J Bacteriol, 1995 Feb, 177(3), 851 - 3 Construction and plasmid-borne complementation of strains lacking the epsilon subunit of the Escherichia coli F1F0 ATP synthase; Xiong H et al.; Two strains of Escherichia coli that lack the epsilon subunit of the F1F0 ATP synthase have been constructed . They are shown to be viable but with very low growth yields (28%) . These strains can be complemented by plasmids carrying wild-type uncC, but not when epsilon is overproduced . These results indicate that epsilon is not essential for growth on minimal glucose medium and that the level of its expression affects the assembly of the ATP synthase. J Bacteriol, 1995 Feb, 177(3), 847 - 50 Redundant transfer of F' plasmids occurs between Escherichia coli cells during nonlethal selections; Peters JE et al.; Surface exclusion is the mechanism by which F plasmids prevent the redundant entry of additional F plasmids into the host cell during exponential growth . This mechanism is relaxed in cells that are in stationary phase . Using genetically marked F' plasmids and host strains, we extend this finding to Escherichia coli populations during extended nonlethal selection in bacterial lawns . We show that a high level of redundant transfer occurs between these nongrowing cells during the selection . This result has implications for the mechanism of adaptive mutagenesis. J Bacteriol, 1995 Feb, 177(3), 810 - 4 Effects of multicopy LeuO on the expression of the acid-inducible lysine decarboxylase gene in Escherichia coli; Shi X et al.; We previously reported that mutations in hns, the structural gene for the histone-like protein H-NS, cause derepressed expression of cadA, which encodes the acid-inducible lysine decarboxylase at noninducing pH (pH 8.0) . This study reports the characterization of a plasmid isolated from an Escherichia coli library that suppresses the effect of an hns mutation on cadA expression . A previously sequenced open reading frame, leuO, proves to be the gene that causes the hns-complementing phenotype . The mechanism for this phenotype appears to be overexpression of leuO from a multicopy plasmid, which drastically reduces production of CadC, the essential activator for cadA induction . These results show an in vivo regulatory phenotype for leuO, consistent with its proposed protein sequence. J Bacteriol, 1995 Feb, 177(3), 783 - 91 Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate; Horiuchi T et al.; To examine the physiological effects of DNA replication arrest at the terminus (Ter), we constructed a replication-blocked Escherichia coli strain so that both bidirectional replication forks would be impeded at two flanking Ter sites, one artificial and the other natural . While the blocked strain grew slightly more slowly than a control strain, it had abnormal phenotypes similar to those of E . coli dam mutants, i.e., hyper-Rec phenotype, recA(+)- and recB+ (C+)-dependent growth, and constitutive SOS induction . The observation that these two apparently unrelated mutants cause similar phenotypes led us to design a model . We propose that the following sequential events may occur in both strains . A double-strand (ds) break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam mutant, through which RecBCD enzyme enters and degrades the ds DNA molecule, and the degradation product serves as the signal molecule for SOS induction . When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to repair the ds end, recombinationally . this model (i) explains the puzzling phenotype of recA and recB (C) mutants and the SOS-inducing phenotype of polA, lig, and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication. J Bacteriol, 1995 Feb, 177(3), 733 - 7 Suppression of thermosensitive initiation of DNA replication in a dnaR mutant of Escherichia coli by a rifampin resistance mutation in the rpoB gene; Sakakibara Y; The thermosensitivity of the Escherichia coli dnaR130 mutant in initiation of DNA replication was suppressed by a spontaneous rifampin resistance mutation in rpoB, the gene for the beta subunit of RNA polymerase . Among the dnaR-suppressing rpoB alleles obtained was rpoB22, which was able to suppress the thermosensitivity of the dnaA46 or dnaA167 mutant, but not that of the dnaA5 mutant, in initiation of replication . Some dnaA-suppressing rpoB alleles obtained from rifampin-resistant derivatives of the dnaA mutants were able to suppress the dnaR defect . The dnaR mutant with the rpoB22 allele was deprived of thermoresistance by the dnaA5 mutation and of viability at low and high temperatures by the dnaA46 but not the dnaA167 mutation . The results show that the rpoB-mediated suppression of the dnaA or dnaR defect depends on the functions of both dnaA and dnaR products . I propose that the dnaR product has a key role in transcriptional activation of the replication origin for the dnaA-dependent initiation of DNA replication. J Bacteriol, 1995 Feb, 177(3), 705 - 15 Mutations in Escherichia coli dnaA which suppress a dnaX(Ts) polymerization mutation and are dominant when located in the chromosomal allele and recessive on plasmids; Gines-Candelaria E et al.; Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene . When these alleles (designated Cs, Sx) were moved into dnaX+ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20 degrees C (J.R . Walker, J.A . Ramsey, and W.G . Haldenwang, Proc . Natl . Acad . Sci . USA 79:3340-3344, 1982) . Detailed localization by marker rescue and DNA sequencing are reported here . One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys . It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K . Skarstad and E . Boye, Biochim . Biophys . Acta 1217:111-130, 1994; W . Messer and C . Weigel, submitted for publication) which must be critical for one of the DnaA activities . The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied . First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location . That is, the dnaA+ allele restored growth to dnaA (Cs, Sx) strains at 20 degrees C independently of which allele was present on the plasmid . The dnaA (Cs, Sx) alleles provided initiator function at 39 degrees C and were dominant in a dnaA(Ts) host at that temperature . On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne . Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele . These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location . Suppression by dnaA (Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin. J Bacteriol, 1995 Feb, 177(3), 699 - 704 Kinetic analysis by in vivo 31P nuclear magnetic resonance of internal Pi during the uptake of sn-glycerol-3-phosphate by the pho regulon-dependent Ugp system and the glp regulon-dependent GlpT system; Xavier KB et al.; When sn-glycerol-3-phosphate (G3P) is taken up exclusively by the pho regulon-dependent Ugp transport system, it can be used as the sole source of Pi but not as the sole source of carbon . We had previously suggested that the inability of G3P to be used as a carbon source under these conditions is due to trans inhibition of G3P uptake by internal Pi derived from the degradation of G3P (P . Brzoska, M . Rimmele, K . Brzostek, and W . Boos, J . Bacteriol . 176:15-20, 1994) . Here we report 31P nuclear magnetic resonance measurements of intact cells after exposure to G3P as well as to Pi, using different mutants defective in pst (high-affinity Pi transport), ugp (pho-dependent G3P transport), glpT (glp-dependent G3P transport), and glpD (aerobic G3P dehydrogenase) . When G3P was transported by the Ugp system and when metabolism of G3P was allowed (glpD+), Pi accumulated to about 13 to 19 mM . When G3P was taken up by the GlpT system, the preexisting internal Pi pool (whether low or high) did not change . Both systems were inversely controlled by internal Pi . Whereas the Ugp system was inhibited, the GlpT system was stimulated by elevated internal Pi. J Bacteriol, 1995 Feb, 177(3), 694 - 8 Identification of receptor binding sites by competitive peptide mapping: phages T1, T5, and phi 80 and colicin M bind to the gating loop of FhuA; Killmann H et al.; Previously we proposed a transmembrane model of the FhuA receptor protein in the outer membrane of Escherichia coli . Removal of the largest loop at the cell surface converted the FhuA transport protein into an open channel and rendered cells resistant to the FhuA-specific phages T1, T5, and phi 80 and to colicin M . In the present study we employed acetylated hexapeptide amides covering the entire surface loop to investigate binding of the phages and of colicin M . Competitive peptide mapping proved to be a powerful technique to uncover three ligand binding sites within a region of 34 amino acid residues . Hexapeptides derived from three specific regions of the surface loop inhibited infection of cells by the phages and killing by colicin M . Two of these regions were common among all four FhuA ligands . Electron microscopy of phage T5 revealed that one inhibitory peptide triggered a strong conformational change leading to the release of DNA from the phage head . These results suggest that the FhuA gating loop is the target for specific binding of phages T1, T5, and phi 80 and colicin M. J Bacteriol, 1995 Feb, 177(3), 642 - 53 Analysis of the Mycobacterium tuberculosis 85A antigen promoter region; Kremer L et al.; A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences . In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species . In contrast to the mycobacteria, the 85A promoter did not function in E . coli . We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis . Insertion and deletion mutations within the promoter region indicated that, unlike most E . coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity . In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis . BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST. J Bacteriol, 1995 Feb, 177(3), 566 - 72 Homologous pairing of single-stranded DNA and superhelical double-stranded DNA catalyzed by RecO protein from Escherichia coli; Luisi-DeLuca C; The recO gene product is required for DNA repair and some types of homologous recombination in wild-type Escherichia coli cells . RecO protein has been previously purified and shown to bind to single- and double-stranded DNA and to promote the renaturation of complementary single-stranded DNA molecules . In this study, purified RecO protein was shown to catalyze the assimilation of single-stranded DNA into homologous superhelical double-stranded DNA, an activity also associated with RecA protein . The RecO protein-promoted strand assimilation reaction requires Mg2+ and is ATP independent . Because of the biochemical similarities between RecO and RecA proteins, the ability of RecO protein to substitute for RecA protein in DNA repair in vivo was also assessed in this study . The results show that overexpression of RecO protein partially suppressed the UV repair deficiency of a recA null mutant and support the hypothesis that RecO and RecA proteins are functionally similar with respect to strand assimilation and the ability to enhance UV survival . These results suggest that RecO and RecA proteins may have common functional properties. J Bacteriol, 1995 Feb, 177(3), 544 - 50 Archaeal grpE: transcription in two different morphologic stages of Methanosarcina mazei and comparison with dnaK and dnaJ; Conway De Macario E et al.; Transcription of the heat shock gene grpE was studied in two different morphologic stages of the archaeon Methanosarcina mazei S-6 that differ in resistance to physical and chemical traumas: single cells and packets . While single cells are directly exposed to environmental changes, such as temperature elevations, cells in packets are surrounded by intercellular and peripheral material that keeps them together in a globular structure which can reach several millimeters in