thiogalactopyranoside about 1.4 mg of the recombinant SsEF-1beta (recSsEF-1beta) and Y54HSsEF-1beta were obtained from 1 liter of cell culture . recSsEF-1beta and Y54HSsEF-1beta were both able to catalyze the GDP/GTP exchange on SsEF-1alpha as observed with the wild-type SsEF-1beta . In addition, the heat inactivation profiles of recSsEF-1beta and Y54HSsEF-1beta were identical, being both half inactivated after 30 min treatment at 105 degrees C . These results suggest that Tyr 54 is not essential for the nucleotide exchange activity and is not involved in the thermostability of SsEF-1beta .

Exp Cell Res, 1998 Feb 1, 238(2), 345 - 53
Physiologically dependent appearance of a low density region that corresponds to the tunnel through the 50S part of the 70S ribosome; Zhang K et al.; Ribosomes have different conformations in cells that are starved for a required amino acid (giving aminoacyl.tRNA starvation), or treated with kirromycin (blocking EF-Tu.GDP release), or are in exponential growth . A tunnel spans the 50S ribosome from a location facing the 70S ribosomal intersubunit space to the back side of the subunit in Escherichia coli cells . Here we have analyzed the internal low density region that corresponds to this tunnel in ribosomes in vivo . The data suggest that the tunnel is opened in connection with spatial separation of the subunits in ribosomes that have an empty A-site due to starvation for aminoacyl.tRNA . A region that corresponds to this tunnel can be found in the more compact structure of ribosomes in kirromycin-treated cells only after a substantial removal of low density material . This region is even less prominent in ribosomes in undefined working modes in growing bacteria . The data suggest that appearance of the tunnel through the 50S ribosomal subunit is working-mode dependent and it is not a characteristic feature of the major fraction of the ribosomal population in growing cells .

Exp Cell Res, 1998 Feb 1, 238(2), 305 - 16
Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95; Eide T et al.; The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs) . A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V . M . Coghlan, L . K . Langeberg, A . Fernandez, N . J . Lamb, and J . D . Scott (1994) J . Biol . Chem . 269, 7658-7665) . The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR . A fragment covering amino acids 414-692 of human AKAP95 was expressed in Escherichia coli and shown to bind RIIalpha . Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIalpha binding to AKAP95 . Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIalpha was excluded from the nucleus . In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate . Furthermore, the subcellular localizations of AKAP95 and RIIalpha overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase . Finally, RIIalpha was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis . The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIalpha may be cell cycle-dependent .

Mol Pharmacol, 1998 Feb, 53(2), 270 - 3
Substrate specificity of human recombinant mitochondrial deoxyguanosine kinase with cytostatic and antiviral purine and pyrimidine analogs; Sjoberg AH et al.; Deoxyguanosine kinase (dGK) is an enzyme responsible for the phosphorylation of purine deoxynucleosides in mitochondria of mammalian cells . Its role in activation of pharmacologically used nucleoside analogs is not well understood, because of the low levels of dGK found in tissue extracts and its inactivation during purification . The cDNA for dGK was recently cloned and expressed in Escherichia coli . Here we present an improved procedure for expression and purification of a highly active form of human recombinant dGK . The enzyme showed a broad substrate specificity toward natural purine and pyrimidine deoxynucleosides as well as toward important nucleoside analogs . The Km and Vmax values for deoxyguanosine, deoxyinosine, deoxyadenosine, and deoxycytidine were 4, 13, 460, 330 microM and 43, 330, 430 and 60 nmol/min/mg of protein, respectively . Antileukemic purine analogs such as arabinosyl guanine, 2-chloro-2'-deoxyadenosine, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, and 2-fluoro-arabinosyl-adenine were phosphorylated as efficiently by dGK as the natural nucleoside substrates . This is the first report in which 2-fluoro-arabinosyl-adenine and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine were shown to be good substrates for dGK . The antiviral analogs dideoxyinosine and arabinosyl adenine also showed significant activity with dGK, as did several pyrimidine analogs (e.g., the cytostatic drugs 5-fluoro-2'-deoxycytidine and difluorodeoxycytidine) . The broad specificity of dGK described here may change our understanding of the mechanisms responsible for the efficacy and mitochondrial toxicity of several nucleoside analogs.

EMBO J, 1998 Feb 2, 17(3), 808 - 16
Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome; Heurgue-Hamard V et al.; Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis . The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase . Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli . Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant . The insertions impaired expression of the frr gene . Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype . Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant . In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling . When present together, the three factors were able to stimulate release up to 12-fold . It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

EMBO J, 1998 Feb 2, 17(3), 786 - 96
CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter; Wang YP et al.; The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription . Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP . CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator . CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation . When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 closed complex via protein-protein contacts . CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites . CRP affects both the final level and the kinetics of activation . The establishment of the repression and its release by the NtrC activator proceed via slow processes . The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate . Only the latter is capable of interacting with an activator to form an open promoter complex . Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the CRP-cAMP complex.

EMBO J, 1998 Feb 2, 17(3), 696 - 705
Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function; Duong F et al.; Preprotein translocase catalyzes membrane protein integration as well as complete translocation . Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer . We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence . Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA . Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states . Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics . In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling . Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself . Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane.

EMBO J, 1998 Feb 2, 17(3), 688 - 95
Mode of interaction of the single a subunit with the multimeric c subunits during the translocation of the coupling ions by F1F0 ATPases; Kaim G et al.; We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na+-translocating Escherichia coli/Propionigenium modestum ATPase hybrid with a Na+-inhibited growth phenotype on succinate . ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl . In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side . A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42 . We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side . Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit . If by mutation of the a subunit this channel becomes impermeable for Na+, N side Na+ ions specifically inhibit the ATPase activity . These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na+ ions . In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na+ ions on H+ transport by the parent hybrid ATPase . If the ATPase carrying the mutated a subunit was incubated with 22NaCl and ATP, 1 mol 22Na+/mol enzyme was occluded . With the parent hybrid ATPase, 22Na+ occlusion was not observed . The occluded 22Na+ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect . Li+ but not Na+ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na+ ions accessible again to the aqueous environment . These results suggest an ion translocation mechanism through F0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na+, Li+, or H+-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 1126 - 30
Tissues of MSH2-deficient mice demonstrate hypermutability on exposure to a DNA methylating agent; Andrew SE et al.; The mutational response of mismatch repair-deficient animals to the alkylating agent N-methyl-N-nitrosourea was evaluated by using a transgenic lacI reporter system . Although the mutations detected in MSH2 heterozygotes were similar to those of controls, MSH2-/- animals demonstrated striking increases in mutation frequency in response to this agent . G:C to A:T transitions at GpG sites, as opposed to CpG sites, dominated the mutational spectrum of both MSH2+/+ and MSH2-/- N-methyl-N-nitrosourea -treated animals . Extrapolating to humans with hereditary non-polyposis colorectal cancer, the results suggest that MSH2 heterozygotes are unlikely to be at increased risk of mutation, even when exposed to potent DNA methylating agents . In contrast, mismatch repair-deficient cells spontaneously arising within individuals with hereditary non-polyposis colorectal cancer would likely exhibit hypermutability in response to such mutagens, an outcome predicted to accelerate the pace of tumorigenesis.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 1120 - 5
Structure-function analysis of integrase interactor 1/hSNF5L1 reveals differential properties of two repeat motifs present in the highly conserved region; Morozov A et al.; Retroviral integrase (IN) catalyzes the integration of retroviral cDNA into host chromosome . Ini1 (integrase interactor 1) is a host protein that specifically binds and stimulates in vitro joining activity of HIV-1 IN . Ini1 has homology to yeast transcription factor SNF5 and is a component of the analogous mammalian SWI/SNF complex that can remodel chromatin . Little is known about the function of Ini1 in mammalian cells . To gain insight into the functional domains of Ini1, and to understand the details of protein-protein interactions of IN and Ini1, a structure-function analysis of Ini1 was initiated . By means of the yeast two-hybrid system, the minimal IN binding domain of Ini1 was characterized . One of the two repeat motifs present in the highly conserved regions of Ini1 was found necessary and sufficient to bind to IN in yeast as well as in vitro . Because IN binds to only one of the two repeat motifs in this conserved region of Ini1, it appears that the IN-Ini1 interaction is very specific and functionally significant . Characterization of DNA-binding properties of Ini1 revealed that Ini1 can bind to plasmid DNA, binding more readily to supercoiled DNA than to the relaxed circular DNA . The minimal domain for DNA binding was localized to a region upstream of repeat 1 . The DNA binding activity of Ini1 is not required for its ability to interact with IN . The finding that the two repeat motifs of Ini1 display differential binding to HIV-1 IN and that this discrete component of mammalian SWI/SNF complex binds to DNA will help understand the role of Ini1 in HIV-1 integration and in cellular process.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 1004 - 9
ATP-enhanced molecular chaperone functions of the small heat shock protein human alphaB crystallin; Muchowski PJ et al.; We report direct experimental evidence that human alphaB-crystallin, a member of the small heat shock protein family, actively participates in the refolding of citrate synthase (CS) in vitro . In the presence of 3.5 mM ATP, CS reactivation by alphaB-crystallin was enhanced approximately twofold . Similarly, 3.5 mM ATP enhanced the chaperone activity of alphaB-crystallin on the unfolding and aggregation of CS at 45 degrees C . Consistent with these findings, cell viability at 50 degrees C was improved nearly five orders of magnitude in Escherichia coli expressing alphaB-crystallin . SDS/PAGE analysis of cell lysates suggested that alphaB-crystallin protects cells against physiological stress in vivo by maintaining cytosolic proteins in their native and functional conformations . This report confirms the action of alphaB-crystallin as a molecular chaperone both in vitro and in vivo and describes the enhancement of alphaB-crystallin chaperone functions by ATP.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 981 - 6
The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli; Yu M et al.; The RecBCD enzyme from Escherichia coli is an ATP-dependent helicase and an ATP-stimulated nuclease . The 3' --> 5' exonuclease activity on double-stranded DNA is suppressed when the enzyme encounters a recombinational hot spot, called chi (chi) . We have prepared a RecB deletion mutant (RecB1-929) by using results of limited proteolysis experiments that indicated that the RecB subunit consists of two main domains . The RecB1-929 protein, comprising the 100-kDa N-terminal domain of RecB, is an ATP-dependent helicase and a single-stranded DNA-dependent ATPase . Reconstitution of RecB1-929 with RecC and RecD leads to processive unwinding of a linearized plasmid . However, the reconstituted RecB1-929CD enzyme has lost the single-strand endo- and exonuclease and the double-strand exonuclease activities of the RecBCD enzyme . These results show that the 30-kDa C-terminal domain of RecB has an important role in the nuclease activity of RecBCD . On the basis of these findings, we propose the RecB C-terminal domain swing model to explain RecBCD's transformation from a 3' --> 5' exonuclease to a helicase when it meets a chi site.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 957 - 62
Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: potential role for coiled coil interaction; Stefancsik R et al.; Troponin T (TnT), a thin filament myofibrillar protein, is essential for the Ca2+ regulation of striated muscle contraction in vertebrates, both in vivo and in vitro . To understand the role of TnT in this process, its interaction with two other troponin components, troponin I (TnI) and troponin C (TnC) was examined by using the yeast two hybrid system, which is a genetic approach to detect protein-protein interactions . Computer assisted analysis of phylogenetically distant TnT amino acid sequences unveiled a highly conserved protein domain that is characterized by a heptad repeat (HR) motif with a potential for alpha-helical coiled coil formation . A similar, potentially coiled coil forming domain is also conserved in all known TnI sequences . These protein motifs appeared to be the regions where TnI-TnT interaction may take place . Deletions and point mutations in TnT, which disrupted its HR motif, severely reduced or abolished TnI binding, but binding to TnC was not affected, indicating that the TnT-TnI and TnT-TnC binary interactions can be uncoupled . Remarkably, the truncated fragments of TnT and TnI in which the HR motifs were retained showed binary interaction in the yeast two hybrid system . It was also observed that the formation of the TnT-TnI heterodimers is favored over the homodimers TnT-TnT and TnI-TnI . These results indicate that the evolutionarily conserved HR motifs may play a role in TnT-TnI dimerization, presumably through the formation of alpha-helical coiled coils.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 945 - 50
Structure of the elongating ribosome: arrangement of the two tRNAs before and after translocation; Nierhaus KH et al.; The ribosome uses tRNAs to translate the genetic information into the amino acid sequence of proteins . The mass ratio of a tRNA to the ribosome is in the order of 1:100; because of this unfavorable value it was not possible until now to determine the location of tRNAs within the ribosome by neutron-scattering techniques . However, the new technique of proton-spin contrast-variation improves the signal-to-noise ratio by more than one order of magnitude, thus enabling the direct determination of protonated tRNAs within a deuterated ribosome for the first time . Here we analyze a pair of ribosomal complexes being either in the pre- or post-translocational states that represent the main states of the elongating ribosome . Both complexes were derived from one preparation . The orientation of both tRNAs within the ribosome and their mutual arrangement are determined by using an electron microscopy model for the Escherichia coli ribosome and the tRNA structure . The mass center of gravity of the (tRNA)2mRNA complex moves within the ribosome by 12 +/- 4 A in the course of translocation as previously reported . The main results of the present analysis are that the mutual arrangement of the two tRNAs does not change on translocation and that the angle between them is, depending on the model used, 110 degrees +/- 10 degrees before and after translocation . The translocational movement of the constant tRNA complex within the ribosome can be described as a displacement toward the head of the 30S subunit combined with a rotational movement by about 18 degrees.

Nucleic Acids Res, 1998 Feb 1, 26(3), 857 - 9
Repair of degraded duplex DNA from prehistoric samples using Escherichia coli DNA polymerase I and T4 DNA ligase; Pusch CM et al.; The most notable feature of DNA extracted from prehistoric material is that it is of poor quality . Amplification of PCR products from such DNA is consequently an exception . Here we present a simple method for the repair of degraded duplex DNA using the enzymes Escherichia coli DNA polymerase I and T4 DNA ligase . Adjacent sequences separated by nicks do not split up into intact strands during the denaturation step of PCR . Thus the target DNA is refractory to amplification . The proposed repair of nicked, fragmented ancient DNA results in an increase of amplification efficiency, such that the correct base order of the respective nuclear DNA segment can be obtained.

Curr Microbiol, 1998 Feb, 36(2), 85 - 9
Characterization of ftsZ, the cell division gene of Buchnera aphidicola (endosymbiont of aphids) and detection of the product; Baumann L et al.; Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, contains the gene ftsZ, which codes for a protein involved in the initiation of septum formation during cell division . With immunological techniques, this protein has been detected in cell-free extracts of the endosymbiont . Nucleotide sequence determination of a 6.4-kilobase B . aphidicola DNA fragment has indicated that, as in E . coli, ftsZ is adjacent to genes coding for other cell division proteins as well as genes involved in murein synthesis (murC-ddlB-ftsA-ftsZ) . Although B . aphidicola ftsZ is expressed in E . coli, it cannot complement E . coli ftsZ mutants . High levels of B . aphidicola FtsZ results in the formation of long filamentous E . coli cells, suggesting that this protein interferes with cell division . The presence of FtsZ indicates that in this, as well as in many other previously described properties, B . aphidicola resembles free-living bacteria.

Mol Biochem Parasitol, 1997 Dec 1, 90(1), 297 - 306
Molecular cloning, expression and enzymatic activity of a thioredoxin peroxidase from Dirofilaria immitis; Klimowski L et al.; A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D . immitis larval infections . The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals . As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity . nDiTPx was expressed in E . coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system . A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D . immitis larval and adult parasite extracts.

Mol Biochem Parasitol, 1997 Dec 1, 90(1), 223 - 34
Merozoite surface protein-1 epitopes recognized by antibodies that inhibit Plasmodium falciparum merozoite dispersal; Lyon JA et al.; Serum antibodies from malaria immune donors can inhibit merozoite dispersal by forming immune complexes through surface-accessible regions of membrane associated antigens . Such merozoite forms are referred to as immune clusters of merozoites (ICM) . Antibodies dissociated from ICM of Plasmodium falciparum identify a restricted subset of antigens, including merozoite surface protein-1 (MSP-1) . We performed epitope mapping by comparing the reactivity of whole immune sera and ICM-derived antibodies in immunoblotting assays, using fourteen overlapping recombinant MSP-1 fragments, and by ELISA, using each of the 1720 octapeptides encoded within MSP-1 . Antibodies in immune sera reacted with thirteen recombinant fragments and hundreds of octapeptides, but antibodies derived from ICM reacted with only six recombinant fragments and twenty octapeptides . Recombinant fragment recognition by ICM-derived antibodies was delimited to three regions 150-200 residues long, with seven of the octapeptide epitopes also mapping to these regions . The octapeptides recognized most strongly by antibodies in whole serum corresponded to the degenerate repeats near the N-terminus of MSP-1, however, neither recombinant fragments, nor octapeptides containing these degenerate repeats, were recognized by ICM-derived antibodies . Compared to reactions with recombinant fragments, the reactions observed with octapeptides were weak and may represent low-affinity mimetopes or cross-reactions . Alternatively, they may represent reactions with a portion of an epitope assembled from more than one non-contiguous peptide . These results suggest that ICM-derived antibodies can be used to map surface-accessible epitopes on MSP-1 and that the recombinant fragments with which they react are appropriate candidates for further evaluation as components of a malaria vaccine.

Mol Biochem Parasitol, 1997 Dec 1, 90(1), 203 - 21
Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis; Paramchuk WJ et al.; We have isolated and characterized two superoxide dismutase (SOD) cDNAs from a Leishmania chagasi promastigote cDNA library using degenerate primers derived from conserved amino acid residues of previously isolated manganese and iron SODs . Comparison of these two L . chagasi SOD deduced amino acid sequences with previously isolated MnSOD and FeSOD amino acid sequences revealed that they have higher homology to, and complete conservation of, invariant residues found in iron-containing SODs . Southern blot analysis showed that one gene, L.c.FeSODA, is a single copy gene, whereas the other gene, L.c.FeSODB, belongs to a multi-gene family . Transcript levels and enzyme activities of L.c.FeSODA and L.c.FeSODB show differential stage expression, with higher levels present in the amastigote stage of the parasite compared to the promastigote stage . Expression of the L.c.FeSODs in an E . coli SOD null strain protected the bacteria against free radical generating agents . Overexpression of these FeSODs in L . chagasi parasites also showed enhanced protection against the free radical generating agents, paraquat and nitroprusside . The cloning, characterization and overexpression of the L.c.FeSODA and L.c.FeSODB genes and proteins demonstrates the possible role of SODs in Leishmania pathogenesis.

Biotechnol Prog, 1998 Jan-Feb, 14(1), 149 - 55
Production and purification of two recombinant proteins from transgenic corn; Kusnadi AR et al.; This study reports the production, purification, and characterization of recombinant Escherichia coli beta-glucuronidase (GUS) and chicken egg-white avidin from transgenic corn seed . The avidin and gus genes were stably integrated in the genome and expressed over seven generations . The accumulation levels of avidin and GUS in corn kernel were 5.7% and 0.7% of extractable protein, respectively . Within the kernel, avidin and GUS accumulation was mainly localized to the germ, indicating possible tissue preference of the ubiquitin promoter . The storage-stability studies demonstrated that processed transgenic seed containing GUS or avidin can be stored at 10 degrees C for at least 3 months and at 25 degrees C for up to 2 weeks without a significant loss of activity . The heat-stability experiments indicated that GUS and avidin in the whole kernels were stable at 50 degrees C for up to 1 week . The buffer composition also had an affect on the aqueous extraction of avidin and GUS from ground kernels . Avidin was purified in one step by using 2-iminobiotin agarose, whereas GUS was purified in four steps consisting of adsorption, ion-exchange, hydrophobic interaction, and size-exclusion chromatography . Biochemical properties of purified avidin and GUS were similar to those of the respective native proteins.

Biotechnol Prog, 1998 Jan-Feb, 14(1), 63 - 74
Two-dimensional fluorescence spectroscopy: a new tool for on-line bioprocess monitoring; Marose S et al.; Two-dimensional fluorescence spectroscopy is presented as a new method for bioprocess monitoring . It covers a wide range of excitation and emission wavelengths and is a further development of the fluorescence measurements performed so far, which concentrated mainly on NAD(P)H culture fluorescence . Biogenic fluorophores such as proteins, coenzymes, and vitamins can simultaneously be detected qualitatively and quantitatively inside and outside the cells . This optical method is noninvasive, suitable for in vivo measurements . One whole spectrum (excitation, 250-550 nm; emission, 260-600 nm) with the described parameters is performed within 1 min, which allows an almost continuous monitoring of the bioprocess . The technique is ideal for on-line, in situ measurements via fiber optical systems . Results are presented for cultivations of Claviceps purpurea, Escherichia coli, Saccharomyces cerevisiae, and Sphingomonas yanoikuyae . Cell growth and the metabolism of the cells (changes from aerobic to anaerobic conditions and uncoupling of the oxidative phosphorylation) could be detected.

Biotechnol Prog, 1998 Jan-Feb, 14(1), 47 - 54
Oxidative renaturation of hen egg-white lysozyme . Folding vs aggregation; De Bernardez Clark E et al.; Since the inception of recombinant DNA technology, different strategies have been developed in the isolation, renaturation, and native disulfide bond formation of proteins produced as insoluble inclusion bodies in Escherichia coli . One of the major challenges in optimizing renaturation processes is to prevent the formation of off-pathway inactive and aggregated species . On the basis of a simplified kinetic model describing the competition between folding and aggregation, it was possible to analyze the effects of denaturant and thiol/disulfide concentrations on this competition . Although higher guanidinium chloride (GdmCl) concentrations resulted in higher renaturation yields, the folding rate was negatively affected, indicating an optimum range of GdmCl for optimum renaturation rates and yields . Similarly, higher total glutathione concentrations resulted in higher yields but decreased rates, also indicating an optimum total glutathione concentration for optimum renaturation rates and yields (6-16 mM), with an optimum ratio of reduced to oxidized glutathione between 1 and 3 . To characterize the nature of aggregates, aggregation experiments were performed under different oxidizing/reducing conditions . It is shown that hydrophobic interactions between partially folded polypeptide chains are the major cause of aggregation . Aggregation is fast and aggregate concentration does not significantly increase beyond the first minute of renaturation . Under conditions which promote disulfide bonding, aggregate size, but not concentration, may increase due to disulfide bond formation, resulting in covalently bonded aggregates.

J Pharmacol Exp Ther, 1998 Mar, 284(3), 1156 - 64
Mast cell chymase-like protease(s) modulates Escherichia coli lipopolysaccharide-induced vasomotor dysfunction in skeletal muscle in vivo; Suzuki H et al.; This study investigated whether short-term exposure to Escherichia coli lipopolysaccharide (LPS) elicits vasomotor dysfunction in skeletal muscle in vivo and, if so, whether perivascular mast cell proteases partly modulate this response . With intravital microscopy, we found that suffusion of E . coli LPS on the in situ hamster spinotrapezius muscle for 60 min elicits immediate vasoconstriction followed by vasodilation . Vasoconstriction is abrogated by SK&F 108566, a selective, nonpeptide angiotensin II (AT II) subtype 1 receptor antagonist, chymostatin and soybean trypsin inhibitor . These compounds also attenuate E . coli LPS-induced vasodilation . By contrast, superoxide dismutase, catalase and indomethacin attenuate only E . coli LPS-induced vasodilation . Endothelin receptor antagonists, lisinopril, leupeptin, Bestatin and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid are ineffective . Histochemical analysis of the spinotrapezius muscle reveals abundant perivascular mast cells with chymostatin-inhibitable chymase-like activity . Pretreatment of hamsters with compound 48/80 for 4 days curtails E . coli LPS-induced vasoconstriction and converts vasodilation to vasoconstriction . On balance, these data indicate that E . coli LPS stimulates perivascular mast cells in the in situ hamster spinotrapezius muscle to release an AT II-producing chymase-like protease(s) . AT II thus produced elicits local vasoconstriction and elaborates reactive oxygen species which, in turn, generate vasodilator prostaglandins.

J Bacteriol, 1998 Mar, 180(5), 1287 - 95
Molecular characterization of the alpha-glucosidase gene (malA) from the hyperthermophilic archaeon Sulfolobus solfataricus; Rolfsmeier M et al.; Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth . To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble alpha-glucosidase (maltase) and flanking sequences from Sulfolobus solfataricus were cloned and characterized . This is the first report of an alpha-glucosidase gene from the archaeal domain . malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa . It is flanked on the 5' side by an unusual 1-kb intergenic region . Northern blot analysis of the malA region identified transcripts for malA and an upstream open reading frame located 5' to the 1-kb intergenic region . The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5' of the malA start codon . Gel mobility shift analysis of the malA promoter region suggests that sequences 3' to position -33, including a consensus archaeal TATA box, play an essential role in malA expression . malA homologs were detected by Southern blot analysis in other S . solfataricus strains and in Sulfolobus shibatae, while no homologs were evident in Sulfolobus acidocaldarius, lending further support to the proposed revision of the genus Sulfolobus . Phylogenetic analyses indicate that the closest S . solfataricus alpha-glucosidase homologs are of mammalian origin . Characterization of the recombinant enzyme purified from Escherichia coli revealed differences from the natural enzyme in thermostability and electrophoretic behavior . Glycogen is a substrate for the recombinant enzyme . Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism . These results indicate a unique role for the S . solfataricus alpha-glucosidase in carbohydrate metabolism.

J Bacteriol, 1998 Mar, 180(5), 1277 - 86
Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon; Haldimann A et al.; Escherichia coli genes regulated by environmental inorganic phosphate (Pi) levels form the phosphate (Pho) regulon . This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the Pi-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU . Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated P(araB) promoter or the rhamnose-regulated P(rhaB) promoter . To do this, we made allele replacement plasmids that may be generally useful for construction of P(araB) or P(rhaB) fusions and for recombination of them onto the E . coli chromosome at the araCBAD or rhaRSBAD locus, respectively . Using strains carrying such single-copy fusions, we showed that a P(rhaB) fusion is more tightly regulated than a P(araB) fusion in that a P(rhaB)-phoR+ fusion but not a P(araB)-phoR+ fusion shows a null phenotype in the absence of its specific inducer . Yet in the absence of induction, both P(araB)-phoB+ and P(rhaB)-phoB+ fusions exhibit a null phenotype . These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action . We also used these fusions to study PhoU . Previously, we had constructed strains with precise delta phoU mutations . However, we unexpectedly found that such delta phoU mutants have a severe growth defect (P . M . Steed and B . L . Wanner, J . Bacteriol . 175:6797-6809, 1993) . They also readily give rise to compensatory mutants with lesions in phoB, phoR, or a pst gene, making their study particularly difficult . Here we found that, by using P(araB)-phoB+, P(rhaB)-phoB+, or P(rhaB)-phoR+ fusions, we were able to overcome the extremely deleterious growth defect of a Pst+ delta phoU mutant . The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.

J Bacteriol, 1998 Mar, 180(5), 1232 - 40
Identification of dnaX as a high-copy suppressor of the conditional lethal and partition phenotypes of the parE10 allele; Levine C et al.; Termination of DNA replication, complete topological unlinking of the parental template DNA strands, partition of the daughter chromosomes, and cell division follow in an ordered and interdependent sequence during normal bacterial growth . In Escherichia coli, topoisomerase IV (Topo IV), encoded by parE and parC, is responsible for decatenation of the two newly formed chromosomes . In an effort to uncover the pathway of information flow between the macromolecular processes that describe these events, we identified dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, as a high-copy suppressor of the temperature-sensitive phenotype of the parE10 allele . We show that suppression derives from overexpression of the gamma, but not the tau, subunit of the holoenzyme and that the partition defect of parE10 cells is nearly completely reverted at the nonpermissive temperature as well . These observations suggest a possible association between Topo IV and the replication machinery.

J Bacteriol, 1998 Mar, 180(5), 1224 - 31
Use of a two-color genetic screen to identify a domain of the global regulator Lrp that is specifically required for pap phase variation; Kaltenbach L et al.; The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation . Unlike most other members of the Lrp regulon such as ilvIH, activation of papBA transcription requires the coregulator PapI and is methylation dependent . We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate ilvIH transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains . Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on ilvIH transcription . In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for pap and ilvIH DNAs similar to those of wild-type Lrp . In addition, both mutant Lrp's were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity for pap Lrp binding sites 4, 5, and 6 . Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited . In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-locked pap derivative containing a mutation in Lrp binding site 3 . Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI . Our data suggest that these mutant Lrp's are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on pap transcription state.