diameter . grpE transcript levels determined by Northern (RNA) blotting peaked after a 15-min heat shock in single cells . In contrast, the highest transcript levels in packets were observed after the longest heat shock tested, 60 min . The same response profiles were demonstrated by primer extension experiments and S1 nuclease analysis . A comparison of the grpE response to heat shock with those of dnaK and dnaJ showed that the grpE transcript level was the most increased, closely followed by that of the dnaK transcript, with that of the dnaJ gene being the least augmented . Transcription of grpE started at the same site under normal and heat shock temperatures, and the transcript was consistently approximately 700 bases long . Codon usage patterns revealed that the three archaeal genes use most codons and have the same codon preference for 61% of the amino acids. J Bacteriol, 1995 Feb, 177(3), 524 - 9 A mutation in the rpoA gene encoding the alpha subunit of RNA polymerase that affects metE-metR transcription in Escherichia coli; Jafri S et al.; The DNA-binding protein MetR belongs to the LysR family of transcriptional activators and is required for expression of the metE and metH promoters in Escherichia coli . However, it is not known if this activation is mediated by a direct interaction of MetR with RNA polymerase . In a search for RNA polymerase mutants defective in MetR-mediated activation of the metE gene, we isolated a mutation in the alpha subunit of RNA polymerase that decreases metE expression independently of the MetR protein . The mutation does not affect expression from the metH promoter, suggesting that the alpha subunit of RNA polymerase interacts differently at these two promoters . The mutation was mapped to codon 261 of the rpoA gene, resulting in a change from a glutamic acid residue to a lysine residue . Growth of the mutant is severely impaired in minimal medium even when supplemented with methionine and related amino acids, indicating a pleiotropic effect on gene expression . This rpoA mutation may identify either a site of contact with an as yet unidentified activator protein for metE expression or a site of involvement by the alpha subunit in sequence-specific recognition of the metE promoter. J Bacteriol, 1995 Feb, 177(3), 517 - 23 The cmk gene encoding cytidine monophosphate kinase is located in the rpsA operon and is required for normal replication rate in Escherichia coli; Fricke J et al.; A gene encoding a polypeptide of 25 kDa is located immediately upstream of the gene for ribosomal protein S1, rpsA . In high gene copy number, this gene, mssA, was previously found to suppress defects in smbA, which is now known to be identical to pyrH, encoding UMP kinase . We show here that the 25-kDa polypeptide comprises CMP kinase and propose that the gene be designated cmk . In a strain deleted for cmk, the pools of CMP and dCMP were elevated approximately 30-fold . We constructed a plasmid from which synthesis of CMP kinase was regulated by the lac promoter-operator and measured the synthesis rates for RNA and DNA after induction in the delta cmk/lacPO-cmk+ strain . A specific increase in the rate of DNA synthesis was observed . Further analyses showed that the replication elongation rate was halved in the delta cmk strain, most likely caused by the reductions of the dCTP and dTTP pools to 30 and 70%, respectively, of the levels in the parental strain, but that this was compensated for by a doubling in the frequency of initiation . The delta cmk strain is viable at 37 degrees C but cold sensitive . The cold sensitivity may be related to defects in the synthesis of phospholipids or lipopolysaccharides . In addition to the physiological studies, the region upstream of cmk was sequenced, and 120 codons with strong homology to an uncharacterized protein of the speB operon were identified. J Bacteriol, 1995 Feb, 177(3), 502 - 7 Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage; Moskovitz J et al.; The Escherichia coli peptide methionine sulfoxide reductase gene (msrA) encodes a single-subunit polypeptide of 212 amino acid residues (M . A . Rahman, H . Nelson, H . Weissbach, and N . Brot, J . Biol . Chem . 267:15549-15551, 1992) . RNA blot analysis showed that the gene is transcribed into an mRNA of about 850 nucleotides . The promoter region was characterized, and the transcription initiation site was identified by primer extension . The synthesis of the MsrA protein increased about threefold in a growth-phase-dependent fashion . In an attempt to define the in vivo role of msrA, a chromosomal disruption was constructed . This mutant was more sensitive to oxidative stress, suggesting that oxidation of methionine in proteins plays an important role in oxidative damage. Gastroenterology, 1995 Feb, 108(2), 463 - 9 Endotoxin-stimulated nitric oxide production increases injury and reduces rat liver chemiluminescence during reperfusion; Ma TT et al.; BACKGROUND/AIMS: Nitric oxide has many physiological functions and may play an important role in modulating tissue injury . However, the mechanism of NO action in ischemia/reperfusion injury is completely unknown . This report investigates the role of NO in hepatic reperfusion injury . METHODS: Rat liver was oxygenated for 30 minutes, followed by 30 minutes of ischemia, and then reperfused for 30 minutes . Perfusate was sampled for aspartate aminotransferase content, as an indication of hepatic injury, and for nitrite, an index of NO production . Spontaneous organ chemiluminescence was continuously monitored as a measure of oxyradical production . RESULTS: NO production by the perfused rat liver was induced in vivo by pretreatment with Escherichia coli lipopolysaccharide . This induction led to an increase in hepatic injury during reperfusion that was partially ameliorated by the NO synthase inhibitor NG-monomethyl-L-arginine . Chemiluminescence during reperfusion, a measure of superoxide production in this system, was also decreased in the lipopolysaccharide-treated animals, and this effect was blunted by NG-monomethyl-L-arginine . CONCLUSIONS: These data suggest that NO may combine with superoxide formed during reperfusion to directly cause hepatocellular injury . In vitro work shows that this chemical product is the highly toxic species peroxynitrite. Cancer Res, 1995 Feb 1, 55(3), 581 - 9 Intratumoral activation and enhanced chemotherapeutic effect of oxazaphosphorines following cytochrome P-450 gene transfer: development of a combined chemotherapy/cancer gene therapy strategy; Chen L et al.; Cyclophosphamide and its isomer ifosfamide are cell cycle-nonspecific alkylating agents that undergo bioactivation catalyzed by liver cytochrome P-450 enzymes . The therapeutic efficacy of these oxazaphosphorine anticancer drugs is limited by host toxicity resulting from the systemic distribution of activated drug metabolites formed in the liver . Since tumor cells ordinarily do not have the capacity to activate oxazaphosphorines, we examined whether introduction into tumor cells of a cDNA encoding CYP2B1, a major catalyst of oxazaphosphorine activation, sensitizes the cells to the cytotoxic effects of cyclophosphamide and ifosfamide . Here we show that 9L gliosarcoma cells stably transfected with a cDNA encoding rat CYP2B1 are highly sensitive to cyclophosphamide and ifosfamide cytotoxicity as compared to parental 9L cells or 9L cells transfected with an Escherichia coli beta-galactosidase gene . The CYP2B1 enzyme inhibitor metyrapone protects the CYP2B1-expressing 9L cells from oxazaphosphorine cytotoxicity, demonstrating that the chemosensitivity of these cells is a direct consequence of intracellular prodrug activation . Moreover, CYP2B1-expressing 9L cells potentiate the cytotoxic effects of cyclophosphamide and ifosfamide toward cocultured CYP2B1-negative 9L tumor cells . This "bystander effect" does not require cell-cell contact, and therefore may have the therapeutic advantage of distributing cytotoxic drug metabolites to a wide area within a solid tumor mass . In vivo experiments using Fischer 344 rats implanted s.c . with CYP2B1-expressing 9L tumor cells demonstrated that intratumoral expression of the CYP2B1 gene provides a substantial therapeutic advantage over that provided by liver cytochrome P-450-dependent drug activation alone; cyclophosphamide treatment resulted in complete growth inhibition of CYP2B1-positive tumors, whereas only a modest growth delay effect was obtained with CYP2B1-negative tumors . These studies establish that drug-activating CYP genes may be useful for the development of novel combined chemotherapy/gene therapy strategies for cancer treatment utilizing established cancer chemotherapeutic agents. Blood, 1995 Feb 1, 85(3), 675 - 84 Expression of TAL-1 proteins in human tissues; Pulford K et al.; Rearrangement of the tal-1 gene (also known as SCL or TCL-5) occurs in at least 25% of T-cell acute lymphoblastic leukemias (T-ALLs) and results in the aberrant expression of tal-1 mRNA in the neoplastic cells . Also, tal-1 mRNA is constitutively expressed in erythroid precursors and megakaryocytes . This report describes a direct immunocytochemical study of the distribution and localization of TAL-1 protein in normal human tissues and cell lines using four monoclonal antibodies raised against recombinant TAL-1 proteins . One of these reagents recognizes a protein of 41 kD molecular weight in in vitro-translated TAL-1 proteins, two others recognize proteins of 39 and 41 kD molecular weight, and the fourth antibody also recognizes a TAL-1 protein of 22 kD in addition to the 39- and 41-kD proteins . These anti-TAL-1 antibodies label the nuclei of erythroid precursor cells and megakaryocytes in fetal liver and adult bone marrow . The punctate pattern of nuclear labeling suggests that TAL-1 may comprise part of a novel nuclear structure, similar to that recently found for the PML protein . The nuclei of T cell lines known to express mRNA encoding the full-length TAL-1 protein (eg, CCRF-CEM, RPMI 8402, and Jurkat) are also labeled . A study of normal human tissues (including thymus) showed labeling of smooth muscle, some tissue macrophages, and endothelial cells . TAL-1 protein is undetectable in other cell types . These reagents may play an important role in the diagnosis of T-ALL and could also be used in the context of lymphoma diagnosis on routinely fixed material. J Invest Dermatol, 1995 Feb, 104(2), 218 - 23 Characterization of two distinct calcium-binding sites in the amino-terminus of human profilaggrin; Presland RB et al.; Profilaggrin is a large phosphorylated protein (approximately 400 kDa in humans) that is expressed in the granular cells of epidermis where it forms a major component of keratohyalin . It consists of multiple copies of similar filaggrin units plus amino- and carboxy-terminal domains that differ from filaggrin . Proteolytic processing of profilaggrin during terminal differentiation results in the removal of these domains and generation of monomeric filaggrin units, which associate with keratin intermediate filaments to form macrofibrils in the stratum corneum . The amino-terminal domain contains two calcium-binding motifs similar to the EF-hands found in the S-100 family of calcium-binding proteins . In this report, we expressed the 293-residue amino-terminal pro-domain of human profilaggrin as a polyhistidine fusion protein in Escherichia coli, and characterized calcium binding by a 45Ca++ binding assay and fluorescence emission spectroscopy . Fluorescence measurements indicated that the profilaggrin polypeptide undergoes conformational changes upon the removal of Ca++ with ethylenediamine tetraacetic acid, demonstrating the presence of two calcium-binding sites with affinities for calcium that differ ninefold (1.4 x 10(-4) M and 1.2 x 10(-3) M) . We suggest that this functional calcium-binding domain at the amino-terminus of human profilaggrin plays a role in profilaggrin processing and in other calcium-dependent processes during terminal differentiation of the epidermis. Mol Cell Biol, 1995 Feb, 15(2), 989 - 96 Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein; McGoldrick JP et al.; A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells . Western blot (immunoblot) analysis using polyclonal antibodies to the E . coli MutY protein detected a protein of 65 kDa in both extracts . Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies . Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes) . A nicking activity that is either associated with or copurified with MYH was also detected . Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity . The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches . No nicking activity was detected on substrates containing other selected mismatches or homoduplexes . Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters . Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates . Binding is sevenfold greater on A/GO substrates than on A/G substrates . The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E . coli MutY protein. Mol Cell Biol, 1995 Feb, 15(2), 1049 - 59 A spectrum of mechanisms for the assembly of the RNA polymerase II transcription preinitiation complex; George CP et al.; To explore the diversity in the mechanisms of basal transcription by RNA polymerase II, we have employed a novel biochemical approach that involves perturbation of the transcription reaction with exogenously added TFIIB or TATA box-binding protein (TBP) . Under these conditions, we observe promoter-selective inhibition of transcription by excess TFIIB or excess TBP . This inhibition occurs at the level of basal transcription, because it is observed with minimal promoters that comprise only the TATA box and initiation site sequences as well as with preparations of basal transcription factors that have been purified to greater than 90% homogeneity . In addition, the excess basal factors inhibit the assembly of a functional preinitiation complex but do not inhibit transcription initiation from preassembled preinitiation complexes . A study of several promoters revealed a reciprocal trend in the promoter specificity of inhibition by excess TFIIB versus that by excess TBP . At opposite ends of this spectrum, promoters are strongly inhibited by excess TFIIB but not excess TBP and vice versa . These results reveal the existence of a spectrum of mechanisms for preinitiation complex assembly at different promoters . The mechanistic preference appears to be specified by the aggregate of basal promoter elements rather than by an individual component, such as the TATA box or initiation site sequence . This spectrum provides a new parameter by which differences in the function of minimal class II promoters can be analyzed in the context of both basal and regulated transcription. Infect Immun, 1995 Feb, 63(2), 676 - 80 Identification of the Chlamydia trachomatis RecA-encoding gene; Zhang DJ et al.; DNA sequencing of the major outer membrane protein (MOMP) gene (omp1) from Chlamydia trachomatis shows that some strains have a mosaic structure suggestive of homologous recombination between two distinct omp1 genes . On the basis of this conjecture, we attempted to clone by complementation and sequence the chlamydial recA homolog from C . trachomatis serovar L2 . Chlamydial genomic DNA was partially restricted with XbaI, and fragments of 2 to 4 kb were ligated into pUC19 . The recombinant plasmid was electroporated into Escherichia coli HB101 (RecA-), and colonies were selected in the presence of methyl methanesulfonate (MMS) . A 2.1-kb fragment of C . trachomatis DNA in pUC19 conferred relative MMS resistance to E . coli HB101 . When this recombinant plasmid (pX203) was electroporated into E . coli JC14604 (RecA- lacZ), lac+ recombinants were isolated . Rabbit polyclonal antibodies produced to purified E . coli RecA were immunoreactive in an immunoblot assay with a 35-kDa antigen in RecA- strains of E . coli transformed with pX203 . The 2.1-kb insert was cycle sequenced by the dideoxy chain termination method . An open reading frame of 1,056 bp encoding 352 amino acids that had 44% sequence identity with E . coli RecA was identified . The finding of a recA homolog in C . trachomatis suggests that homologous recombination may occur in this organism . The cloned C . trachomatis RecA-encoding gene will be useful for the construction of a recA mutant once a gene transfer system is developed for chlamydiae. Exp Parasitol, 1995 Feb, 80(1), 36 - 45 Schistosoma mansoni hexokinase: cDNA cloning and immunogenicity studies; Shoemaker CB et al.; DNA encoding a Schistosoma mansoni hexokinase (SHEX) was amplified from cDNA by the polymerase chain reaction using opposing oligonucleotide primers designed to hybridize with two short segments of hexokinase coding sequences that are well-conserved through evolution . The resulting DNA fragment was then used as a probe to identify a full-length hexokinase cDNA clone . SHEX cDNA encodes a 50-kDa protein that is approximately 46% homologous to rat hexokinase, 40% to rat glucokinase, and 34% to yeast hexokinase A . SHEX coding DNA was expressed within Escherichia coli cells and the 50-kDa recombinant product (rSHEX) was partially purified . Mice repeatedly immunized with rSHEX produced antibodies which recognize rSHEX but this offered no significant protection against subsequent cercarial challenge . On Western blots, rSHEX is weakly recognized by antisera against rat brain hexokinase but not by sera from three strains of mice experimentally infected with S . mansoni parasites or from numerous human schistosomiasis patients . Thus, unlike other reported S . mansoni glycolytic enzymes, hexokinase appears to be poorly immunogenic during schistosome infection and of limited potential as a vaccine candidate. East Afr Med J, 1995 Feb, 72(2), 103 - 9 Prevalence and risk factors of parasitic infections among under-five Sudanese children: a community based study; Karrar ZA et al.; A community based prospective study was conducted among randomly selected 300 children aged less than five years selected from three camps of the police force in Khartoum from 534 households representing a total population of 4962 individuals . The study was planned to determine the prevalence and type of parasitic infestations and the related risk factors in that community . From the 300 children, 298 stools specimens were examined: 116 were positive for a single parasite, while samples from 15 children showed ova and cysts for two types of parasites giving a prevalence rate of 44% . The commonest infestations were Giardiasis (21.1%), Taeniasis (10.4%) and Enterobiasis (7.4%) . Non pathogenic E . coli, E . histolytica and Taenia saginata were detected in 2.7%, 0.7% and 1.7% of stools specimen respectively . Children aged between 3 years and above were the most affected group and the infection rate was highest among the illiterate, overcrowded and large sized families . Malnourished children comprised 9.4% of the study group but there was no significant association between undernutrition and the overall prevalence of intestinal infestations, although Giardia lamblia significantly affected the undernourished groupPIP: Infection with intestinal parasites is a common problem among poor, urban populations in African countries and the Middle East . The authors assessed the prevalence of infection with intestinal parasites among children younger than five years old in an urban community in Khartoum and the factors involved . The community-based prospective study was conducted from March 1990 to February 1991 . Soldiers and their families comprise a total of 25,400 individuals residing in ten camps in different areas of Khartoum . Each family has a two-room brick house with kitchen, piped water, and a pit latrine . Most families, however, have no refrigerator, so food is prepared daily and kept in covered containers . Drinking water is kept in large clay pots . 298 stool specimens were examined from 300 randomly selected children under five years old from three police force residential camps in Khartoum representing a total population of 4962 individuals . 116 of the samples were positive for a single parasite, while samples from 15 children showed ova and cysts for two types of parasites, giving a prevalence rate of 44% . The most common infections were giardiasis (21.1%), taeniasis (10.4%), and enterobiasis (7.4%) . Nonpathogenic E . coli, E . histolytica, and Taenia saginata were detected in 2.7%, 0.7%, and 1.7% of stool specimens, respectively . Children aged 3 years and older were the most affected group, with the infection rate highest among the illiterate, overcrowded, and large-sized families . Malnourished children comprised 9.4% of the study group, but no significant association was found between undernutrition and the overall prevalence of intestinal infestations, although Giardia lamblia significantly affected the undernourished group . Bioorg Med Chem, 1995 Feb, 3(2), 195 - 205 The mechanism of Escherichia coli tryptophan indole-lyase: substituent effects on steady-state and pre-steady-state kinetic parameters for aryl-substituted tryptophan derivatives; Lee M et al.; We have examined the reaction of Escherichia coli tryptophan indole-lyase with fluoro, chloro, methyl and hydroxytryptophans using steady-state kinetics, rapid-scanning and single wavelength stopped-flow spectrophotometry, and rapid chemical quench methods . All of the 16 tryptophan derivatives examined are substrates for alpha, beta-elimination catalyzed by tryptophan indole-lyase . The steady-state kinetic parameter, kcat/Km, did not show a consistent trend with the steric bulk of the substituent, but Km increased for larger substituents . Rapid-scanning stopped-flow spectra show that all tryptophan analogues undergo covalent reaction with the pyridoxal-5'-phosphate cofactor to give equilibrating mixtures of external aldimine and quinonoid intermediates, but the relative amounts of each intermediate are strongly dependent on the nature and position of the substituent . The dissociation constants for external aldimine formation, Kd, obtained from single-wavelength stopped-flow experiments decreased for most substituted tryptophans, which suggests that part of the binding energy is derived from hydrophobic interactions between the enzyme and the indole ring of tryptophan . In contrast, the rate constants of quinonoid intermediate formation and reprotonation and of indole elimination were quite variable, depending on the position and the nature of the substituent . Overall, 6-substituted tryptophans have the most consistent reactivity, which indicates that there may be space in the enzyme active site near the 6-position . There is a good linear correlation between log (kcat/Km) and log (kf/Kd) (apparent second order rate constant for quinonoid intermediate formation), with a slope of 0.66 . This suggests that quinonoid intermediate formation contributes only about 66% of the activation energy for the reaction, and thus a later step in the reaction must be partially rate-limiting . Rapid chemical quench experiments demonstrate a 'burst' of indole in the reaction of L-tryptophan under single turnover conditions, confirming that a step subsequent to the elimination is partially rate-determining . In contrast, 5-methyl-L-tryptophan does not exhibit a significant 'burst', suggesting that 5-methylindole elimination is nearly completely rate-determining . These results support the proposed mechanism and demonstrate that there are significant effects of aryl substituents on the distribution of covalent intermediates and on the rate-determining step in the alpha, beta-elimination reaction catalyzed by E . coli tryptophan indole-lyase. Prostaglandins Leukot Essent Fatty Acids, 1995 Feb-Mar, 52(2-3), 163 - 6 Fatty acyl CoA esters as regulators of cell metabolism; Shrago E et al.; Long chain fatty acyl CoA esters have the ability to interact with certain proteins and thereby serve as effectors in cell metabolism . In particular, they can displace nucleotides from specific nucleotide dependent or binding proteins and interfere with their action . The ADP/ATP carrier and uncoupling protein are two examples where the interplay of nucleotide and acyl CoA binding to the proteins regulate their function . Other proteins such as glucokinase can be considered in this group . In certain tissues like liver they are affected during fasting and insulin deficiency, and when serum fatty acids and liver acyl CoA levels are elevated . More recently, an acyl CoA binding protein in E . coli has been found to be a transcription factor for gene regulation of fatty acid metabolism enzymes . There appears to be some consensus in the amino acid sequence for acyl CoA binding sites on these proteins which serve a variety of important roles in cellular metabolism. Mol Microbiol, 1995 Feb, 15(4), 617 - 26 Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells; Rockey DD et al.; Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle . Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane . To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae . Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera . Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product . The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein . Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C . psittaci elementary bodies . Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion . Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inclusion membrane protein A) and its gene product, IncA . In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells . Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification . This pattern was not observed in immunoblots of RBs or in the E . coli expressing IncA . Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells. Mol Microbiol, 1995 Feb, 15(3), 573 - 80 Identification of phosphoproteins in Escherichia coli; Freestone P et al.; The substrates of ion- and lipid-stimulated protein kinase activity in extracts of Escherichia coli were purified by chromatography . Subsequent N-terminal sequencing suggests that these substrates include the following: a novel 80 kDa protein co-purifying with RNA polymerase but partially homologous to elongation factor G; a protein with an apparent molecular weight of 65 kDa identified as the ribosomal protein S1; and a 32 kDa protein identified as succinyl CoA synthetase, a key enzyme in the tricarboxylic acid cycle . The phosphorylation of these three proteins was markedly stimulated by the addition of manganese, and occurred on threonine, serine or tyrosine residues as indicated by the stability of the phosphoresidues during acid treatment . In addition, a calcium-stimulated protein of 70 kDa was identified as the heat-shock protein DnaK, and a 17 kDa lipid-stimulated phosphoprotein as nucleotide diphosphate kinase. Mol Microbiol, 1995 Feb, 15(3), 519 - 29 Purification, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR-aceEF-lpd operon of Escherichia coli; Quail MA et al.; The repressor of the pdhR-aceEF-lpd operon of Escherichia coli, PdhR, was amplified to 23% of total cell protein and purified to homogeneity by heparin-agarose and cation-exchange chromatography . The purified protein is a monomer (M(r) 29,300) which binds specifically to DNA fragments containing the pdh promoter (Ppdh) in the absence of pyruvate . The pdh operator was identified by DNase I footprinting as a region of hyphenated dyad symmetry, +11AATTGGTaagACCAATT+27, situated just downstream of the transcript start site . In vitro transcription from Ppdh was repressed > 1000-fold by PdhR and this repression was antagonized in a concentration-dependent manner by its co-effector, pyruvate . Studies on RNA polymerase binding at Ppdh showed that RNA polymerase protects the -44 to +21 region in the absence of PdhR, but no RNA polymerase binding or protection upstream of +9 could be detected in the presence of PdhR . It is concluded that PdhR represses transcription by binding to an operator site centred at +19 such that effective binding of RNA polymerase is prevented. Mol Microbiol, 1995 Feb, 15(3), 473 - 82 Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr; Park SJ et al.; Succinate dehydrogenase (SDH) of Escherichia coli, the sole membrane-bound enzyme of the tricarboxylic acid cycle, participates in the aerobic electron-transport pathway to generate energy via oxidative phosphorylation reactions . Previous studies have established that succinate dehydrogenase (SDH) synthesis is elevated by aerobiosis and suppressed during growth with glucose . To examine how the sdhCDAB genes that encode SDH are regulated by changes in the environment, sdh-lacZ fusions were constructed and analysed in vivo following cell growth under a variety of alternative culture conditions . Expression of sdh-lacZ was highest under aerobic conditions and was decreased 10-fold in the absence of oxygen . The fnr and arcA gene products are required for this oxygen control and each acts to repress sdhC-lacZ expression . Expression of sdh-lacZ also varied 10- to 14-fold depending on the type of carbon substrate used or the medium richness . This control was shown to be independent of the crp and fruR gene products, and indicates that some other regulatory element exists in the cell to adjust SDH enzyme levels accordingly . Iron and haem availability affected sdhC-lacZ expression by two- to three-fold . Lastly, sdhC-lacZ expression was shown to vary with the cell growth rate during aerobic and anaerobic conditions. Proteins, 1995 Feb, 21(2), 91 - 104 Three-dimensional structure prediction of the NAD binding site of proton-pumping transhydrogenase from Escherichia coli; Fjellstrom O et al.; A three-dimensional structure of the NAD site of Escherichia coli transhydrogenase has been predicted . The model is based on analysis of conserved residues among the transhydrogenases from five different sources, homologies with enzymes using NAD as cofactors or substrates, hydrophilicity profiles, and secondary structure predictions . The present model supports the hypothesis that there is one binding site, located relatively close to the N-terminus of the alpha-subunit . The proposed structure spans residues alpha 145 to alpha 287, and it includes five beta-strands and five alpha-helices oriented in a typical open twisted alpha/beta conformation . The amino acid sequence following the GXGXXG dinucleotide binding consensus sequence (residues alpha 172 to alpha 177) correlates exactly to a typical fingerprint region for ADP binding beta alpha beta folds in dinucleotide binding enzymes . In the model, aspartic acid alpha 195 forms hydrogen bonds to one or both hydroxyl groups on the adenosine ribose sugar moiety . Threonine alpha 196 and alanine alpha 256, located at the end of beta B and beta D, respectively, create a hydrophobic sandwich with the adenine part of NAD buried inside . The nicotinamide part is located in a hydrophobic cleft between alpha A and beta E . Mutagenesis work has been carried out in order to test the predicted model and to determine whether residues within this domain are important for proton pumping directly . All data support the predicted structure, and no residue crucial for proton pumping was detected . Since no three-dimensional structure of transhydrogenase has been solved, a well based tertiary structure prediction is of great value for further experimental design in trying to elucidate the mechanism of the energy-linked proton pump. J Med Virol, 1995 Feb, 45(2), 146 - 50 Comparison of the kinetics of Puumala virus specific IgM and IgG antibody responses in nephropathia epidemica as measured by a recombinant antigen-based enzyme-linked immunosorbent assay and an immunofluorescence test; Elgh F et al.; Immunoglobulin M and G (IgM and IgG) responses were followed up to 6 months in patients with nephropathia epidemica (NE) by an enzyme-linked immunosorbent assay (ELISA) using a recombinant Puumala virus (PUU) nucleocapsid protein as antigen and an immunofluorescence test (IF) using PUU infected, acetone-treated cells as antigen . The recombinant protein was produced by cloning and expressing the nucleocapsid encoding gene of PUU as a polyhistidine fusion protein in Escherichia coli . The product was purified over a metal chelating ion affinity column . On admission, all 17 patients had an IgM response by both methods . The IgM titers decreased significantly by both methods 3 months after onset (ELISA P < 0.05 and IF P < 0.05) . Four of six still had detectable IgM, however at low levels, after 6 months . Presence of specific IgG differed significantly on admission between the two methods: by ELISA 8 of 17 had detectable specific IgG, whereas by IF 15 of 17 had specific IgG (P < 0.02) . There were 10 significant titer rises between acute and convalescent serum samples in the same patients by both methods . It is concluded that the IgG antibody response differs in the early phase of NE as measured by a method using a recombinant PUU nucleocapsid protein and a method using PUU infected acetone-treated cells as antigens . Furthermore, the results suggest that it is of importance to rely on specific IgM for serodiagnosis of NE during the acute phase. Electrophoresis, 1995 Feb, 16(2), 279 - 85 Genomic mismatch scanning: current progress and potential applications; Nelson SF; Genomic mismatch scanning (GMS) is a new method of genetic mapping which attempts to purify and map the regions of identity between two complex genomes in a single test . Identical DNA fragments from two genomic sources are enriched in two steps: (i) after reannealing of the two genomes, heterohybrids are purified by using a combination of a restriction methylase and methylation-sensitive endonucleases, (ii) heterohybrids that contain mismatches are nicked in vitro by the E . coli MutHLS mismatch repair system and are eliminated subsequently from the pool, leaving only mismatch-free heterohybrids . The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step onto metaphase chromosomes or ordered DNA arrays . The principal advantages of GMS are (i) it approaches the theoretical limit of mapping power and resolution offered by an arbitrarily dense set of completely informative polymorphic markers and (ii) it results in a great increase in the effective number of informative markers without a corresponding increase in the number of individual tests . Thus, it should provide an efficient method for affected-relative-pair linkage mapping and for linkage disequilibrium mapping . In addition, a variation of GMS may allow rapid genomic scanning for regions of homozygosity-by-descent or somatic loss-of-heterozygosity . The feasibility of GMS has been validated in the 15 mb genome of Saccharomyces cerevisiae . This article discusses the principles of GMS, the application to more complex genomes, and the possible uses of GMS. FEMS Immunol Med Microbiol, 1995 Feb, 10(3-4), 207 - 18 Detection of attachment of enterotoxigenic Escherichia coli (ETEC) to human small intestinal cells by enzyme immunoassay; Mynott TL et al.; Simple immunoassays were developed to study the binding between enterocytes of the small intestine and other cell types, and enterotoxigenic Escherichia coli (ETEC) . CFA/I or CFA/II pilus protein or CFA-positive E . coli bacteria were immobilised in wells of microtitre plates and incubated with vesicles or crude mucus prepared from human brush border enterocytes . Binding of the cell