J Bacteriol, 1998 Mar, 180(5), 1200 - 6
The narA locus of Synechococcus sp . strain PCC 7942 consists of a cluster of molybdopterin biosynthesis genes; Rubio LM et al.; The narA locus required for nitrate reduction in Synechococcus sp . strain PCC 7942 is shown to consist of a cluster of genes, namely, moeA, moaC, moaD, moaE, and moaA, involved in molybdenum cofactor biosynthesis . The product of the moaC gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from Escherichia coli and in its C-terminal half to MoaB or Mog . Overexpression of the Synechococcus moaC gene in E . coli resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of E . coli . Insertional inactivation of the moeA, moaC, moaE, and moaA genes showed that the moeA-moa gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942 . The moaCDEA genes constitute an operon which is transcribed divergently from the moeA gene . Expression of the moeA gene and the moa operon was little affected by the nitrogen source present in the culture medium.

J Bacteriol, 1998 Mar, 180(5), 1159 - 65
Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution; Boyd EF et al.; We studied the ancestry of virulence-associated genes in Escherichia coli by examining chromosomal regions specific to pathogenic isolates . The four virulence determinants examined were the alpha-hemolysin (hly) loci hlyI and hlyII, the type II capsule gene cluster kps, and the P (pap) and S (sfa) fimbria gene clusters . All four loci were shown previously to be associated with pathogenicity islands of uropathogenic E . coli isolates . The hly, kps, sfa, and pap regions each have an unexpected clustered distribution among the E . coli collection of reference (ECOR) strains, but all these regions were absent from a collection of diarrheagenic E . coli isolates . Strains in the ECOR subgroup B2 typically had a combination of at least three of the four loci, and all strains in subgroup D had a copy of the kps and pap clusters . In contrast, only four strains in subgroup A had either hly, kps, sfa, or pap, and no subgroup A strains had all four together . Strains of subgroup B1 were devoid of all four virulence regions, with the exception of one isolate that had a copy of the sfa gene cluster . This phylogenetic distribution of strain-specific sequences corresponds to the ECOR groups with the largest genome size, namely, B2 and D . We propose that the pathogenicity islands are ancestral to subgroups B2 and D and were acquired after speciation, with subsequent horizontal transfer into some group A, B1, and E lineages . These results suggest that the hly, kps, sfa, and pap pathogenicity determinants may play a role in the evolution of enteric bacteria quite apart from, and perhaps with precedence over, their ability to cause disease.

J Bacteriol, 1998 Mar, 180(5), 1095 - 102
Multiple regions on the Escherichia coli heat shock transcription factor sigma32 determine core RNA polymerase binding specificity; Joo DM et al.; We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoH gene, which encodes sigma32 in Escherichia coli . All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2 . Based on the estimated K(s)s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes . The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants' effect on core RNAP interaction was only clearly seen in the presence of sigma70 competitor . The class I mutant behaved nearly identically to the wild type in core RNAP binding . Two point mutations in class III altered residues that were distant from one another . One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors . These data suggest that there is more than one core RNAP binding region in sigma32 and that differences in contact sites probably exist among sigma factors.

J Bacteriol, 1998 Mar, 180(5), 1053 - 62
Control of ftsZ expression, cell division, and glutamine metabolism in Luria-Bertani medium by the alarmone ppGpp in Escherichia coli; Powell BS et al.; Inactivation of transcription factor sigma54, encoded by rpoN (glnF), restores high-temperature growth in Luria-Bertani (LB) medium to strains containing the heat-sensitive cell division mutation ftsZ84 . Mutational defects in three other genes involved in general nitrogen control (glnD, glnG, and glnL) also suppress lethal filamentation . Since addition of glutamine to LB medium fully blocks suppression by each mutation, the underlying cause of suppression likely derives from a stringent response to the limitation of glutamine . This model is supported by several observations . The glnL mutation requires RelA-directed synthesis of the nutrient alarmone ppGpp to suppress filamentation . Artificially elevated levels of ppGpp suppress ftsZ84, as do RNA polymerase mutations that reproduce global effects of the ppGpp-induced state . Both the glnF null mutation and an elevated copy number of the relA gene similarly affect transcription from the upstream (pQ) promoters of the ftsQAZ operon, and both of these genetic conditions increase the steady-state level of the FtsZ84 protein . Physiological suppression of ftsZ84 by a high salt concentration was also shown to involve RelA . Additionally, we found that the growth of a glnF or glnD strain on LB medium depends on RelA or supplemental glutamine in the absence of RelA function . These data expand the roles for ppGpp in the regulation of glutamine metabolism and the expression of FtsZ during cell division.

Clin Chim Acta, 1997 Dec 10, 268(1-2), 41 - 60
In vivo kinetics as a sensitive method for testing physiologically intact human recombinant apolipoprotein A-I: comparison of three different expression systems; Schmidt HH et al.; In order to assess the structural and functional integrity of recombinant human apoA-I, we expressed apoA-I using three different expression systems: Baculovirus transfected Spodoptera frugiperda (Sf9) cells, stably transfected Chinese hamster ovary (CHO) cells, and transformed Escherichia coli (E . coli) . Purified apoA-I from the three expression systems was radioiodinated and their catabolism was compared in normolipemic rabbits . The kinetic turnover studies of radiolabelled apoA-I in normolipemic rabbits revealed that highly purified recombinant apoA-I had an identical decay curve compared to native apoA-I, regardless whether it was purified from Sf9 cells, CHO cells, or E . coli . We also determined the association of the three recombinant apoA-I forms with both rabbit and human HDL . All three recombinant apoA-I forms were associated with HDL2 and HDL3 after injection into the rabbits and after incubation with human serum using both a Superose 6 column separation system and density gradient ultracentrifugation . The addition of the pro-segment or the addition of methionine at the amino-terminal end of apoA-I did not alter its metabolism and association to HDL . In conclusion, all studied expression systems are capable of producing high levels of physiologically intact recombinant human apoA-I . The aminoterminal addition of the prosegment of apoA-I or methionine did not alter the in vivo metabolism of apoA-I or its association to HDL.

Brain Res Mol Brain Res, 1997 Dec 15, 52(2), 182 - 93
Structure-function relationship of different domains of the rat corticotropin-releasing factor receptor; Sydow S et al.; The significance of different domains of corticotropin-releasing factor receptor, type 1, (CRFR1) for ligand binding and cAMP accumulation was investigated with C-terminally truncated forms of rat CRFR1 (rCRFR1) tagged by a sequence of six histidine residues (His-tag) to facilitate protein purification and identification . These different forms of the receptor were N-glycosylated and transported properly to the membranes of transfected mammalian cells as indicated by Western blot analysis and immunocytochemical staining with two polyclonal antibodies developed against the N- and C-terminus of rCRFR1 . The N-terminal fragment, rCRFR1(23-121), expressed in Escherichia coli bound oCRF specifically, but with low affinity . Several mutants lacking transmembrane domain (TM) 7 and the C-terminus exhibited similarly low affinities to oCRF after expression in transfected mammalian cells . None of these cells produced significant amounts of cAMP after exposure to oCRF . Only mutants containing the N-terminus, all loops and TMs bound oCRF and produced cAMP with high affinity (Kd = 62 nM) and efficacy (EC50 = 0.8 nM) . The additional presence of the C-terminus provided similar characteristics (Kd = 5 nM, EC50 = 0.3 nM) as known for the native receptor . It is suggested on the basis of these data that the last extracellular loop is involved in ligand binding.

Poult Sci, 1998 Feb, 77(2), 256 - 65
Antibody responses and body weights of chicken lines selected for high and low humoral responsiveness to sheep red blood cells . 2 . Effects of separate application of Freund's Complete and Incomplete Adjuvant and antigen; Parmentier HK et al.; Antibody (Ab) responses to SRBC, BSA, Mycobacterium butyricum, and Escherichia coli lipopolysaccharide (LPS) were measured in two chicken lines divergently selected for high and low Ab responses to SRBC, and in a randombred control line . Levels of Ab binding SRBC, BSA, and Mycobacterium protein, but not LPS were higher in the high Ab producing (H) line than in the control (C) and low Ab producing (L) lines (P < 0.05), and at almost every time, the L line showed significantly lower titers than the H and C lines . In the H and C lines, Ab responses to SRBC were enhanced when Complete Freund's Adjuvant (CFA) or Incomplete Freund's Adjuvant (IFA) were simultaneously administered on a separate location than SRBC . In the L line, Ab titers to SRBC and BSA were enhanced when antigen was administered emulsified in CFA . At all times until 28 d after sensitization the C and L line birds were significantly heavier than birds of the H line . Body weight, body growth, and percentage body growth were impaired in birds that received antigen emulsified in CFA, which suggested a negative relationship between BW gain and immune responses to Mycobacteria protein . Prolonged divergent selection for Ab responses to SRBC resulted into two lines that not only differ in Ab responses to T cell-dependent antigens but also in BW . In contrast to previous findings with the current lines, line differences with respect to Ab responses were not abolished by CFA treatment.

Poult Sci, 1998 Feb, 77(2), 248 - 55
Antibody responses and body weights of chicken lines selected for high and low humoral responsiveness to sheep red blood cells . 1 . Effect of Escherichia coli lipopolysaccharide; Parmentier HK et al.; Antibody (Ab) responses to i.m . administered SRBC and BSA, and i.p . administered Escherichia coli lipopolysaccharide (LPS), and BW at various times after treatment, were measured in chicken lines divergently selected for high (H) and low (L) Ab responses to SRBC, and in a randombred control line (C) . The Ab responses to SRBC and BSA, but not LPS, were significantly affected by line by treatment interactions . Levels of antibodies to SRBC and BSA were higher in the H line than in either the C or L line (P < 0.05) . Administration of LPS did not affect Ab responses to SRBC, but Ab responses to BSA were decreased in birds that received BSA and LPS simultaneously . Body weights of C and L lines were significantly higher than BW of H line birds at all times . Lipopolysaccharide injection induced an acute, but transient reduction of BW gain, which was not affected by line . Antibody responses to SRBC and BSA were negatively correlated with BW . During the experimental period, however, percentage BW gain and humoral responsiveness were positively correlated . A higher percentage BW gain growth was seen in H line birds at the end of the experimental period . The present results confirm the hypothesized acute cachectin nature of LPS, but the relationship between live BW (gain) and immune responsiveness in chickens remains to be further clarified.

Circulation, 1998 Feb 24, 97(7), 645 - 50
Induction of neoangiogenesis in ischemic myocardium by human growth factors: first clinical results of a new treatment of coronary heart disease; Schumacher B et al.; BACKGROUND: The present article is a report of our animal experiments and also of the first clinical results of a new treatment for coronary heart disease using the human growth factor FGF-I (basic fibroblast growth factor) to induce neoangiogenesis in the ischemic myocardium . METHODS AND RESULTS: FGF-I was obtained from strains of Escherichia coli by genetic engineering, then isolated and highly purified . Several series of animal experiments demonstrated the apathogenic action and neoangiogenic potency of this factor . After successful conclusion of the animal experiments, it was used clinically for the first time . FGF-I (0.01 mg/kg body weight) was injected close to the vessels after the completion of internal mammary artery (IMA)/left anterior descending coronary artery (LAD) anastomosis in 20 patients with three-vessel coronary disease . All the patients had additional peripheral stenoses of the LAD or one of its diagonal branches . Twelve weeks later, the IMA bypasses were selectively imaged by intra-arterial digital subtraction angiography and quantitatively evaluated . In all the animal experiments, the development of new vessels in the ischemic myocardium could be demonstrated angiographically . The formation of capillaries could also be demonstrated in humans and was found in all cases around the site of injection . A capillary network sprouting from the proximal part of the coronary artery could be shown to have bypassed the stenoses and rejoined the distal parts of the vessel . CONCLUSIONS: We believe that the use of FGF-I for myocardial revascularization is in principle a new concept and that it may be particularly suitable for patients with additional peripheral stenoses that cannot be revascularized surgically.

Prostate, 1998 Feb 15, 34(3), 195 - 202
Effects of combined treatment of chemotherapeutics and hyperthermia on survival and the regulation of heat shock proteins in Dunning R3327 prostate carcinoma cells; Roigas J et al.; BACKGROUND: Hyperthermia can enhance the clinical response of chemotherapeutic agents in prostate cancer, but optimal sequencing of this combination therapy needs to be developed . Given the role of heat shock proteins (HSPs) in the development of resistance (thermotolerance) to subsequent hyperthermic stresses as well as to certain chemotherapeutics, the study of HSP regulation is important in the establishment of effective schedules in multimodal treatment strategies . METHODS: In this study we evaluated the effects of the chemotherapeutic agents cisplatin, 5-fluorouracil, and adriamycin in combination with hyperthermia . (43 degrees C, 1 h) on clonogenic survival and inducible HSP70 regulation in Dunning rat adenocarcinoma of the prostate . HSP70 was analyzed by Western blot and by measuring beta-galactosidase produced by cells stably transfected with a gene construct containing the E . coli beta-galactosidase gene driven by the Drosophila HSP70 promoter . RESULTS: Colony formation assays revealed a sensitizing effect of hyperthermia when simultaneously combined with each chemotherapeutic agent, resulting in a potentiated cytotoxicity compared to subsequenced treatments . Thermotolerant cells showed a significantly better survival when treated with adriamycin alone, but also when each chemotherapeutic agent was combined with hyperthermia . This enhanced survival was correlated with inducible HSP70 accumulation . The chemotherapeutics modified the HSP70 promoter activation induced by hyperthermia, suggesting changes in the development of cellular thermotolerance . CONCLUSIONS: Our data reveal synergistic cytotoxic effects of the synchronous application of chemotherapeutic agents and hyperthermia on this model of prostate cancer . Furthermore, they demonstrate that the induction of HSPs in thermotolerant cells, as measured by HSP70 induction, results in a modulation the chemotherapeutic-mediated cytotoxicity . Therefore, HSP70 is a useful marker of cellular resistance in multimodal approaches combining hyperthermia and chemotherapeutic agents in the treatment of locally advanced prostate carcinoma.

Circulation, 1998 Feb 17, 97(6), 521 - 4
Adenosine inhibits lipopolysaccharide-induced secretion of tumor necrosis factor-alpha in the failing human heart; Wagner DR et al.; BACKGROUND: The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of congestive heart failure . Recent studies have shown that adenosine inhibits lipopolysaccharide (LPS)-induced expression of TNF-alpha in macrophages and rat cardiomyocytes . The aim of this study was to determine whether adenosine has a similar effect in the failing human heart . METHODS AND RESULTS: Left ventricular muscle strips were obtained from seven patients with end-stage congestive heart failure undergoing heart transplantation or insertion of a left ventricular assist device . The muscle strips were incubated at 37 degrees C in 95% O2/5% CO2 and stimulated with LPS (10 microg/mL) . TNF-alpha release in the supernatant was measured with ELISA, and muscle sections were stained for TNF-alpha . Muscle strips released TNF-alpha in the absence of LPS (0.22+/-0.05 pg x mL(-1) x mg wet wt{-1}) . TNF-alpha was immunolocalized to the cardiac myocyte, suggesting that the myocyte is a source for TNF-alpha production . Adenosine (10 micromol/L) decreased TNF-alpha by 40% (P<.05) . The selective adenosine A2 receptor agonist DPMA (10 micromol/L) decreased TNF-alpha release by 87% (P<.001), whereas ITu (10 micromol/L), an adenosine-regulating agent that increases endogenous adenosine concentration, inhibited TNF-alpha release by 93% (P<.001) . CONCLUSIONS: Adenosine can significantly diminish TNF levels in the failing human heart and may represent a new pharmacological intervention in congestive heart failure.

Biochem Cell Biol, 1997, 75(4), 369 - 75
Preferential localization of DNA damage induced by depurination and bleomycin in a plasmid containing a scaffold-associated region; Legault J et al.; Recent evidence suggests that DNA damage of various origins is not randomly distributed in the genome but appears to be clustered in unidentified hypersensitive regions of the chromatin . A model was proposed that stipulates that unpaired DNA stretches, such as those found in scaffold- (or matrix)-associated regions (SARs) under torsional strain, are candidate regions of hypersensitivity to DNA damage in vivo . In this study, we assessed in vitro the relative susceptibility of supercoiled plasmids containing a SAR or chromatin loop DNA segment to DNA damage induced by acid-catalyzed depurination or FeIII-bleomycin . Single-strand specific S1 nuclease was used in combination with 3'-end-labeling to detect single-strand breaks or gaps, after cleavage of abasic sites or removal of 3'-phosphoglycolates by Escherichia coli endonuclease IV . The optimal conditions of DNA cleavage specificity by S1 nuclease were determined . Using these conditions, the DNA cleavage patterns obtained showed (i) a preferential localization of S1 hypersensitive sites in the SAR DNA as compared with plasmid or chromatin loop DNA and (ii) a strikingly similar localization of DNA damage with the two clastogenic treatments.

Biochem Cell Biol, 1997, 75(4), 327 - 36
The apurinic-apyrimidinic endonuclease IV family of DNA repair enzymes; Ramotar D; Apurinic-apyrimidinic (AP) sites are DNA lesions that lack template information and are produced either spontaneously or by a variety of DNA damaging agents . AP sites must therefore be repaired; otherwise they are mutagenic . All cells investigated to date possess the DNA repair enzyme AP endonuclease that can repair AP sites . There are two discrete families of AP endonucleases, Exo III and Endo IV, which are exemplified by Escherichia coli exonuclease III and endonuclease IV, respectively . These AP endonucleases have evolved not only to repair AP sites but also to correct distinct DNA lesions.

Microbiology, 1998 Feb, 144 ( Pt 2), 561 - 7
The recombinant antigen ASPND1r from Aspergillus nidulans is specifically recognized by sera from patients with aspergilloma; Calera JA et al.; A 996 bp Aspergillus nidulans cDNA encoding the ASPND1 immunodominant antigen was cloned and expressed in Escherichia coli as a fusion protein with the enzyme glutathione S-transferase (GST) from Schistosoma japonicum . The GST-ASPND1 fusion protein was purified from isolated bacterial inclusion bodies by preparative SDS-PAGE . After cleavage with thrombin, the ASPND1 recombinant antigen (ASPND1r) and the GST protein were separated by SDS-PAGE and immunoblotted with a number of different human sera . The sera from 22 (88%) of 25 patients with an aspergilloma recognized the ASPND1r recombinant antigen on immunoblots . Forty-nine normal human sera and 14 sera from patients with other infections were unreactive . The ASPND1r expressed in E . coli could therefore be used, in combination with previously reported recombinant antigens, as a standardized antigen for serological and clinical diagnosis of Aspergillus-associated diseases.

Microbiology, 1998 Feb, 144 ( Pt 2), 361 - 73
Requirement for ubiquinone downstream of cytochrome(s) b in the oxygen-terminated respiratory chains of Escherichia coli K-12 revealed using a null mutant allele of ubiCA; Soballe B et al.; An Escherichia coli knockout ubiCA mutant has been constructed using a gene replacement method and verified using both Southern hybridization and PCR . The mutant, which was unable to synthesize ubiquinone (Q), showed severely diminished growth yields aerobically but not anaerobically with either nitrate or fumarate as terminal electron acceptors . Low oxygen uptake rates were demonstrated in membrane preparations using either NADH or lactate as substrates . However, these rates were greatly stimulated by the addition of ubiquinone-1 (Q-1) . The rate of electron transfer to those oxidase components observable by photodissociation of their CO complexes was studied at sub-zero temperatures . In the ubiCA mutant, the reduced form of haemoproteins--predominantly cytochrome b595--was reoxidized significantly faster in the presence of oxygen than in a Ubi+ strain, indicating the absence of Q as electron donor . Continuous multiple-wavelength recordings of the oxidoreduction state of cytochrome(s) b during steady-state respiration showed greater reduction in membranes from the ubiCA mutant than in wildtype membranes . A scheme for the respiratory electron-transfer chain in E . coli is proposed, in which Q functions downstream of cytochrome(s) b.

Microbiology, 1998 Feb, 144 ( Pt 2), 353 - 9
Appearance of a stress-response protein, phage-shock protein A, in Escherichia coli exposed to hydrophobic organic solvents; Kobayashi H et al.; A 28 kDa protein associated with the inner membrane was induced strongly in Escherichia coli K-12 cells grown in the presence of a hydrophobic organic solvent, n-hexane or cyclooctane . These organic solvents suppressed the growth (growth rate and yield) of E . coli . A partial amino acid sequence showed that this protein was the phage-shock protein PspA . PspA is known to be induced in E . coli cells under extreme stress conditions . The results suggest that E . coli cells are subject to strong stress in the presence of organic solvents . Introduction of a multi-copy plasmid vector carrying the psp operon into E . coli improved the survival frequency of cells exposed suddenly to n-hexane but not the growth rate of cells growing in the presence of n-hexane.

Structure, 1998 Jan 15, 6(1), 11 - 21
Functional analyses of the domain structure in the Holliday junction binding protein RuvA; Nishino T et al.; BACKGROUND: Homologous recombination is crucial for genetic diversity and repairing damaged chromosomes . In Escherichia coli cells, the RuvA, RuvB and RuvC proteins participate in the processing of an important intermediate, the Holliday junction . The RuvA-RuvB protein complex facilitates branch migration of the junction, depending on ATP hydrolysis . The atomic structure of RuvA should enable critical questions to be addressed about its specific interactions with the Holliday junction and the RuvB protein . RESULTS: The crystal structure of RuvA shows the tetrameric molecules with a fourfold axis at the center . Each subunit consists of three distinct domains, some of which contain important secondary structure elements for DNA binding . Together with the detailed structural information, the biochemical assays of various mutant RuvA proteins and domains, isolated by partial proteolysis, allowed us to define the functional roles of these domains in Holliday junction binding and the RuvB interaction . CONCLUSIONS: The RuvA molecule is formed by four identical subunits, each with three domains, I, II and III . The locations of the putative DNA-binding motifs define an interface between the DNA and the Holliday junction . Domain III is weakly attached to the core region, comprising domains I and II; the core domains can form a tetramer in the absence of domain III . Functional analyses of the mutant proteins and the partial digestion products, including Holliday junction binding and branch-migration assays, revealed that domain III and the preceding loop are crucial for RuvB binding and branch migration, although this region is not required for the junction-DNA binding.

Mikrobiologiia, 1997 Nov-Dec, 66(6), 790 - 5
{Comparative study of properties of two extracellular protectors, isolated from Escherichia coli at elevated temperature}; Nikolaev IuA; The regularities of the formation and action, as well as some physicochemical properties, of two extracellular protectants secreted by Escherichia coli cells under the condition of heat shock (a temperature increase from 37 to 46-49 degrees C), were investigated . Both factors, "antilysin" (factor X(I)), either protecting from, or diminishing, cell lysis under the action of 9 microM N-ethylmaleimide (NEM), and "mu-reducer" (factor X(II)), reducing the rate of culture growth after a 60-min incubation and increasing culture viability in the presence of 200 microM NEM, were shown to be low-molecular-weight substances (less than 10 kDa) stable at extreme (pH values pH 1 and pH 11) . Factors X(I) and X(II) differed in the dynamics of their accumulation in the culture liquid under unfavorable conditions and in the dependence pattern of their action upon concentration . Unlike factor X(II), factor X(I) was highly unstable during heating and storage . By their characteristics, X(I) should be considered a protectant, or an adaptogen of direct action, and X(II) to an inductor activating the cellular system to enhance cell viability.

Genetika, 1997 Dec, 33(12), 1655 - 63
{Correlation of changes in bone marrow cell sensitivity to the clastogenic effect of thiotepa and fertility in CBA/LacY mice in successive inbred generations}; Malashenko AM et al.; The causes of the significant increases in the sensitivity to of clastogenic effect of thio-TEPA in bone marrow cells of inbred CBA/LacY mice were studied . The increases were shown to be associated with decreased fertility . The fluctuations of fertility and the association of fertility and sensitivity with thio-TEPA were analyzed in foundation stocks (FSs) for 24 and 6 inbred generations, respectively . The stocks examined were obtained in the Laboratory of Experimental Biological Models and the Stolbovaya breeding facility . The correlation analysis revealed a significant association of fluctuations of the coefficient of fertility in two FSs (eta = 0.78 +/- 0.13) and a high negative correlation between fertility and mutagen sensitivity in inbred generations (r = -0.91 +/- 0.21) . The comparison between the fluctuations of fertility for four generations revealed the same fluctuation period in CBA/LacY, DBA/2Y, and C57Bl/6JY mice . The detected phenomena were assumed to result from genetically determined regular fluctuations in genome functioning . This explained a number of observations and allowed several prognoses to be made.

Genetika, 1997 Dec, 33(12), 1621 - 8
{A mobile genetic element on a Bordetella pertussis chromosome stimulates cointegrate formation in Escherichia coli strains}; Kirillov MIu et al.; This work continues a series of reports devoted to the study of a repeated sequence isolated from the Bordetella pertussis chromosome (RSBP--Repeated Sequence of Bordetella Pertussis) . The RSBP was earlier demonstrated to have a structure similar to IS elements and exhibit some properties of mobile genetic elements . The results of this work demonstrate the ability of this novel genetic element to stimulate the formation of cointegrates between independent replicons . We have studied the structure of five cointegrates formed by integration of a resident RSBP-containing plasmid into three different sites of a recipient replicon . The studied cointegrates are capable of resolution with the release of a resident plasmid independent of the host cell RecA function.

Biochemistry, 1998 Feb 17, 37(7), 1736 - 42
Molten globule unfolding monitored by hydrogen exchange in urea; Chamberlain AK et al.; The molten globule state of Escherichia coli ribonuclease H1 was studied by hydrogen exchange in order to understand how the energetics of individual regions react to the presence of denaturant . Hydrogen exchange rates were monitored (1) directly by NMR spectroscopy and (2) indirectly by quenching the exchange process and returning to the native state for NMR detection . Direct hydrogen exchange on the molten globule state demonstrated that the observed protons exchange 3-100-fold more slowly than in an unfolded peptide . The quenched hydrogen exchange experiments were modeled after the recently developed "native state hydrogen exchange" experiment and were carried out as a function of urea concentration . The free energy of hydrogen exchange varied linearly with denaturant concentration for all 29 measurable protons, suggesting that each amide's exchange behavior can be modeled with only one type of opening transition . The free energy of unfolding measured by hydrogen exchange and corresponding m values varied for each residue implying a noncooperative molten globule structure . These results are in contrast to similar exchange experiments on native proteins which generally display more than one type of exchange behavior . The single type of exchange seen in the molten globule is probably due to its larger conformational freedom and noncooperative nature.

Schweiz Arch Tierheilkd, 1998, 140(2), 60 - 4
{Reasons for condemnation of slaughtered broilers from two large Swiss producers}; Jakob HP et al.; 30 broiler flocks from two majors Swiss broiler producing companies were examined for lesions relevant to meat inspection between November 1995 and April 1996 . 71.8% of condemned carcasses and viscera were assigned to ascites syndrome, bacterial infections and runting . Other diseases and lesions due to injury or processing were rare . Escherichia coli was responsible for the majority of infections . The average condemnation rate (1.0%) corresponded to findings in literature.

Eur J Biochem, 1998 Jan 15, 251(1-2), 510 - 5
Modulation of the substrate specificity of Escherichia coli dimethylsulfoxide reductase; Simala-Grant JL et al.; X-ray crystallographic studies of the Rhodobacter sphaeroides dimethyl sulfoxide (Me2SO) reductase {Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K . V., & Rees, D . C . (1996) Science, 272, 1615-1620} indicated that the active site is at the bottom of a 25-A funnel . Substrates must travel to the bottom of the funnel for reduction to occur . The homologous DmsA subunit of the trimeric Escherichia coli Me2SO reductase, was subjected to site-directed mutagenesis of residues potentially lining the bottom of the funnel, based on sequence alignment of the E . coli and Rhodobacter Me2SO reductases . Sixteen E . coli DmsA mutants were characterized . Mutants G167N, A178Q, Q179I and R217Q showed functional impairment, as indicated by abnormal anaerobic growth with Me2SO as the sole terminal acceptor, in a recombinant strain deleted for chromosomal dmsABC . The kinetic parameters of the mutant enzymes were examined using the artificial electron donor benzyl viologen and the quinone analogue dimethylnaphthoquinone, with Me2SO and pyridine N-oxide as electron acceptors . Mutants A178Q and R217Q showed dramatic alterations of their electron-acceptor Km, with values at least 35-fold less or greater than wild-type values, respectively, for Me2SO and pyridine N-oxide . T148S showed altered kinetic parameters for pyridine N-oxide and Me2SO, with Km and k(cat) decreasing and increasing approximately fourfold, respectively . Other mutants showed less drastic alterations in kinetic parameters . This analysis has identified amino acids important in substrate binding and catalysis.