preparations was detected by adding specific rabbit anti-brush border IgG followed by urease-labelled goat anti-rabbit IgG and urea substrate . The binding of purified CFA/I to human or rabbit small intestine, human oral epithelial cells or Caco-2 cells was detected with specific anti-CFA/I IgG . Both human brush border and mucus-derived preparations were able to attach to ETEC . The binding was CFA-specific and strong enough to withstand several washings . In contrast, CFA/I did not bind to small intestinal cells of non-human small intestinal origin, indicating that there may be important differences in affinity between receptors present on human small intestinal cells and cells of non-human small intestinal origin . Antibodies directed against human small intestinal and non-small intestinal cells did not cross-react with either preparation, indicating that receptors between these different cell sources are different . The EIA proved useful during the identification of a newly-recognised 15 kDa bacterial surface component of ETEC strain H10407P, which may function as a putative attachment factor . The EIAs developed in this study were easy to perform and multiple tests could be performed on small samples, including biopsy samples obtained during endoscopy. Jpn J Genet, 1995 Feb, 70(1), 35 - 46 DNA sequence changes in mutations in the tonB gene on the chromosome of Escherichia coli K12: insertion elements dominate the spontaneous spectra; Kitamura K et al.; To obtain insight into the nature and mechanisms of spontaneous mutations, Escherichia coli K12 strain TM31 was constructed to determine, by DNA sequencing, the mutational spectrum of the tonB gene on the chromosome . We inserted the chloramphenicol resistant gene 1.6 kb upstream of the tonB gene, thus making it possible to retrieve the mutated tonB gene from the chromosome by shotgun cloning using a drug-resistant marker . The spontaneous mutation frequency in the tonB gene, which was judged by its colicin B-resistant phenotype, is 3-10 x 10(-7) . Spontaneous mutations were dominated by large insertions that are identified by DNA sequencing to be IS elements; IS1 dominated, but IS2, IS5, and IS10 were also obtained . In uvrA- strain, transposition of both IS10-R and IS10-L are equally increased, suggesting the interaction of the UvrA protein and IS10 transposition . The base substitutions are the second largest group of mutations, among which G:C-->A:T transition is predominant . Deletions also contribute significantly in wild type with regard to DNA repair and uvrA- strains, but not recA- strain, suggesting that the RecA protein is involved to some extent in deletion formation . Endpoints of these deletions do not always correlate with the presence of repeated sequences, indicating the absence of homologous recombination for deletion formation. J Mol Endocrinol, 1995 Feb, 14(1), 79 - 90 Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli; Upton Z et al.; Recombinant chicken (c)IGF-II has been produced in Escherichia coli after first modifying a plasmid that coded for a human (h)IGF-II fusion protein . The cIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography and refolded . Recombinant cIGF-II was then released from the fusion protein using a genetically engineered serine protease and purified to homogeneity by reverse-phase HPLC . In vitro analysis of recombinant cIGF-II revealed differences between cIGF-II and its human counterpart . Recombinant cIGF-II was less potent than hIGF-II in stimulating protein synthesis in rat myoblasts . This appeared to be due to a decreased affinity for the type-1 IGF receptor . The human and chicken peptides were similar, however, in studies assessing binding to the type-2 IGF receptor and to IGF-binding proteins . Moreover, recombinant cIGF-II and hIGF-II were equipotent in both biological and receptor binding studies in chick embryo fibroblasts, suggesting that there may be a difference between mammalian and avian type-1 IGF receptors. Mol Biochem Parasitol, 1995 Feb, 69(2), 281 - 8 Biochemical analysis of recombinant glutathione S-transferase of Fasciola hepatica; Salvatore L et al.; Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses . The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE . Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions . The rGST proteins are recognised by antisera raised to the native GST of F . hepatica . All four rGST proteins from F . hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene . The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST . No rGST were active with EPNP . rGST47 and 51 showed activity with the other four substrates . rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal . The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors . These results show that F . hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles. J Cell Sci, 1995 Feb, 108 ( Pt 2), 581 - 93 Effects of cofilin on actin filamentous structures in cultured muscle cells . Intracellular regulation of cofilin action; Nagaoka R et al.; The previous investigation (Abe et al . (1989) J . Biochem . 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle . In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR) . When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated . Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions . 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin . Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands . At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin . FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time . The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2) . These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding. Immunopharmacology, 1995 Feb, 29(1), 47 - 52 In vitro inhibition of murine IFN gamma production by phosphorothioate deoxyguanosine oligomers; Halpern MD et al.; Phosphorothioate (PT) oligonucleotides are designed as specific agents for antisense therapy although they have been reported to exert non-specific immunomodulatory effects . To elucidate further their actions, the effect of PT deoxyguanosine oligomers (S-oligo(dG)) on in vitro cytokine production by mouse splenocytes was studied . S-oligo(dG)20 inhibited production of INF gamma induced by Con A, E . coli DNA or the combination of PMA and calcium ionophore A23187 . The diester analogue was inactive, and of PT homo-oligomers tested, S-oligo(dG)20 was the most active . PT compounds with as few as 5 dG residues could also block INF gamma production . These results indicate that base composition and length, as well as the PT backbone, contribute to the inhibition of INF gamma production and extend the range of immunomodulatory effects of PT compounds. Hybridoma, 1995 Feb, 14(1), 9 - 18 Two-phase approach for the expression of high-affinity human anti-human immunodeficiency virus immunoglobulin Fab domains in Escherichia coli; Takeda S et al.; We describe here a two-phase approach for the development of high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system . The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs) . Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160 . One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4 x 10(-10) M . A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 with a dissociation constant on the order of 8 x 10(-10) M, but was unable to recognize cell surface antigen . In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector . mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA . The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector . The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ) . DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage . The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS) Biosci Biotechnol Biochem, 1995 Feb, 59(2), 294 - 7 Hairpin ribozyme-mediated cleavage of the full-length beta-glucuronidase (GUS) mRNA; Hisamatsu S et al.; We designed three hairpin ribozymes to cleave Escherichia coli beta-glucuronidase (GUS) mRNA and tested those activities in vitro . One of the ribozymes was designed to form 9 base pairs in total with the target GUS mRNA, and the other two ribozymes had longer substrate binding sites . All ribozymes cleaved the model substrate (100 bases long) at the predicted target site . Two ribozymes containing longer substrate binding sites cleaved the substrate much more efficiently than the other ribozyme containing shorter substrate binding site . Also, the ribozymes with long substrate binding sites had high activity against the full-length GUS mRNA (1.9 kilobases) and maintained the activity even at a low temperature, 26 degrees C, a general growth condition of plant cells . Effects of the substrate binding site length of the ribozyme on cleavage activity are discussed. Biosci Biotechnol Biochem, 1995 Feb, 59(2), 184 - 9 Molecular analysis of growth inhibition caused by overexpression of the biotin operon in Escherichia coli; Ifuku O et al.; Constitutive overexpression of the biotin operon (type 9 mutation) in a multicopy plasmid resulted in growth inhibition in Escherichia coli . Deletion analysis of the biotin operon indicated that overexpression of the bioB gene alone, the product of which is believed to catalyze the conversion of dethiobiotin to biotin, is sufficient for growth inhibition . This growth inhibition was still observed when the wild-type bioB gene was replaced by several mutant-type bioB genes derived from biotin auxotrophs that have base-pair substitutions creating amino acid substitutions in the bioB gene product . However, the modification of Ala 143 and Gly 99 of the bioB gene product resulted in recovery from growth inhibition . These results suggest that this phenotype of growth inhibition by overexpression of the bioB gene in E . coli is independent of the biotin-forming activity itself, but is caused by some function involving a specific conformation of the bioB gene product. Hum Mol Genet, 1995 Feb, 4(2), 275 - 8 A molecular defect in coproporphyrinogen oxidase gene causing harderoporphyria, a variant form of hereditary coproporphyria; Lamoril J et al.; Hereditary coproporphyria (HC) is an acute hepatic porphyria with autosomal dominant inheritance caused by a deficient activity of coproporphyrinogen IX oxidase (CPX) . We previously described harderoporphyria, a homozygous variant form of coproporphyria in three siblings, characterized by a massive excretion of harderoporphyrin and a marked decrease of coproporphyrinogen IX oxidase activity . In this kindred, the transmission of the disease was autosomal recessive . In the present study, sequencing of cDNA and genomic DNA from these patients revealed a point mutation resulting in a lysine to glutamic acid substitution (K304E) in exon 6 of the gene and the absence of the normal allele, suggesting a homozygous state for the mutation . Expression studies of normal and mutated cDNAs in E . coli demonstrated that this amino acid substitution was responsible for the important decrease in the enzyme activity and for the accumulation of harderoporphyrin . The Michaelis constant of the mutated enzyme was 10-fold higher than normal suggesting that the lysine at position 304 is important for binding the substrate: a slightly increased sensitivity to thermal denaturation was also observed. Protein Sci, 1995 Feb, 4(2), 258 - 67 Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase; Baker DP et al.; Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits . Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain . Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158) . In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100-->Ala mutation was introduced into the Glu-50-->Ala holoenzyme, a mutant in which domain closure is impaired . The Glu-50/Asp-100-->Ala enzyme is fourfold more active than the Glu-50-->Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate . In addition, the Asp-100-->Ala mutation restores the ability of the Glu-50-->Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA) . At subsaturating concentrations of aspartate, the Glu-50/Asp-100-->Ala enzyme is activated more by ATP than the Glu-50-->Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50-->Ala enzyme . As opposed to the wild-type enzyme, the Glu-50/Asp-100-->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate . Structural analysis of the Glu-50/Asp-100-->Ala enzyme by solution X-ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA . However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100-->Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50-->Ala and wild-type enzymes . Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100-->Ala enzyme. Protein Sci, 1995 Feb, 4(2), 167 - 77 Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR; Jones BE et al.; The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates . The first forms within 5 ms and has substantial secondary structure but little stability . The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789) . Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period . A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms) . The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet . The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices . Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms . Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms . Only approximately half of the population of molecules form this set of stable hydrogen bonds . Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction. Protein Sci, 1995 Feb, 4(2), 159 - 66 Rearranging the domains of pepsinogen; Lin X et al.; Most eukaryotic aspartic protease zymogens are synthesized as a single polypeptide chain that contains two distinct homologous lobes and a pro peptide, which is removed upon activation . In pepsinogen, the pro peptide precedes the N-terminal lobe (designated pep) and the C-terminal lobe (designated sin) . Based on the three-dimensional structure of pepsinogen, we have designed a pepsinogen polypeptide with the internal rearrangement of domains from pro-pep-sin (native pepsinogen) to sin-pro-pep . The domain-rearranged zymogen also contains a 10-residue linker designed to connect sin and pro domains . Recombinant sin-pro-pep was synthesized in Escherichia coli, refolded from 8 M urea, and purified . Upon acidification, sin-pro-pep autoactivates to a two-chain enzyme . However, the emergence of activity is much slower than the conversion of the single-chain zymogen to a two-chain intermediate . In the activation of native pepsinogen and sin-pro-pep, the pro region is cleaved at two sites between residues 16P and 17P and 44P and 1 successively, and complete activation of sin-pro-pep requires an additional cleavage at a third site between residues 1P and 2P . In pepsinogen activation, the cleavage of the first site is rate limiting because the second site is cleaved more rapidly to generate activity . In the activation of sin-pro-pep, however, the second site is cleaved slower than the first, and cleavage of the third site is the rate limiting step.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Sci, 1995 Feb, 4(2), 141 - 8 Ecotin: lessons on survival in a protease-filled world; McGrath ME et al.; Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has been shown to be a potent inhibitor of a group of homologous serine proteases with widely differing substrate recognition . It is highly effective against a number of enzymes, including both pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein . Recent structural and functional studies on ecotin and its interactions with different serine proteases have clarified these initial observations and revealed the remarkable features of this protein in inhibiting a strikingly large subset of the chymotrypsin family of serine proteases . The structures of the ecotin:serine protease complexes provide the first examples of protein-protein recognition where the concept of specificity of interactions needs to be reexamined . The binding sites show a fluidity of protein contacts derived from ecotin's innate flexibility in fitting itself to proteases while strongly interfering with their function. Protein Expr Purif, 1995 Feb, 6(1), 79 - 90 Overexpression and characterization of human tetrameric pyruvate dehydrogenase and its individual subunits; Korotchkina LG et al.; Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, is the first component of the pyruvate dehydrogenase complex which catalyzes a two-step oxidative decarboxylation of pyruvic acid . To overexpress human E1 and its subunits individually, cDNAs for the mature forms of human E1 alpha and E1 beta were subcloned either individually or together into a plasmid pQE-9 and expressed in Escherichia coli M15 . A polyhistidine extension was added at the NH2-termini of the recombinant E1 alpha and E1 beta for the rapid purification of the proteins by Ni-nitrilotriacetic-agarose chromatography . The polyhistidine extension on either E1 alpha or E1 beta subunit did not affect the activity of the recombinant tetrameric E1 . Highly purified recombinant human E1 catalyzed the partial reactions of the oxidative and nonoxidative conversion of pyruvic acid with the same efficiency as E1 purified from bovine kidney . Recombinant human E1 interacted with thiamin pyrophosphate by forming a charge transfer complex band at 330 nm that changed during the catalytic cycle . Recombinant human E1 was phosphorylated by E1-kinase (with concomitant inactivation) by incorporating nearly three phosphoryl groups per mole of E1 . When expressed individually, E1 alpha and E1 beta subunits lacked any catalytic activity in the oxidative or nonoxidative reactions . Spectral studies demonstrated that there was no thiamin pyrophosphate binding to either recombinant E1 alpha or E1 beta subunit . The E1 alpha subunit retained the ability to be phosphorylated; however, the incorporation of phosphoryl groups into recombinant E1 alpha alone was only about 12% of that observed with the tetrameric E1 . These findings show that both subunits are required for formation of the active center and catalysis. Protein Expr Purif, 1995 Feb, 6(1), 56 - 62 In vitro refolding of cyclomaltodextrin glucanotransferase from cytoplasmic inclusion bodies formed upon expression in Escherichia coli; Hellman J et al.; The recovery of active cyclomaltodextrin glucanotransferase (CGT) from inclusion bodies formed upon expression of a signal sequence deletion mutant (cgt delta ss) gene in Escherichia coli was studied . Under the conditions examined the in vitro renaturation yield of active enzyme was up to 81% of the maximum activity from urea solubilized and up to 3.7% from guanidinium hydrochloride (GdmCl) solubilized inclusion bodies . Refolding from GdmCl solutions resulted in the reaggregation of delta ss CGT . Although native (folded) CGT showed enzymatic activity at urea concentrations as high as 10 M, the inclusion bodies could be solubilized with 3 M urea solution, but the preparation had no enzymatic activity prior to reduction of the denaturant concentration . This suggests that the delta ssCGT inclusion bodies are composed of molecules that are trapped in an inactive state . The individual molecules may have extensive secondary structure, since inclusion bodies solubilized with 4.5 M urea gave maximum yield of activity in the renaturation step . With higher urea concentration the yield decreased . Thus, a "mild" solubilization technique seems to increase the yield of active delta ssCGT from the inclusion bodies . The stability of the refolded delta ssCGT was identical to that of extracellularly produced wild type CGT, whereas a disulfide bond mutant, Ser-70-CGT, showed reduced stability under identical conditions . This suggests that the single disulfide bond in delta ssCGT is formed during refolding and contributes to the stability of the molecule . A fusion of maltose binding protein to the NH2-terminus of delta ssCGT did not significantly affect the in vitro folding of the delta ssCGT portion when tested by a denaturation-renaturation experiment. Protein Expr Purif, 1995 Feb, 6(1), 45 - 55 Hamster monomorphic arylamine N-acetyltransferase: expression in Escherichia coli and purification; Bergstrom CP et al.; N-Acetylation is a major pathway in the metabolism of hydrazine and arylamine drugs, and has been associated with carcinogen bioactivation . Monomorphic hamster liver N-acetyltransferase (NAT1) cDNA was cloned from hamster liver cells by reverse transcriptase-coupled polymerase chain reaction . The determined nucleotide sequence was identical to that reported for NAT1 . The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient acetyltransferase activity . In addition, cDNA was subcloned into the expression vectors pFLAG-ATS and pFLAG-MAC . The latter vectors encoded a tac promoter and appended a low-molecular weight (1 kDa) hydrophilic FLAG marker peptide to the amino terminus of NAT1 . Unexpectedly, periplasmic export of FLAGATS-NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity . High acetyltransferase activity, however, was obtained when the fusion protein was expressed in the cytosol . Enzyme purified to homogeneity by immunoaffinity chromatography exhibited substrate specificity comparable to that of the hamster-derived protein. Protein Expr Purif, 1995 Feb, 6(1), 39 - 44 High-level expression of Trypanosoma brucei fructose-1,6-bisphosphate aldolase in Escherichia coli and purification of the enzyme; Chevalier N et al.; A procedure has been developed for high-level expression of Trypanosoma brucei fructose-bisphosphate aldolase in Escherichia coli . Therefore, a specific restriction site was introduced by mutagenesis at the front of the gene, enabling its ligation in an expression plasmid, immediately downstream of the regulatory sequences . Growth conditions were established for production of high amounts of soluble and active enzyme . Aldolase was purified to near-homogeneity from the soluble fraction of the bacterial lysate by nuclease treatment, differential precipitation steps, and passage over a CM-Sepharose column . From a 1-liter culture of E . coli cells, 60-120 mg of purified protein that is essentially indistinguishable in physicochemical and kinetic properties and in stability from the enzyme purified from trypanosomes grown in infected laboratory animals was reproducibly obtained. Protein Expr Purif, 1995 Feb, 6(1), 33 - 8 Purification and properties of thyroid hormone receptor beta 1 expressed in Escherichia coli as a fusion protein; Ball EH et al.; Thyroid hormone receptor binds to specific DNA sequences and acts as a hormone-dependent transcriptional regulator . The protein can form homodimers, or heterodimers with the related 9-cis-retinoic acid receptor (RXR) or retinoic acid receptor (RAR) receptor families, leading to complex patterns of regulation . To obtain relatively large quantities of the receptor for biochemical studies, we have inserted the cDNA for human thyroid receptor beta into a variant of the pGEX vector (pGEX-KG) and produced the protein in Escherichia coli as a fusion with glutathione-S-transferase . Conditions for protein production, isolation on glutathione agarose, and thrombin cleavage to generate active receptor were developed . Final yields were approximately 1 mg/liter of culture . Scatchard plots of 125I-triiodothyronine binding data revealed a single class of sites with a Kd of 0.1 nM . An overlay assay was established to measure protein-protein binding and used to show a direct interaction with bacterially expressed RXR receptor . Binding of the purified receptor to DNA response elements measured in a DNA binding assay