Eur J Biochem, 1998 Jan 15, 251(1-2), 443 - 7
Cloning and characterisation of a group II allergen from the dust mite Tyrophagus putrescentiae; Eriksson TL et al.; The complete cDNA encoding a major allergen from the dust mite Tyrophagus putrescentiae, Tyr p 2, has been sequenced and expressed . A degenerate primer was designed to the N-terminal amino acid sequence of the 16-kDa protein . The complete cDNA sequence was achieved by using reverse transcriptase PCR, PCR+1, standard cloning and sequencing techniques . The cDNA of Tyr p 2 is 552 nucleotides in length from the start codon including 126 nucleotides after the stop codon up to the beginning of the poly(A) tail . The leader sequence consists of 15 amino acids . Regarding the predicted amino acid sequence, there are no potential N-glycosylation sites (N-X-S/T) . The sequence showed similarity to group II allergens from other mite species, and some regions are completely conserved . To show that the cloned cDNA sequence was coding for an allergen, Tyr p 2 was expressed in Escherichia coli and shown to react with a T . putrescentiae-positive serum pool.

Eur J Biochem, 1998 Jan 15, 251(1-2), 413 - 7
Metabolic compartmentation of plastid prenyllipid biosynthesis--evidence for the involvement of a multifunctional geranylgeranyl reductase; Keller Y et al.; The addition of phytyl side chain to chlorophylls, tocopherols and phylloquinone is prerequisite to their integration into plastid membranes . We have cloned a cDNA encoding a pre-geranylgeranyl reductase from Arabidopsis thaliana . The deduced primary structure predicts a mature size with a molecular mass of 47 kDa and displays a characteristic dinucleotide binding domain . Geranylgeranyl reductase expressed in Escherichia coli sequentially catalyzes the reduction of geranylgeranyl-chlorophyll a into phytyl-chlorophyll a as well as the reduction of free geranylgeranyl diphosphate into phytyl diphosphate . Due to its multifunctionality and weak hydrophobicity, we suggest that in plastid the same geranylgeranyl reductase is recruited into the chlorophyll, the tocopherol and the phylloquinone pathways . The geranylgeranyl reductase gene is up-regulated during etioplast to chloroplast and chloroplast to chromoplast development.

Eur J Biochem, 1998 Jan 15, 251(1-2), 389 - 97
Cloning, sequencing, and expression of the PSA genes from Leishmania infantum; Jimenez-Ruiz A et al.; We describe the molecular cloning of the PSA genes from the Leishmania infantum parasite, which show high sequence similarity with the L . major PSA-2 and L . amazonensis GP46/M2 genes . The PSA genes in L . infantum are arrayed in tandem with a repetition unit of 6 kb . A single-size class of PSA mRNA of 4 kb was detected . The characterised L . infantum PSA genes code for a protein lacking the glycosylphosphatidylinositol addition signal described in other Leishmania species due to the presence of a stop codon located upstream from the DNA sequence coding for the signal . The data obtained after immunoprecipitation of PSA indicate that the protein is present as a water-soluble form, but that also a membrane-anchored form can be detected . The amino acid sequence derived from the isolated PSA gene shows that 60% of the deduced protein is formed by 13 leucine-rich repeats, each one of which is 24 amino acids long . The analysis of the consensus sequence of the repeats revealed that the L . infantum PSA as well as the described L . major PSA-2 and L . amazonensis GP46/M2 proteins may be classified as new members of the leucine-rich repeat-containing protein superfamily . The number of leucine-rich motifs, however, varies considerably between the PSA protein from L . infantum and from the other Leishmania species . The PSA protein is a major antigen determinant during L . infantum infections since 87% of the sera from naturally infected dogs recognise the recombinant PSA purified from Escherichia coli.

Eur J Biochem, 1998 Jan 15, 251(1-2), 343 - 52
Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L . as revealed by sequence-specific antibodies; Albrecht T et al.; Higher plants possess two types of glucan phosphorylase (EC 2.4.1.1) . One isozyme type, designated as Pho1, is located in the plastid whereas the other type, Pho2, is restricted to the cytosol . For Solanum tuberosum L . two Pho1 type phosphorylases have been sequenced {Nakano, K . & Fukui, T . (1986) J . Biol . Chem . 261, 8230-8236; Sonnewald, U., Basner, A., Greve, B . & Steup, M . (1995) Plant Mol . Biol . 27, 567-576} . Both proteins (referred to as Pho1a and Pho1b, respectively) are highly similar (81-84% amino acid identity over most parts of the two sequences) with the exception of the N-terminal transit peptide and the large insertion located between the N- and the C-terminal domains . In this communication antibodies that bind specifically to either Pho1a or Pho1b were used to study both isoforms at the protein level . The antibodies were applied to both potato tuber and leaf extracts following either denaturing or non-denaturing electrophoresis . Pho1a but not Pho1b was immunochemically detectable in tuber extracts whereas leaf extracts contained both the Pho1a and Pho1b protein . During denaturing electrophoresis the two antigens comigrated . When the leaf Pho1 isoforms were separated by affinity electrophoresis three bands of activity were resolved; all of them were recognized by the anti-Pho1a antibodies, but only two of these reacted with the anti-Pho1b antibodies . The isoform binding exclusively to the anti-Pho1a antibodies comigrated with the Pho1 isozyme from potato tubers . Immunoprecipitation experiments performed with anti-Pho1a antibodies removed the entire Pho1 phosphorylase activity from both tuber and leaf extracts . Addition of anti-Pho1b antibodies to tuber extracts did not affect the enzyme pattern, whereas in leaf extracts one isoform remained unchanged but the two other bands were strongly retarded . This indicates that the Pho1a protein is present in all three forms and Pho1b is associated with Pho1a . Association of Pho1a and Pho1b was further demonstrated by cross-linking experiments using bis(sulfosuccinimidyl)suberate as linker . Immunoprecipitation experiments were also performed using extracts of transformed Escherichia coli cells that expressed either Pho1a or Pho1b or both simultaneously . Under these conditions a homodimeric Pho1b phosphorylase was observed that had a lower electrophoretic mobility than the heterodimer from leaves . In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a)2 more strongly than that of the heterodimer . Thus, in leaves, Pho1a exists both as a homodimer, (Pho1a)2 and as heterodimer, (Pho1a-Pho1b); a part of it appears to be covalently modified . Pho1b, in the homodimeric form, is often below the limit of detection . In tubers the homodimer, (Pho1a)2, is the only detectable Pho1-type enzyme . To our knowledge this is the first report on a heterodimeric structure of plant phosphorylase.

Eur J Biochem, 1998 Jan 15, 251(1-2), 181 - 8
GroEL/ES chaperonins protect interferon-gamma against physicochemical stress--study of tertiary structure formation by alpha-casein quenching and ELISA; Vandenbroeck K et al.; Interferon-gamma (IFN-gamma) is a structurally labile cytokine that rapidly denatures upon exposure to acid or heat . Here we show that both acid-denatured (pH 2) and thermally inactivated (50 degrees C) porcine IFN-gamma can be rescued with the Escherichia coli GroEL/ES chaperonin system and ATP, and reassembled into bioactive dimers . At 35 degrees C, spontaneous refolding of acid-denatured IFN-gamma was found to be dependent on the presence of guanidinium hydrochloride (0.15-0.25 M) or NaCl (0.1-0.2 M) . Under non-permissive reaction conditions for regain of native structure (low-ionic-strength buffer at 35 degrees C), the yield of IFN-gamma refolded with GroEL/ES/ATP increased about 30-fold above the level of spontaneous refolding . In the absence of GroES, GroEL captured IFN-gamma in a folding-competent complex . Under these conditions, both ATP and alpha-casein induced release of IFN-gamma from GroEL but with the released protein tending to partition into sedimentable aggregates . Only in the presence of GroES, did ATP induce complete discharge of IFN-gamma from GroEL, with the released protein refolded into a conformation that is (a) immunoreactive/bio-active, (b) resistant to precipitation and (c) in a dimeric configuration . Chicken egg albumin and 90-kDa heat-shock protein were inactive in the exertion of any protective effect against physicochemical stress . The precise amount of protein refolded to the native state at different times of the folding reaction was determined by alpha-casein quenching and ELISA . The former is based on the conversion by excess alpha-casein of any population of unfolded IFN-gamma into one that escapes antibody recognition by subsequent ELISA . Since the native dimers, however, are not affected by alpha-casein quenching, immunoreactivity is directly proportional to the yield of correctly refolded protein . The validity of this approach was confirmed by measurement of biological activity . GroEL/ES-meditated reactivation amounted to > 80% both by ELISA and antiviral assay.

Eur J Biochem, 1998 Jan 15, 251(1-2), 122 - 32
Topology of subunit a of the Escherichia coli ATP synthase; Jager H et al.; The antigenic determinants of mAbs against subunit a of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic decapeptides . For two of the mAbs the epitopes are E4NMTPQD10 (GDH 14-5C6) and V29DPQ32 (GDH 8-8B3) . Binding of these mAbs to membrane vesicles of different orientation revealed that both epitopes are accessible in vesicles with inside-out orientation . These results demonstrate that at least the N-terminal amino acids 1-32 of subunit a are located at the cytoplasmic side of the membrane . A further determination of the topology of subunit a was performed by inserting the reporter epitope DYKDDDDK (FLAG epitope) at different positions of the polypeptide chain . 10 of 13 insertions led to a functional F0F1 ATP synthase and allowed specific detection of the modified subunit a by immunoblotting using an mAb against the FLAG epitope . In addition, polyclonal anti-FLAG IgG was applied for the recognition of the mutant FLAG epitope DYKDDVDK . Cells carrying this mutant FLAG epitope at the C terminus of subunit a were able to grow on succinate as sole carbon and energy source, revealing a functional ATP synthase, in contrast to those carrying the original FLAG epitope at the same position . Binding studies with membrane vesicles of different orientation and anti-FLAG Ig demonstrated that both termini of the protein are located at the cytoplasmic side of the membrane, indicating that an even number of membrane-spanning segments is present in subunit a . In addition, insertion of two FLAG epitopes in tandem after K66, or one epitope after H95, and Q181 revealed that the polypeptide regions including these residues are accessible from the cytoplasmic surface of the membrane . These results support the view that the polypeptide chain of subunit a traverses the membrane six times.

Eur J Biochem, 1998 Jan 15, 251(1-2), 98 - 106
Purification, characterisation and reaction mechanism of monofunctional 2-hydroxypentadienoic acid hydratase from Escherichia coli; Pollard JR et al.; 2-Hydroxypentadienoic acid hydratase is found on many bacterial catabolic pathways responsible for the degradation of aromatic compounds . Monofunctional 2-hydroxypentadienoic acid hydratase from Escherichia coli has been purified 3800-fold to homogeneity, using enzymatically generated 2-hydroxypentadienoic acid as substrate . The purified 28-kDa protein requires a divalent metal ion for activity, optimum activity being obtained with Mn2+ . Steady-state kinetic parameters were measured (Km = 41 +/- 4 microM, k(cat) = 450 s(-1)), the enzyme exhibiting substrate inhibition at high substrate concentrations . The pH/rate profile and inhibition by group-specific reagents were examined, and evidence was obtained for essential cysteine and tryptophan residues . An amino acid sequence alignment of the inferred amino acid sequence with nine other sequences was carried out and revealed several conserved sequence motifs . The substrate for the enzymatic reaction was found to be the dienol tautomer of 2-hydroxypentadienoic acid . Analysis of the reaction products by HPLC confirmed the identity of the 4-hydroxy-2-ketopentanoic acid product . Analogues of possible reaction intermediates were tested as inhibitors, and sodium oxalate was found to act as a potent enzyme inhibitor (Ki = 4.9 +/- 0.7 microM) . The potent inhibition by oxalate is consistent with a mechanism in which tautomerisation to 2-ketopent-3-enoic acid takes place at the active site, followed by conjugate addition of water.

Eur J Biochem, 1998 Jan 15, 251(1-2), 36 - 44
Characterization of a methyljasmonate-inducible lipoxygenase from barley (Hordeum vulgare cv . Salome) leaves; Voros K et al.; We found three methyl jasmonate-induced lipoxygenases with molecular masses of 92 kDa, 98 kDa, and 100 kDa (LOX-92, -98 and -100) {Feussner, I., Hause, B., Voros, K., Parthier, B . & Wasternack, C . (1995) Plant J . 7, 949-957} . At least two of them (LOX-92 and LOX-100), were shown to be localized within chloroplasts of barley leaves . Here, we describe the isolation of a cDNA (3073 bp) coding for LOX-100, a protein of 936 amino acid residues and a molecular mass of 106 kDa . By sequence comparison this lipoxygenase could be identified as LOX2-type lipoxygenase and was therefore designated LOX2:Hv:1 . The recombinant lipoxygenase was expressed in Escherichia coli and characterized as linoleate 13-LOX and arachidonate 15-LOX, respectively . The enzyme exhibited a pH optimum around pH 7.0 and a moderate substrate preference for linoleic acid . The gene was transiently expressed after exogenous application of jasmonic acid methyl ester with a maximum between 12 h and 18 h . Its expression was not affected by exogenous application of abscisic acid . Also a rise of endogenous jasmonic acid resulting from sorbitol stress did not induce LOX2:Hv:1, suggesting a separate signalling pathway compared with other jasmonate-induced proteins of barley . The properties of LOX2:Hv:1 are discussed in relation to its possible involvement in jasmonic acid biosynthesis and other LOX forms of barley identified so far.

Thyroid, 1998 Jan, 8(1), 3 - 7
Expression of a biotinylated human thyrotropin receptor in HeLa cells using recombinant vaccinia virus and its application for the detection of Graves' autoantibodies; Minich WB et al.; We have prepared a biotinylated thyrotropin receptor (TSHR-BIO), and characterized its activity in cells and when bound to solid phase (streptavidin agarose) . TSHR-BIO consists of the N-terminal 725 amino acids of the human thyrotropin (TSH) receptor linked to 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 post-translational biotinylation of the protein . TSHR-BIO was expressed using a vaccinia virus expression system . HeLa cells infected with recombinant virus produced large amounts of TSH receptor of approximately 120,000 molecules per cell . Vaccinia virus produced TSHR-BIO was fully functional interacting with TSH (Kd of 2.3+/-0.1 x 10(-10) M) and coupling to cyclic adenosine monophosphate (cAMP) second messenger system . The expressed protein was biotinylated with high efficiency; more than 90% of TSHR-BIO was bound to streptavidin . We have shown the application of streptavidin agarose immobilized TSHR-BIO for the detection of thyroid-binding inhibiting immunoglobulines in unfractionated sera . There was a good positive correlation between the results obtained in this assay and the commercially available TRAK assay performed with solubilized porcine TSH receptor (r = 0.71; p < 0.001, in 45 sera of patients with Graves' disease and 17 normal sera).

AIDS Res Hum Retroviruses, 1998 Feb 10, 14(3), 275 - 83
Partial protection by vaccination with recombinant feline immunodeficiency virus surface glycoproteins; Leutenegger CM et al.; In an effort to induce a strong immune response that might protect against feline immunodeficiency virus (FIV) challenge infection, three groups of five specified pathogen-free (spf) cats each were immunized subcutaneously with different FIV antigen preparations . Immunizations were done at weeks 0, 2, and 4 with 100 microg of recombinant SU from an FIV Zurich 2 (FIV Z2) strain expressed by E . coli (group 1) or the baculovirus expression system (groups 2 and 3) adsorbed on aluminum hydroxyde and administered with QS-21 (groups 1 and 2) or Freund's adjuvant together with the recombinant nucleocapsid protein (protein NC) of rabies virus (group 3) . Protein NC was described to act as an exogenous superantigen . Group 3 cats demonstrated the highest detectable antibody response to the vaccine antigen as determined by ELISA and Western blot analysis . All immunized cats together with seven control animals were challenged with 20 CID50 of cat lymphocyte-grown FIV Z2 3 weeks following the last immunization . Whereas virus was readily recovered from peripheral blood lymphocytes of seven of seven nonvaccinated control cats following this challenge dose, virus was not recovered from two cats of groups 1 and 2 . All cats in groups 2 and 3 showed a provirus load significantly decreased to 3% of that of controls up to week 8 after challenge infection . Eleven of 15 vaccinated cats and 5 of 7 control cats developed virus-neutralizing antibodies by week 8 after challenge infection . The two cats negative on virus isolation remained seronegative, developed no detectable virus-neutralizing activities, but were repeatedly positive in provirus PCR . Moreover, starting at week 1 after challenge, both cats showed the lowest provirus load in their respective groups . These results indicate that immunization with recombinant FIV SU in conjunction with appropriate adjuvants may lead to partial protection against FIV challenge infection.

Cell, 1998 Feb 20, 92(4), 451 - 62
A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA; Wei P et al.; The HIV-1 Tat protein regulates transcription elongation through binding to the viral TAR RNA stem-loop structure . We have isolated a novel 87 kDa cyclin C-related protein (cyclin T) that interacts specifically with the transactivation domain of Tat . Cyclin T is a partner for CDK9, an RNAPII transcription elongation factor . Remarkably, the interaction of Tat with cyclin T strongly enhances the affinity and specificity of the Tat:TAR RNA interaction, and confers a requirement for sequences in the loop of TAR that are not recognized by Tat alone . Moreover, overexpression of human cyclin T rescues Tat activity in nonpermissive rodent cells . We propose that Tat directs cyclin T-CDK9 to RNAPII through cooperative binding to TAR RNA.

Cell, 1998 Feb 20, 92(4), 441 - 50
Crystal structure of the tyrosine phosphatase SHP-2; Hof P et al.; The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 angstroms resolution, shows how its catalytic activity is regulated by its two SH2 domains . In the absence of a tyrosine-phosphorylated binding partner, the N-terminal SH2 domain binds the phosphatase domain and directly blocks its active site . This interaction alters the structure of the N-SH2 domain, disrupting its phosphopeptide-binding cleft . Conversely, interaction of the N-SH2 domain with phosphopeptide disrupts its phosphatase recognition surface . Thus, the N-SH2 domain is a conformational switch; it either binds and inhibits the phosphatase, or it binds phosphoproteins and activates the enzyme . Recognition of bisphosphorylated ligands by the tandem SH2 domains is an integral element of this switch; the C-terminal SH2 domain contributes binding energy and specificity, but it does not have a direct role in activation.

Mol Biol Evol, 1998 Jan, 15(1), 1 - 5
Activation of the bgl operon by adaptive mutation; Hall BG; In growing Escherichia coli K12 cells, the cryptic bgl operon is activated 98% of the time by insertions of IS1 or IS5 into the control region, designated bglR . The activated bgl operon permits utilization of the beta-glucoside sugar arbutin as a sole carbon and energy source . The bgl operon is also activated by late-occurring mutations during prolonged selection on arbutin . The late-occurring mutations that occurred during prolonged carbon starvation in the presence of arbutin were "adaptive mutations" because they were specific to the presence of arbutin, and they did not occur during prolonged starvation in the absence of arbutin . The spectrum of late-arising mutations differed from that of early-arising, growth-dependent mutations in that 20% of the late-arising mutants resulted from mutations at the hns locus . This provides the first direct evidence for adaptive mutagenesis mediated by the insertion of IS elements . Because no special genetic background is required to select Bgl+ mutants, this affords the opportunity to study IS-element-mediated adaptive mutagenesis in a variety of genetic backgrounds, including the backgrounds of natural isolates of E . coli.

Parasitol Res, 1998, 84(1), 38 - 40
Molecular cloning and expression of an anti-idiotype antibody mimicking a protective oligosaccharide of the parasite Schistosoma mansoni; Petitprez K et al.; Genes encoding the heavy and light chains of an anti-idiotype antibody (AB2) mimicking a protective oligosaccharide of Schistosoma mansoni were cloned and expressed as a single-chain Fv fragment . The expression in a functional state was tested using the AB1 . A specific binding between sFv and AB1 was observed . Immunization with the recombinant AB2 indicates its capacity to elicit anti-S . mansoni antibodies.

Mutagenesis, 1998 Jan, 13(1), 9 - 18
The mutational specificity of 1-nitroso-6-nitropyrene in the lacI gene of Escherichia coli strains deficient in nucleotide excision repair; Lambert IB et al.; We have examined the mutational specificity of 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene, in the lacI gene of Escherichia coli strains which are deficient in nucleotide excision repair (strain NR6113, delta uvrB; strain CM6114, delta uvrB, plasmid pKM101) . Separate collections of lacI- mutations and dominant lacI-d mutations, which contain DNA sequence alterations in the region of the lacI gene that encodes the DNA binding domain of the lacI repressor, were made following 1,6-NONP treatment . The DNA sequence of 418 mutations was determined, of which 228 were lacI- mutations and 190 were lacI-d mutations . Ninety three percent of the induced point mutations occurred at G:C residues.0 -(G:C) frameshifts were the dominant mutational class in the lacI- collections of both NR6113 and CM6114, and in the lacI-d collection of NR6113 . The frameshift mutations occurred preferentially in runs of guanine residues and their frequency increased markedly with the length of the reiterated sequence . In strain CM6114, which contained the plasmid pKM101, there was a marked stimulation in the frequency of G:C-->T:A transversions that was particularly apparent in the lacI-d collection . We discuss models which might account for the apparent differences in mutational specificity resulting from the presence of the UmuD/C and MucA/B proteins . The results suggest that major classes of mutation are recovered in both the lacI- and lacI-d collections . However, the proportions of the major classes of mutations within the two collections can differ significantly . Depending on the genetic background of the host strain, the relative ratios of base substitutions to frameshift mutations in the lacI-d target can differ by almost an order of magnitude as compared with the lacI- target . This is primarily a function of the relative mutational target size of the different classes of mutation.

Proteins, 1998 Feb 1, 30(2), 183 - 92
Specific fluorescent labeling of two functional domains in RNA polymerase alpha subunit; Ozoline ON et al.; A monomercury derivative of fluoresceine acetate (FMMA) was previously suggested as a specific reagent reacting with only one of four cysteine (Cys) residues in the alpha . subunit of Escherichia coli RNA polymerase . Here, we analyzed the reactivity against FMMA of both isolated alpha subunit and alpha subunit assembled in the holoenzyme . In both cases, the highest reactivity was identified for Cys-269 positioned in the regulatory helix of C-terminal domain (CTD) which includes the contact sites for both class-I transcription factors and DNA UP elements . Substitution of Ala for both Cys-269 and Cys-176 completely eliminates the reactivity of alpha subunit against the fluorescent dye, supporting the prediction that another reactive amino acid under native conformation is Cys-176, which is positioned within or near the region important for alpha dimerization and its binding of beta' subunit . In the isolated alpha subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176 . Mutant alpha-subunits, bearing only one Cys residue at either 269 or 176, could be reconstituted into locally modified and active enzymes . This FMMA modification system may provide a tool suitable for studies of intra- and intermolecular interactions of this subunit.

Blood, 1998 Feb 15, 91(4), 1418 - 25
The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells; Hoatlin ME et al.; The Fanconi anemia (FA) complementation group C (FAC) protein gene encodes a cytoplasmic protein with a predicted Mr of 63,000 . The protein's function is unknown, but it has been hypothesized that it either mediates resistance to DNA cross-linking agents or facilitates repair after exposure to such factors . The protein also plays a permissive role in the growth of colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-erythroid (CFU-E) . Attributing a specific function to this protein requires an understanding of its intracellular location . Recognizing that prior study has established the functional importance of its cytoplasmic location, we tested the hypothesis that FAC protein can also be found in the nucleus . Purified recombinant Escherichia coli-derived FAC antigens were used to create antisera able to specifically identify an Mr = 58,000 protein in lysates from human Epstein-Barr virus (EBV)-transformed cell lines by immunoblot analysis . Subcellular fractionation of the cell lysates followed by immunoblot analysis revealed that the majority of the FAC protein was cytoplasmic, as reported previously; however, approximately 10% of FAC protein was reproducibly detected in nuclear fractions . These results were reproducible by two different fractionation methods, and included markers to control for contamination of nuclear fractions by cytoplasmic proteins . Moreover, confocal image analysis of human 293 cells engineered to express FAC clearly demonstrated that FAC protein is located in both cytoplasmic and nuclear compartments, consistent with data obtained from fractionation of the FA cell lines . Finally, complementation of the FAC defect using retroviral-mediated gene transfer resulted in a substantial increase in nuclear FAC protein . Therefore, while cytoplasmic localization of this protein appears to be functionally important, it may also exert some essential nuclear function.

Bioorg Khim, 1997 Aug, 23(8), 630 - 4
{Functional expression of bovine retina adenylate cyclase b fragment cDNA in Escherichia Coli cells}; Solov'eva OV et al.; A 1488-bp fragment of bovine retina guanylate cyclase B gene encoding the catalytic and dimerizing domains as well as part of the protein kinase domain was expressed in Escherichia coli cells . The expression product was obtained as inclusion bodies and solubilized in 6 M guanidine hydrochloride . The fragment of guanylate cyclase B is a dimer close in catalytic activity to the native enzyme.

Bioorg Khim, 1997 Oct, 23(10), 800 - 4
{Monoclonal antibodies to the alpha-subunit of the putative human H+,K+-ATPase encoded by the atp1al1 gene}; Korneenko TV et al.; The N-terminal fragment of ATP1AL1, the possible catalytic subunit of human ouabain-sensitive H+,K(+)-ATPase, was expressed in Escherichia coli cells as two recombinant proteins: the Ser14-Ile104 fragment or the same fragment containing His6 sequence at its N-end . The second protein was purified by metal-affinity chromatography and used as an antigen to construct two hybridoma lines producing antibodies of the IgM class . These monoclonal antibodies were shown to recognize not only the starting antigen but also the full-size recombinant ATP1AL1 protein and do not react with Na+,K(+)-ATPase.

Eur J Biochem, 1998 Feb 1, 251(3), 998 - 1004
The effect of acylated polyamine derivatives on polyamine uptake mechanism, cell growth, and polyamine pools in Escherichia coli, and the pursuit of structure/activity relationships; Karahalios P et al.; Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with {14C}putrescine or {14C}spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools . Spermine uptake follows simple Michaelis-Menten kinetics (Kt = 24.58 +/- 2.24 microM) . In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (Kt = 3.63 +/- 0.43 microM, Kt' = 0.61 +/- 0.10 microM) . From the Ki values, it is inferred that N1-5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N1-acetylspermine, and then N1,N12-diacetylspermine . N1-acetylspermidine and N8-acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor . In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine . The order of analogue potency, regarding the other carrier of putrescine is as follows: N1,N12-diacetylspermine approximately N1-5-amino-2-nitro-benzoylspermine > N1-acetylspermine . Both N1-acetylspermidine (Ki = 753 +/- 25 microM, Ki' = 128 +/- 5 microM) and N8-acetylspermidine (Ki = 22.4 +/- 0.4 microM, Ki' = 279 +/- 3 microM) also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers . Neither N4,N9-diacetylspermine, nor N1,N4-bis(beta-alanyl)diaminobutane affect the uptake of any polyamine . Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells . Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.

Eur J Biochem, 1998 Feb 1, 251(3), 854 - 62
Mutational analysis of disulfide bonds in the trypsin-reactive subdomain of a Bowman-Birk-type inhibitor of trypsin and chymotrypsin--cooperative versus autonomous refolding of subdomains; Philipp S et al.; It is widely believed that protein folding is a hierarchical process proceeding from secondary structure via subdomains and domains towards the complete tertiary structure . Accordingly, protein subdomains should behave as independent folding units . However, this prediction would underestimate the well-established structural significance of tertiary context and domain interfaces in proteins . The principal objective of this work was to distinguish between autonomous and cooperative refolding of protein subdomains by means of mutational analysis . The double-headed Bowman-Birk inhibitor of trypsin and chymotrypsin of known crystal structure was selected for study . The relative orientation of the two subdomains is stabilized by intramolecular and water-mediated hydrogen bonds and close ion pairs across a polar domain interface . The binary arrangement of a trypsin-reactive and a chymotrypsin-reactive subdomain facilitates the distinction of local and global irregularities in the mutants of this protein by means of functional assays . The functional consequences of five replacements in the S-S bond framework of the trypsin-reactive subdomain are analyzed in the present report . The mutants were subjected to refolding experiments in a refolding buffer and on trypsin-Sepharose as a template with complementary structure leading into a fully active state . The stability of the variants was assessed by means of subsequent equilibration experiments in solution . The mutants may be grouped into the following two classes: the class-I mutations located within beta-strand A are characterized by a breakdown of the trypsin- and the chymotrypsin-reactive subdomain upon refolding in solution and a complicated behavior in the equilibration experiments; by contrast, the Class-II mutations (beta-strand B) display rather local perturbations and a reversible return to the initial ratio of the two subdomains . This points to a significance of polar interactions connecting the beta-strand A of the trypsin-reactive with the chymotrypsin-reactive subdomain . In conclusion, the polar domain interface appears as a major refolding unit of the Bowman-Birk inhibitor.