was increased by RXR to different extents, depending on the DNA sequence . This preparation will be useful in exploring the mechanisms of receptor activity. Protein Expr Purif, 1995 Feb, 6(1), 1 - 9 Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites; Kupper D et al.; EcoRII was the first restriction endonuclease (ENase) reported needing the cooperative interaction with at least two DNA sites for activity . We constructed an EcoRII-overproducing strain of Escherichia coli by placing the coding sequence under control of the T7 gene 10 regulatory elements . The yield of EcoRII expression could be increased to about 10% of total soluble cellular protein . Inclusion bodies are formed that mainly consist of insoluble EcoRII molecules . After solubilization by 6 M guanidine hydrochloride refolding of the enzyme was achieved by dilution into appropriate buffer . The endonuclease was purified to homogeneity from both the soluble protein fraction and the protein renatured from inclusion bodies . Their identity was proven by circular dichroism and analysis of enzyme activity with respect to the special substrate requirements of EcoRII . It is shown that EcoRII cleavage of oligodeoxyribonucleotide duplexes (oligo duplexes) with only one recognition site follows a sigmoidal concentration dependence, i.e., they cannot be cleaved below a distinct low DNA concentration where simultaneous interaction with two substrate molecules is no longer possible . We demonstrate that the restriction of oligo duplexes containing two recognition sites does not show this concentration dependence, confirming an intramolecular site cooperativity. Shock, 1995 Feb, 3(2), 132 - 6 Effect of lipopolysaccharide, leukocytes, and monoclonal anti-lipid A antibodies on erythrocyte membrane elastance; Bellary S et al.; The micropipette aspiration technique was used to quantify membrane deformability of individual red blood cells (RBCs) before and after exposing whole blood and blood free of leukocytes to lipopolysaccharide (LPS) . The ability of an anti-lipid A monoclonal antibody (E5) to inhibit the effects of LPS was also investigated . In the LPS/whole blood studies, a significant increase in elasticity modulus was observed following incubation with LPS . An increase in elasticity modulus indicates a decrease in RBC membrane deformability . The effect depended on the incubation time but was not concentration-dependent in the range studied (25, 40, or 170 micrograms/mL) . When incubating blood free of leukocytes with LPS, the elasticity moduli of erythrocytes did not change . Results also showed that preincubation of the LPS with E5 prior to incubation with whole blood partially inhibited the effect of LPS, suggesting a possible mechanism of the beneficial actions of monoclonal antibodies in septic shock. J Dairy Sci, 1995 Feb, 78(2), 285 - 90 Effects of an Escherichia coli J5 vaccine on mild clinical coliform mastitis; Hogan JS et al.; Efficacy of an Escherichia coli (O111:B4) J5 bacterin was tested in an experimental challenge trial . Nineteen cows were vaccinated with an E . coli J5 bacterin, and 10 cows were injected with a placebo containing adjuvant only . Vaccine and placebo were administered at drying off, 30 d after drying off, and within 48 h after calving . Cows were challenged approximately 30 d after calving by intramammary infusion with a smooth heterologous strain of E . coli previously shown to cause mild clinical mastitis . Vaccination with the J5 bacterin reduced duration of IMI and local signs of clinical mastitis . Concentrations of BSA in milk 24 h after challenge were greater in control cows than in cows vaccinated with J5 . The SCC at 7 d postchallenge were greater for cows vaccinated with the placebo than for cows vaccinated with J5 . Bacterial counts were lower for cows vaccinated with the placebo than for cows vaccinated with J5 at 3, 6, and 9 h postchallenge . In contrast, cows vaccinated with J5 had lower bacterial counts at 2, 3, and 4 d postchallenge than did cows vaccinated with placebo . Systemic signs of clinical mastitis were relatively mild and similar between treatment groups . Rectal temperature, DMI, and milk production did not differ between control and cows vaccinated with J5 following challenge. Free Radic Biol Med, 1995 Feb, 18(2), 365 - 71 Apparent inhibition of superoxide dismutase activity in vitro by diesel exhaust particles; Kumagai Y et al.; The inhibitory effects of diesel exhaust particles (DEP) on superoxide dismutase (SOD) activity were examined in vitro because intratracheal administration of DEP to mice resulted in a suppression of the pulmonary enzyme activity (Sagai et al., Free Radic . Biol . Med . 14:37-47; 1993) . Superoxide production, based on the reduction of cytochrome c, was suppressed considerably by the soluble fraction of mouse lung and by purified SOD from bovine erythrocytes, but the suppression was drastically diminished in the presence of methanol-extractable compounds of DEP . Inhibition of SOD by diethyldithiocarbamate was irreversible, but that by 1,2-naphthoquinone (1,2-NQ) and the methanol extract of DEP was removed by dialysis . Inhibition of superoxide mediated cytochrome c reduction by Tiron, a scavenging agent for superoxide, was blocked by the methanol extract and 1,2-NQ in a concentration-dependent manner . In contrast, addition of a large amount of SOD to the reaction mixture resulted in an almost complete disappearance of inhibitory action of not only 1,2-NQ but also the methanol extract . The existence of carbonyl compounds in the DEP was confirmed by thin-layer chromatography (TLC) with 2,4-dinitrophenylhydrazine reagent . Electron spin resonance (ESR) spectra of an incubation mixture of oxidized 1,2-dihydroxynaphthalene in the absence and presence of cytochrome c indicated a reaction between the semiquinone radical of 1,2-NQ and cytochrome c . These results indicate that the apparent reduction in SOD activity by DEP is due to the chemical reaction of superoxide with components like quinones, which reduce levels of superoxide. Curr Biol, 1995 Feb 1, 5(2), 97 - 9 Evolutionary genetics . Directed mutations slip-sliding away? Lenski RE, Sniegowski PD. Adaptive frameshift mutations in the lacZ gene of Escherichia coli are, unusually, nearly all short deletions, perhaps caused by slipped-strand mispairings in mononucleotide runs . But are they directed? Biol Pharm Bull, 1995 Feb, 18(2), 372 - 6 Soluble expression of a synthetic gene for human translation initiation factor 4E in Escherichia coli; Morino S et al.; In order to obtain the active form of recombinant human initiation factor (eIF) 4E effectively, an artificial synthetic gene was cloned into an expression vector (pMAL-p2) and the soluble expression was attempted in Escherichia coli under the control of a tac promoter . Two expression systems were finally constructed as a fusion protein with maltose-binding protein, which contain a recognition sequence for the site specific protease alpha-thrombin and factor Xa, respectively . Most of the fusion protein was induced as a soluble form . The soluble human eIF-4E digested from the fusion protein showed binding specificity for the m7GTP affinity column. Vet Microbiol, 1995 Feb, 43(2-3), 131 - 41 Neutralizing antibodies against Shiga-like toxins from Escherichia coli in colostra and sera of cattle; Pirro F et al.; Previous or present infection with Shiga-like toxin producing E . coli (SLTEC) was detected by an indirect neutralization assay of antibody titer . Bovine colostra and sera blocked the cytotoxic effects of Shiga-like toxin on Vero cell monolayers . SLT neutralizing antibodies were present in 84.0% (189/225) of the colostrum samples from randomly chosen cows in Bavaria, Germany . While all of the colostra with neutralizing activity reacted with SLT-I, only 14.7% neutralized both SLT-I and -II . Approximately 93.0% (37/40) of sera from heifers had SLT neutralizing activity . To quantify the neutralizing antibodies, colostra were tested in the Vero cell assay for their capability to reduce the 50% cytotoxic dose (CD50) of SLT standards, where the neutralizing units/ml (nu/ml) equal the log10 of CD50 reduction . Almost half of reactive colostra (48.7%) reduced the CD50 of the SLT-I standard by 10(4) to 10(5) (4-5 nu/ml) . Higher reactivity (5-7 nu/ml) was found in 46.5% of positive colostra . The remaining colostra samples had over 7 nu/ml . To determine if the colostra were blocking receptors for SLT on Vero cells, cells were preincubated with colostra, and SLT was later added . No neutralizing activity was detected, indicating the reactivity of colostra was directed against SLT . When the colostra were subjected to ammonium sulphate precipitation and DEAE anion exchange chromatography, high levels of neutralizing activity were found in the IgG1 containing fractions . Colostrum fractions were tested for SLT-I binding antibodies in a capture ELISA, based on the binding of SLT-I to the toxin receptor analogue P1-glycoprotein . Only fractions from colostra with over 5 nu/ml were reactive in this assay, indicating the ELISA was less sensitive than the Vero cell assay . The results support the theory that SLTEC exposure of cows in Germany is more widespread than expected from epidemiological studies based on bacterial isolation . This possibly indicates a higher risk of human SLTEC infection via beef and milk products. Vet Microbiol, 1995 Feb, 43(2-3), 123 - 9 Presence of F107, 2134P and Av24 fimbriae on strains of Escherichia coli isolated from Swedish piglets with diarrhoea; Kennan R et al.; A total of 109 Escherichia coli isolates from piglets with diarrhoea, that had previously been shown to be enterotoxin producers, but negative for the adhesive fimbriae K88, K99, 987P and F41 were tested for the presence of more recently characterised fimbriae . Testing was done by immunodot assay with absorbed polyclonal antisera against Av24 and F107 fimbriae, and unabsorbed polyclonal antiserum and monoclonal antiserum against 2134P fimbriae . Strains were also tested by polymerase chain reaction for the presence of genes encoding the major subunit of F107 fimbriae . After elimination of possible non-specific reactions, antisera testing produced 10 strains positive with all 4 antisera, 1 strain that reacted with all antisera except F107, 2 strains that reacted with all antisera except the 2134P monoclonal, 3 strains that reacted with 2134P polyclonal and F107 and 2 that reacted with F107 only . The PCR testing confirmed the results of the antisera, but also produced an additional 14 positive strains, giving a total of 30% of the strains tested reacting positively by PCR . Furthermore, all 33 isolates positive by PCR came from pigs that were older than 1 week, which is 45% of the 72 isolates tested which came from older pigs. J Virol Methods, 1995 Feb, 51(2-3), 297 - 304 Molecular cloning of Indian tomato leaf curl virus genome following a simple method of concentrating the supercoiled replicative form of viral DNA; Srivastava KM et al.; DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and leaf curl in tomato (ITLCV) were cloned following a simple procedure for isolation of the double stranded replicative form of viral DNA from infected tomato plants . The method is based on extraction of total DNA from infected plants followed by concentration of the double stranded replicative form of viral DNA by an alkaline denaturation procedure identical to that used for isolation of plasmid DNA from Escherichia coli . The attempted cloning of DNA showed that 95% of the transformants contained plasmids with an insert of either DNA-A (2.75 kb) or DNA-B (2.55 kb) . Cloned DNA-A and DNA-B when used as probes could detect DNA-A/DNA-B in total nucleic acid obtained from fresh diseased tissue . Both DNA-A and DNA-B are needed for infection and they have a common region of 166 bases with about 94% nucleotide sequence homology, a characteristic of all bipartite geminiviruses . Comparison of the amino acid sequence of the putative coat protein product of ITLCV with some other mono- and bipartite geminiviruses revealed a maximum of 86% homology with Indian cassava mosaic virus. Jpn J Cancer Res, 1995 Feb, 86(2), 155 - 9 Induction of DNA recombination by activated 3-amino-1-methyl-5H-pyrido{4,3-b}indole; Hiramoto K et al.; To investigate the genotoxic properties of a food-derived carcinogen, 3-amino-1-methyl-5H-pyrido-{4,3-b}indole (Trp-P-2), we have tested whether Trp-P-2 and its metabolically transformed products can induce DNA recombinations . Trp-P-2 is a strong mutagen and its activated form, the N-hydroxylated derivative, Trp-P-2(NHOH), is known to form DNA adducts and cause DNA chain cleavage . Using a system in which phage lambda undergoes recombination inside host Escherichia coli, we have found that Trp-P-2(NHOH), but not Trp-P-2 itself, can induce recombination . A nitroso derivative of Trp-P-2, Trp-P-2(NO), which can be reduced intracellularly to form Trp-P-2(NHOH), also induced recombination . Active oxygens are implicated in this recombinogenic action, since Trp-P-2(NHOH) is known to undergo spontaneous oxidative degradation, generating active oxygen radicals which can cause DNA chain cleavages . 