Eur J Biochem, 1998 Feb 1, 251(3), 724 - 8
Cooperation of the DnaK and GroE chaperone systems in the folding pathway of plant ferredoxin-NADP+ reductase expressed in Escherichia coli; Dionisi HM et al.; The DnaK system is required for the productive folding of pea chloroplast ferredoxin-NADP+ reductase (FNR) expressed in Escherichia coli . The formation of a mature active enzyme was severely impaired in E . coli dnaK, dnaJ or grpE mutants expressing either the cytosolic precursor of the reductase (preFNR) or the mature apoenzyme, and these forms aggregated extensively in these cells . Coexpression of dnaK from a multicopy plasmid in the dnaK-null mutants restored preFNR processing and folding of FNR, rendering a mature-sized active enzyme . Overexpression of GroESL chaperonins failed to prevent preFNR aggregation, but it restored productive folding of FNR in dnaK-null mutants expressing the mature enzyme . Expression of preFNR in OmpT-protease-deficient E . coli cells resulted in the accumulation of the unprocessed precursor in the soluble fraction of the cells . The interaction of this soluble preFNR, but not the mature reductase, with DnaK and GroEL was evidenced by immunoprecipitation studies . We conclude that, in addition to the GroE chaperonins {Carrillo, N., Ceccarelli, E . A., Krapp, A . R., Boggio, S., Ferreyra, R . G . & Viale, A . M . (1992) J . Biol . Chem . 267, 15537-15541}, the DnaK chaperone system plays a crucial role in the folding pathway of FNR.

Eur J Biochem, 1998 Feb 1, 251(3), 682 - 90
Autonomous folding of the recombinant large cytoplasmic loop of sarcoplasmic reticulum Ca2+-ATPase probed by affinity labeling and trypsin digestion; Moutin MJ et al.; Recombinant large cytoplasmic loop (LCL, residues 329-740) of sarcoplasmic reticulum Ca2+-ATPase, expressed in and purified from Escherichia coli, comprises most of the active site and binds ATP {Moutin, M.-J., Cuillel, M., Rapin, C., Miras, R., Anger, M., Lompre, A.-M . & Dupont, Y . (1994) J . Biol . Chem . 269, 11147-11154} . In this study, we show that fluorescein-5' isothiocyanate (FITC) specifically labels the same lysine residue as in the native Ca2+-ATPase (Lys515), with similar kinetics and pH dependence . ATP blocks the reaction with the lysine residue, but at higher concentrations compared with those for the native pump, in agreement with the lower ATP-binding affinity found previously . Graded tryptic digestion of LCL shows that favored cleavage is at the T1 site and that the N-terminal 75% of LCL are resistant to trypsin, as is native Ca2+-ATPase . Other experiments reveal differences to the native pump . (a) FITC derivatizes some -SH groups of LCL . (b) The C-terminal 25% of the polypeptide is susceptible to end-clipping by trypsin . (c) 2',3'-O-(2,4,6-trinitrophenyl)-ATP fails to specifically label the LCL (on the equivalent of Lys492), although it binds tightly (KD = 1.3 microM) and (d) Glutaraldehyde does not specifically cross-link LCL (between the equivalent of Lys492 and Arg678) . These results could be explained by a flexible and loose structure of the hinge region of LCL (C-terminal 25%) . Anchoring this region in the membrane and/or interaction with the missing beta-strand domain may be required for its compact folding and proper interaction with the rest of LCL . The results suggest that the N-terminal 75% of LCL expressed in E . coli folds autonomously to a fairly stable unit and native-like structure, encompassing the phosphorylation and central ATP binding sections . The hinge region does not appear to be part of the FITC-binding site but constitutes portions of the 2',3'-O-(2,4,6-trinitrophenyl)-ATP and, probably, ATP-binding site.

Eur J Biochem, 1998 Feb 1, 251(3), 631 - 40
Molecular cloning of the peroxisome proliferator-induced 46-kDa cytosolic acyl-CoA thioesterase from mouse and rat liver--recombinant expression in Escherichia coli, tissue expression, and nutritional regulation; Lindquist PJ et al.; Feeding clofibrate to rats and mice results in a strong induction of acyl-CoA thioesterase activity in the liver that is mainly due to increases in the enzyme activities in mitochondria and cytosol . The cytosolic acyl-CoA thioesterase protein of about 40 kDa, referred to as CTE-I, is strongly induced by the treatment . We report here the molecular cloning of the cDNA corresponding to the rat and mouse enzymes, and the further characterization of the mouse CTE-I by recombinant expression in bacteria and regulation of expression of the enzyme . The cDNAs corresponding to the rat and mouse enzymes contained open reading frames encoding proteins of 419 amino acids with calculated molecular masses of 45938 Da and 46135 Da, respectively . Sequence analysis revealed an active site serine consensus sequence commonly found in lipases and carboxylesterases . Recombinant expression of the mouse CTE-I cDNA in Escherichia coli resulted in production of immunoreactive protein that was mainly active with long-chain acyl-CoAs . Northern blot analysis showed that the full-length CTE-I cDNA probe hybridized to two major transcripts corresponding to CTE-I and MTE-I (mitochondrial acyl-CoA thioesterase I), respectively . The expression of both mRNA species was found to be highly regulated . As expected, both CTE-I and MTE-I were strongly upregulated (> 50-fold) by clofibrate treatment . Interestingly, fasting for 48 h resulted in a similar magnitude of induction as two days of clofibrate feeding . In addition, feeding a fat-free diet resulted in down-regulation of CTE-I mRNA . CTE-I mRNA was strongly expressed in kidney and brown adipose tissue and MTE-I mRNA was expressed mainly in brown adipose tissue and heart but was also expressed in kidney and white adipose tissue . Dietary regulation and tissue-specific expression suggest that CTE-I and MTE-I play important roles in lipid metabolism.

FEBS Lett, 1998 Jan 30, 422(2), 259 - 64
The first constant domain (C(H)1 and C(L)) of an antibody used as heterodimerization domain for bispecific miniantibodies; Muller KM et al.; Bispecific miniantibodies were constructed by genetically fusing the C(H)1 domain of an IgG1 to the C-terminus of a single-chain Fv fragment (scFv-425), specific for the EGF receptor, and fusing the C(L) domain of a kappa light chain to the C-terminus of a scFv specific for CD2 (scFv-M1) . An efficient dicistronic gene arrangement for functional expression in Escherichia coli was constructed . Immunoblots demonstrated correct domain assembly and the formation of the natural C(H)1-C(L) disulfide bridge . Gel filtration confirmed the correct size, sandwich ELISAs demonstrated bispecific functionality, and SPR biosensor measurements determined binding to EGF-R in comparison to bivalent constructs . Bispecific anti-EGF-R/anti-CD2 miniantibodies are candidates for the immunotherapy of cancer.

FEBS Lett, 1998 Jan 30, 422(2), 218 - 20
Mutations in the proteolytic domain of Escherichia coli protease Lon impair the ATPase activity of the enzyme; Starkova NN et al.; Conserved residues of the proteolytic domain of Escherichia coli protease Lon, putative members of the classic catalytic triad (H665, H667, D676, and D743) were identified by comparison of amino acid sequences of Lon proteases . Mutant enzymes containing substitutions D676N, D743N, H665Y, and H667Y were obtained by site-directed mutagenesis . The mutant D743N retained the adenosine triphosphate (ATP)-dependent proteolytic activity, thereby indicating that D743 does not belong to the catalytic site . Simultaneously, the mutants D676N, H665Y, and H667Y lost the capacity for hydrolysis of protein substrates . The ATPase activity of these three mutants was decreased by more than an order of magnitude, which suggests a close spatial location of the ATPase and proteolytic active sites and their tight interaction in the process of protein degradation.

FEBS Lett, 1998 Jan 30, 422(2), 189 - 92
Interaction of EF-Tu with EF-Ts: substitution of His-118 in EF-Tu destabilizes the EF-Tu x EF-Ts complex but does not prevent EF-Ts from stimulating the release of EF-Tu-bound GDP; Jonak J et al.; Elongation factor Tu from Escherichia coli with His-118 substituted by glycine (EF-TuH118G) was found to be defective in complex formation with EF-Ts . EF-Ts in excess failed to dissociate kirromycin from the EF-TuH118G x kirromycin complex and to form a stable complex with EF-TuH118G on column chromatography . However, the stimulatory effect of EF-Ts on GDP dissociation from EF-TuH118G x GDP and on poly(U)-directed poly(Phe) synthesis catalyzed by EF-TuH118G was only partially influenced . These results indicate that His-118, while very important for the formation of a stable EF-Tu-EF-Ts complex, is not essential for the transmission of the EF-Ts-dependent signal accelerating the release of the EF-Tu-bound GDP.

Cell Mol Biol (Noisy-le-grand), 1997 Dec, 43(8), 1165 - 9
An efficient liposome-mediated gene transfer into the branchial arch, neural tube and the heart of chick embryos; a strategy to elucidate the organogenesis; Yamada G et al.; Hemagglutinating virus of Japan (HVJ)-liposome mediated gene-transfer permitted to clarify the mechanisms of embryonic organogenesis of the branchial arches, neural tubes and heart by micro-injecting reporter-plasmid DNA, containing the Escherichia coli LacZ gene, in embryos at several stages.

Cell, 1998 Jan 9, 92(1), 117 - 29
Crystal structure of a G:T/U mismatch-specific DNA glycosylase: mismatch recognition by complementary-strand interactions; Barrett TE et al.; G:U mismatches resulting from deamination of cytosine are the most common promutagenic lesions occurring in DNA . Uracil is removed in a base-excision repair pathway by uracil DNA-glycosylase (UDG), which excises uracil from both single- and double-stranded DNA . Recently, a biochemically distinct family of DNA repair enzymes has been identified, which excises both uracil and thymine, but only from mispairs with guanine . Crystal structures of the mismatch-specific uracil DNA-glycosylase (MUG) from E . coli, and of a DNA complex, reveal a remarkable structural and functional homology to UDGs despite low sequence identity . Details of the MUG structure explain its thymine DNA-glycosylase activity and the specificity for G:U/T mispairs, which derives from direct recognition of guanine on the complementary strand.

Mol Microbiol, 1998 Feb, 27(3), 573 - 85
An Escherichia coli protein that exhibits phosphohistidine phosphatase activity towards the HPt domain of the ArcB sensor involved in the multistep His-Asp phosphorelay; Ogino T et al.; The Escherichia coli sensory kinase, ArcB, possesses a histidine-containing phosphotransfer (HPt) domain, which is implicated in the His-Asp multistep phosphorelay . We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity . Using in vivo screening, we first identified a gene that appeared to interfere with the His-Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid . The cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine-histidine-glycine (RHG) signature, at its N-terminus . Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose-2,6-bisphosphatase, E . coli periplasmic phosphatase and E . coli glucose-1-phosphate phosphatase, and ubiquitous phosphoglycerate mutase . Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA . It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not . Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling . These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His-Asp phosphorelay through regulating the phosphorylation state of the HPt domain.

Mol Microbiol, 1998 Feb, 27(3), 563 - 71
The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli; MacLean MJ et al.; The glyoxalase I gene (gloA) of Escherichia coli has been cloned and used to create a null mutant . Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity . Potassium efflux via KefB is also enhanced in the overexpressing strain . Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells . The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile . Activation of KefB and KefC is diminished in the absence of functional glyoxalase I . These data suggest that the glutathione-dependent glyoxalase I is the dominant detoxification pathway for MG in E . coli and that the product of glyoxalase I activity, S-lactoylglutathione, is the activator of KefB and KefC.

Mol Microbiol, 1998 Feb, 27(3), 553 - 62
From famine to feast: the role of methylglyoxal production in Escherichia coli; Totemeyer S et al.; The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa . Database analysis identified an open reading frame in the E . coli genome, YccG, corresponding to a protein of 16.9 kDa . When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity . MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression . This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM . High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol . A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium . However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.

Br J Pharmacol, 1998 Jan, 123(2), 281 - 91
Characterization of the receptor and the mechanisms underlying the inflammatory response induced by des-Arg9-BK in mouse pleurisy; Vianna RM et al.; 1 The characterization of the B1 kinin receptor, and some mediators involved in the inflammatory response elicited by intrathoracic (i.t.) administration of des-Arg9-bradykinin (BK) in the mouse model of pleurisy, was investigated . 2 An i.t . injection of des-Arg9-BK (10-100 nmol per site), a selective B1 agonist, caused a significant and dose-related increase in the vascular permeability observed after 5 min, which peaked at 1 h, associated with an increase in cell influx, mainly neutrophils, and, to a lesser extent, mononuclear cell influx, peaking at 4 h and lasting for up to 48 h . The increase in fluid leakage caused by des-Arg9-BK was completely resolved 4 h after peptide injection . I.t . injection of Lys-des-Arg9-BK (30 nmol per site) caused a similar inflammatory response . 3 Both the exudation and the neutrophil influx elicited by i.t . injection of des-Arg9-BK were significantly antagonized (P<0.01) by an i.t . injection of the selective B1 antagonists des-Arg9-{Leu8}-BK (60 and 100 nmol per site) or des-Arg9-NPC 17731 (5 nmol per site), administered in association with des-Arg9-BK (P<0.01), or 30 and 60 min before the cellular peak, respectively . In contrast, an i.t . injection of the B2 bradykinin selective receptor antagonist Hoe 140 (30 nmol per site), at a dose which consistently antagonized bradykinin (10 nmol per site)-induced pleurisy, had no significant effect on des-Arg9-BK-induced pleurisy . 4 An i.t . injection of the selective tachykinin receptor antagonists (NK1) FK 888 (1 nmol per site), (NK2) SR 48968 (20 nmol per site) or (NK3) SR 142801 (10 nmol per site), administered 5 min before pleurisy induction, significantly antagonized neutrophil migration caused by i.t . injection of des-Arg9-BK . In addition, FK 888 and SR 142801, but not SR 48968, also prevented the influx of mononuclear cells in response to i.t . injection of des-Arg9-BK (P<0.01) . However, the NK3 receptor antagonist SR 142801 (10 nmol per site) also significantly inhibited des-Arg9-BK-induced plasma extravasation . An i.t . injection of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP8-37 (1 nmol per site), administered 5 min before pleurisy induction, inhibited des-Arg9-BK-induced plasma extravasation (P<0.01), without significantly affecting the total and differential cell migration . 5 The nitric oxide synthase inhibitors L-NOARG and L-NAME (1 pmol per site), administered 30 min beforehand, almost completely prevented des-Arg9-BK (i.t.)-induced neutrophil cell migration (P<0.01), and, to a lesser extent, mononuclear cell migration (P<0.01) . The D-enantiomer D-NAME had no effect on des-Arg9-BK-induced pleurisy . At the same dose range, L-NOARG and L-NAME inhibited the total cell migration (P<0.01) . L-NAME, but not L-NOARG caused significant inhibition of des-Arg9-BK-induced fluid leakage . Indomethacin (1 mg kg(-1), i.p.), administered 1 h before des-Arg9-BK (30 nmol per site), inhibited the mononuclear cell migration (P<0.05), but, surprisingly, increased the neutrophil migration at 4 h without interfering with plasma extravasation . The administration of terfenadine (50 mg kg(-1), i.p.), 30 min before des-Arg9-BK (30 nmol per site), did not interfere significantly with the total cell migration or with the plasma extravasation in the mouse pleurisy caused by i.t . injection of des-Arg9-BK . 6 Pretreatment of animals with the lipopolysaccharide of E . coli (LPS; 10 microg per animal, i.v.) for 24 h did not result in any significant change of the inflammatory response induced by i.t . injection of des-Arg9-BK compared with the saline treated group . However, the identical treatment of mice with LPS resulted in a marked enhancement of des-Arg9-BK induced paw oedema (P<0.01) . 7 In conclusion, we have demonstrated that the inflammatory response induced by i.t . injection of desArg9-BK, in a murine model of pleurisy, is mediated by stimulation of constitutive B1 receptors . (ABSTRACT TRUNCATED)

Plant Physiol, 1998 Feb, 116(2), 733 - 42
Cloning of a tobacco apoplasmic invertase inhibitor . Proof of function of the recombinant protein and expression analysis during plant development; Greiner S et al.; Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development . We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity . Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction . Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene . Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants . The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected . However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells . Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein . RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

Parasitology, 1997 Dec, 115 ( Pt 6), 581 - 90
Characterization of a cDNA-clone encoding Nc-p43, a major Neospora caninum tachyzoite surface protein; Hemphill A et al.; Neospora caninum is an apicomplexan parasite of veterinary importance which invades many different cell types and tissues . N . caninum tachyzoites proliferate intracellularly by endodyogeny . Eventually the massive proliferation of tachyzoites leads to host cell lysis and the newly formed parasites are released and invade neighbouring cells . Tachyzoite cell surface molecules could serve as ligands, mediating host cell adhesion and invasion . Nc-p43 is a recently identified N . caninum tachyzoite surface protein which is functionally involved in the processes leading to host cell invasion in vitro . Affinity-purified antibodies directed against Nc-p43 were used to screen a lambda gt22A-cDNA expression library constructed from N . caninum tachyzoites . The cDNA insert of one immunoreactive clone was subcloned and expressed in E . coli as a poly-histidine fusion protein . The identity of the resulting recombinant antigen termed recNc-p43 was confirmed by immunoblotting, immunofluorescence and electron microscopy using affinity-purified antibodies . The sequence of the cDNA insert encoding recNc-p43 was determined . Analysis of the deduced amino acid sequence revealed that Nc-p43 exhibited similarity to SAG1 (p30) and SAG3 (p43), 2 major surface antigens of Toxoplasma gondii tachyzoites . These similarities were not reflected on the immunochemical level, since no cross-antigenicity between SAG1, SAG3 and Nc-p43 was observed.

Biochemistry, 1998 Feb 3, 37(5), 1430 - 7
Cardiolipin binding a light chain from lupus-prone mice; Pereira B et al.; Autoantibodies in systemic lupus erythematosus react with multiple epitopes on highly conserved molecules such as nucleic acids, cytoskeletal proteins, phospholipids, and phospholipid-binding proteins . Analysis of the heavy- and light-chain variable sequences (VH and VL) has shown that a restricted set of V genes gives rise to these autoantibodies . Several monoclonal antibodies were developed from a strain of mouse prone to lupus (F1 male NZW x BXSB) . Two of these antibodies, A1.72 and A1.84, reacted directly with cardiolipin and their VH and VL sequences were analyzed . Surprisingly, these two antibodies had identical light-chain variable sequences despite having substantially different heavy-chain variable sequences . This VL sequence, VL 72/84 was 97% identical with the germ-line sequences with only four single nucleotide substitutions . When this VL sequence was shuffled with the VH sequence of other monoclonal antibodies and expressed as single chain variable fragment (scFv) in Escherichia coli, it imparted cardiolipin-binding activity to the hybrids . Furthermore, the VL 72/84 sequence, when expressed alone without any VH sequence, also bound to cardiolipin . The antibodies and their recombinant fragments were immunoaffinity-purified on cardiolipin liposomes . The dissociation constant of the light chain for cardiolipin was similar to the intact molecule (21 +/- 0.01 vs 20 +/- 0.03 nM) . These studies demonstrate that the VL sequence alone, in the absence of any other immunoglobulin domains, can mediate cardiolipin binding, raising the possibility that antigen specificity of certain antibodies may exclusively reside in their light-chain sequences.

Biochemistry, 1998 Feb 3, 37(5), 1350 - 6
Regions of 16S ribosomal RNA proximal to transfer RNA bound at the P-site of Escherichia coli ribosomes; Bullard JM et al.; Unmodified uridines have been randomly replaced by 4-thiouridines in transfer RNAPhe (tRNAPhe) transcribed in a T7 RNA polymerase system . These 4-thiouridines serve as conjugation sites for attachment of the cleavage reagent 5-iodoacetamido-1,10-o-phenanthroline (IoP) . In a reducing environment, when complexed with Cu2+, 1,10-o-phenanthroline causes cleavage of nearby nucleic acids . We show here that tRNA-phenanthroline (tRNA-oP) conjugates, when bound at the P-site of 70S ribosomes and 30S ribosomal subunits, caused cleavage of ribosomal RNA (rRNA) mainly in domains I and II of 16S rRNA . Some positions were cleaved only when tRNA-oP was bound to 70S ribosomes or to 30S ribosomal subunits . In domain I, most cleavage sites occurred in or near the 530 pseudoknot region . In domain II, most nucleotides cleaved were near the 690 region and the 790 region . The only positions cleaved in domain III were near the 1050 region . There were no discernible nucleotides cleaved near the 1400 (decoding) region . Our results corroborated results of others, which have shown these sites to be protected from chemical modification by tRNA binding or to be cross-linked to P-site-bound tRNA . Use of cleavage reagents tethered to tRNA provides evidence for additional regions of rRNA that may be proximal to bound tRNA.

Biochemistry, 1998 Feb 3, 37(5), 1344 - 9
Dimeric association of Escherichia coli RNA polymerase alpha subunits, studied by cleavage of single-cysteine alpha subunits conjugated to iron-(S)-1-{p-(bromoacetamido)benzyl}ethylenediaminetetraacetate; Miyake R et al.; Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269) . These mutant alpha subunits were conjugated with the cutting reagent iron-(S)-1-{p-(bromoacetamido)benzyl}ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys . Analysis of the cleavage sites identified segments within approximately 12 A of the conjugation site . These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another . Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.

Biochemistry, 1998 Feb 3, 37(5), 1315 - 21
Formation of CLC-0 chloride channels from separated transmembrane and cytoplasmic domains; Maduke M et al.; CLC-0, a member of the CLC family of Cl(-)-conducting ion channels, consists of an N-terminal hydrophobic core and a C-terminal region that is thought to be cytoplasmic . This study provides evidence that the C-terminal region is a mechanistically relevant cytoplasmic domain of the CLC-0 ion channel . Both a point mutation and a 37-residue deletion in this region cause drastic alterations in voltage-dependent gating of CLC-0 current expressed in Xenopus oocytes . CLC-0 current is not observed when the entire C-terminal region is deleted, but functional channels are efficiently reconstituted by co-injection of separate cRNA constructs encoding the N-terminal transmembrane and the C-terminal cytoplasmic domains . Moreover, reconstitution of CLC-0 can be achieved by co-injection of cRNA encoding the transmembrane domain along with Escherichia coli-expressed C-terminal domain polypeptide.

Biochemistry, 1998 Feb 3, 37(5), 1235 - 44
Interdomain information transfer during substrate activation of yeast pyruvate decarboxylase: the interaction between cysteine 221 and histidine 92; Baburina I et al.; Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at two cysteines on the beta domain (221 and 222) and at H92 on the alpha domain, across the domain divide from C221 . While C221 has been shown to provide the trigger for substrate activation {Baburina, I., et al . (1994) Biochemistry 33, 5630-5635}, the information must be transmitted from the substrate bound at this site {Arjunan, D., et al . (1996) J . Mol . Biol . 256, 590-600} to the active center thiamin diphosphate located at the interface of the alpha and gamma domains . Substitution at H92 with G, A, or C leads to great reduction of the Hill coefficient (from 2.0 in the wild-type enzyme to 1.2-1.3), while substitution for Lys affords an active enzyme with a Hill coefficient of 1.5-1.6 . Iodoacetate at 10 mM reduced the Hill coefficient from 2.0 to 1.1, while also causing significant inactivation of the enzyme, presumably by carboxymethylation of C221 . 1,3-Dibromoacetone, a potential cross-linker when added to the H92C/C222S variant at 0.1 mM, abolished substrate activation while reducing the activity only by 30% . Therefore, 1,3-dibromoacetone may cross-link C92 and C221 . It was concluded that H92 is on the information transfer pathway during the substrate activation process and the interaction between C221 on the beta domain and H92 on the alpha domain is required for substrate activation . Extensive pH studies of the steady-state kinetic constants provide support for the interaction of C221 and H92 and the transmission of regulatory information to the active center via this pathway and pKaS for the two groups . This important interaction between the C221-bound pyruvate and His92 probably has both electrostatic and steric components.

Biochemistry, 1998 Feb 3, 37(5), 1199 - 203
Complementary truncations of a hydrogen bond to ribose involved in transition-state stabilization by cytidine deaminase; Carlow DC et al.; The crystal structure of the complex formed between Escherichia coli cytidine deaminase and the transition-state analogue inhibitor 3,4-dihydrouridine {Betts, L., Xiang, S., Short, S . A., Wolfenden, R., & Carter, C . W . (1994) J . Mol . Biol . 235, 635} shows the presence of an H-bond between Glu-91 and the 3'-OH group of substituent ribose, a part of the substrate that is not directly involved in its chemical transformation . To test the contribution of this interaction to transition-state stabilization, Glu-91 was converted to alanine . The mutant enzyme is very much less active than the wild-type enzyme, with a 500-fold increase in Km and a 32-fold reduction in kcat using cytidine as substrate . No change in secondary structure is evident in the circular dichroic spectrum . As measured by kcat/Km, Glu-91 thus appears to stabilize the transition state for cytidine deamination by an overall factor of 1.7 x 10(4), equivalent to -5.8 kcal/mol in free energy . To test the contribution of this interaction in the opposite sense, the 3'-OH group of the substrate was replaced by a hydrogen atom . Comparing 3'-deoxycytidine with cytidine, the native enzyme shows a 17-fold increase in Km and a 400-fold decrease in kcat, indicating that the 3'-hydroxyl group of cytidine stabilizes the transition state for deamination by an overall factor of 6.3 x 10(3), equivalent to -5.2 kcal/mol in free energy, as measured by kcat/Km . After one binding partner has been removed, however, the effect of removing the remaining partner is relatively slight . For the mutant enzyme E91A, removal of the 3'-hydroxyl group from substrate cytidine reduces kcat/Km by a factor of only 3 . Complete removal of substituent ribose reduces the wild-type enzyme's kcat/Km by a factor of more than 10(8); thus, substituent ribose, although distant from the site of chemical transformation of the substrate, contributes at least 11 kcal to the free energy of stabilization of the transition state for cytidine deamination, matching the apparent contribution to transition state binding made by the 4-OH group of the pyrimidine ring, which is at the site of substrate transformation {Frick, L., Yang, C., Marquez, V . E., & Wolfenden, R . (1989) Biochemistry 28, 9423}.

J Biol Chem, 1998 Feb 6, 273(6), 3461 - 9
A biochemical and genetic model for parasite resistance to antifolates . Toxoplasma gondii provides insights into pyrimethamine and cycloguanil resistance in Plasmodium falciparum; Reynolds MG et al.; We have exploited the experimental accessibility of the protozoan parasite Toxoplasma gondii and its similarity to Plasmodium falciparum to investigate the influence of specific dihydrofolate reductase polymorphisms known from field isolates of drug-resistant malaria . By engineering appropriate recombinant shuttle vectors, it is feasible to examine mutations by transient or stable transformation of T . gondii parasites, in bacterial and yeast complementation assays, and through biochemical analysis of purified enzyme . A series of mutant alleles that mirror P . falciparum variants reveals that the key mutation Asn-108 (Asn-83 in T . gondii) probably confers resistance to pyrimethamine by affecting critical interactions in the ternary complex . Mutations such as Arg-59 (T . gondii 36) have limited effect in isolation, but in combination with other mutations they enhance the competitive ability of folate by increasing the speed of product turnover . Val-16 (T . gondii 10) confers low level resistance to cycloguanil but hypersensitivity to pyrimethamine . This mutation precludes Asn-108, probably because compression of the folate binding pocket introduced by this combination is incompatible with enzyme function . These studies permit detailed biochemical, kinetic, and structural analysis of drug resistance mutations and reconstruction of the probable phylogeny of antifolate resistance in malaria.

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