4-Hydroxyaminoquinoline N-oxide and phenyl-hydroxylamine also showed recombinogenic actions in this assay system; hence, it is suspected that aromatic amine-type carcinogens have this property in common. Clin Pediatr (Phila), 1995 Feb, 34(2), 86 - 9 Cephalhematomas revisited . When should a diagnostic tap be performed? LeBlanc CM, Allen UD, Ventureyra E. The purpose of this article is to review the most appropriate method for investigating cephalhematomas for possible infection and to clarify the indications for diagnostic aspiration . MEDLINE searches were conducted for the period from 1972 to 1993, and all English-language reports were obtained . A summary of the findings from the reports identified was supplemented by a patient report . Eleven articles reporting 13 infected cephalhematomas were identified in the literature from 1972 to 1993 . Escherichia coli was isolated from approximately 50% of the cephalhematomas that were aspirated . Most patients presented with obvious clinical signs of scalp infection, sepsis, meningitis, and/or osteomyelitis . Plain radiographs, bone scans, and enhanced CT scans were limited in their ability to determine if a cephalhematoma was infected unless associated osteomyelitis existed . Aspiration is the diagnostic procedure of choice for cephalhematomas suspected of being infected, as indicated by an increase in size, development of erythema, development of fluctuance, relapse of systemic infection, or a delay in the resolution of clinical symptoms of infection. Antimicrob Agents Chemother, 1995 Feb, 39(2), 427 - 30 Molecular characterization of nine different types of mutants among 107 inhibitor-resistant TEM beta-lactamases from clinical isolates of Escherichia coli; Henquell C et al.; DNA-DNA hybridization and sequencing were performed to determine the molecular basis of resistance to clavulanic acid in 107 inhibitor-resistant TEM (IRT) enzymes produced by Escherichia coli clinical isolates . These beta-lactamases derived from TEM-1 enzyme focused at pI 5.2 (n = 68) or 5.4 (n = 39) and were very poorly inhibited by clavulanic acid compared with TEM-1 enzyme . Results showed that the amino acid sequences of 84 of the 107 enzymes differ from TEM-1 by one or two substitutions previously described: Arg-244-->Ser (IRT-2) in 22 strains, Met-69-->Leu (TEM-33) in 17 strains, Met-69-->Val (TEM-34) in 14 strains, Met-69-->Ile (IRT-3) in 6 strains, Met-69-->Leu associated with Asn-276-->Asp (IRT-4) in 13 strains, and Met-69-->Val associated with Asn-276-->Asp (TEM-36) in 12 strains . A new combination, Met-69-->Ile with Asn-276-->Asp, was found in 20 strains and was called IRT-8 . Two IRT enzymes not previously described were characterized . The substitution Met-69-->Val associated with a novel substitution Arg-275-->Leu occurred in one strain . The combination Met-69-->Leu and Asn-276-->Asp was associated with the novel substitution Trp-165-->Arg in two strains . These two novel enzymes were called IRT-9 and IRT-10, respectively . The implication of these novel mutated positions, 165 and 275, in resistance to inactivation by clavulanate was supported by crystallographic data on the TEM-1 enzyme and results of site-directed mutagenesis . Molecular characterization of these mutants showed great diversity among the genes coding for inhibitor-resistant TEM enzymes produced by clinical E . coli isolates. Genetika, 1995 Feb, 31(2), 276 - 7 {Determination of the direction of transcription of Escherichia coli K-12 structural genes of the fructose regulon in vivo}; Dobrynina OIu et al.; The direction of transcription of specific components of the fructose phosphotransferase system, the fruA, fruK, and fruB genes, was determined in vivo by plasmid F'ts1141ac . Transcription from each of these genes was shown to run in the same direction, counterclockwise with respect to the E . coli chromosomal map. Cell Immunol, 1995 Feb, 160(2), 193 - 8 Utilization of soluble fusion proteins for induction of T cell proliferation; Kirschmann DA et al.; A peptide display library was evaluated as a means to identify peptide binding motifs for class II molecules . Peptides expressed as part of a soluble fusion protein with a maltose binding protein (malE) were produced by Escherichia coli . Constructs containing the high-affinity binding influenza hemagglutinin peptide 307W-319 (mal-HA) or the low-affinity binding tetanus toxoid peptide 830-843 (mal-TT) were used as controls . mal-HA, but not mal-TT, inhibited synthetic biotinylated-HA peptide from binding to purified DR4 Dw4 molecules in a dose-dependent manner . The fusion-peptide presentation system was also evaluated for its ability to induce antigen-specific T cell proliferation . DR4 Dw4+ B cells pulsed with mal-HA, but not mal-TT, induced dose-dependent proliferation of an HA-specific DR4 Dw4-restricted T cell line to the same extent as synthetic HA peptide . Using this type of peptide display library, it may be possible to determine the antigenic specificity of T cell clones isolated from patients with autoimmune diseases. Can J Microbiol, 1995 Feb, 41(2), 170 - 6 Induction of resistance to hydrogen peroxide and radiation in Deinococcus radiodurans; Wang P et al.; Though bacteria of the radiation-resistant genus Deinococcus have a high resistance to the lethal and mutagenic effects of many DNA-damaging agents, the mechanisms involved in the response of these bacteria to oxidative stress are poorly understood . To investigate antioxidant enzyme responses in Deinococcus spp., the catalase activity produced by these bacteria was measured and the sensitivity of these bacteria to hydrogen peroxide was tested . Deinococcus spp . had higher levels of catalase and were more resistant to hydrogen peroxide than Escherichia coli K12 . The high levels of catalase produced by Deinococcus radiodurans were, in part, regulated by growth phase . Cultures of D . radiodurans, when pretreated with sublethal levels of hydrogen peroxide, became relatively resistant to the lethal effects of hydrogen peroxide and exhibited higher levels of catalase than untreated control cultures . These pretreated cells were also resistant to lethality mediated by ultraviolet light and gamma-rays . These results suggest that Deinococcus spp . possess inducible defense mechanism(s) against the deleterious effects of oxidants and ionizing and ultraviolet radiation. Biokhimiia, 1995 Feb, 60(2), 315 - 27 {Interaction of Escherichia coli cytochrome bd with hydrogen peroxide}; Borisov VB et al.; The absorption spectrum of the cytochrome bd complex from Escherichia coli in the "as isolated" state is characterized by an intense band at approximately 648 nm belonging to reduced heme d oxycomplex (d2+-O2) . This band is often accompanied by a small shoulder around 680 nm . Treatment of the oxycomplex with hydrogen peroxide results in the loss of the 648 nm band and increased absorbance at 680 nm . The peak at 680 nm also appears in the difference absorption spectrum after addition of hydrogen peroxide to the oxidized form of the enzyme and can be attributed to formation of a peroxy or an oxoferryl complex of heme d . The increase in extinction at 680 nm is accompanied by a small red shift of the Soret band; the corresponding difference spectrum with lambda min = 405-410 nm and lambda max = 430-440 nm is of a magnitude similar to the changes in the visible region (delta A440-410 approximately equals 10 mM-1.cm-1) . This circumstance favours H2O2 interaction with heme d rather than b595 . The lineshape of the H2O2-induced spectral changes does not vary throughout the hydrogen peroxide concentration range studied (5 microM-5 mM) . The H2O2 concentration dependence on the 680 nm peak magnitude follows a saturation curve with apparent Kd of 30-40 microM . The product of cytochrome bd interaction with H2O2 reacts with cyanide approximately tenfold slower than the free oxidized enzyme . Addition of excess catalase to the hydrogen peroxide-treated cytochrome bd complex fully reverses the H2O2-induced spectral changes . However, the rate of disappearance of these changes (keff approximately equals 10(-3) s-1) is ca . 10-fold slower than expected for the dissociation rate constant, koff, for the peroxy adduct, assuming reversible H2O2 binding with Kd approximately equal to 30-40 microM and kon > 500 M-1.s-1 . This may point to H2O2 interaction with cytochrome bd, being essentially irreversible . The initial addition of H2O2 to heme d is likely to be followed by cleavage of the O--O bond, giving rise to the oxoferryl state (Fe4+ = O) of heme d which disappears upon removal of H2O2 by catalase due to reduction by endogenous electron sources. Biokhimiia, 1995 Feb, 60(2), 297 - 307 {Directed cleavage of the 16S rRNA molecule at a single internucleotide bond}; Bogdanova SL et al.; Cleavage of 16S ribosomal RNA (rRNA) from E . coli "hammerhead" type ribozymes as well as by RNAase iI in the presence of "hymeric" (2'-deoxy-F-thymidine containing) oligonucleotides has been studied . The conditions for the cleavage of a desired single internucleotide bond have been found for a large molecule with a very complicated secondary and three-dimensional structure. Appl Biochem Biotechnol, 1995 Feb, 50(2), 145 - 59 The effect of cellular energetics on foreign protein production; Ko YF et al.; Escherichia coli strain F-122 was used to determine if there are additional physiological effects, other than decreasing energetic efficiency accompanied by the excretion of the acetate, on foreign protein production . This organism was the host for expressing HIV582-beta-galactosidase fusion protein under the control of the trp promoter, with ampicillin resistance . By comparing parallel batch cultures with and without acetate addition, it was found that the presence of acetate in the media did not influence beta-galactosidase activity . In these experiments, it appears that the low protein productivity often observed during acetate formation is the result of inefficient cell metabolism, rather than acetate acting as a specific inhibitor of protein production. Physiol Behav, 1995 Feb, 57(2), 401 - 5 Behavioral conditioning of lipopolysaccharide-induced anorexia; Exton MS et al.; One manifestation of the acute phase response, sickness behavior, is now considered an important response in the organism's overall attempt to reinstate homeostasis . This report aimed to determine whether the sickness behavior of anorexia was conditionable using the conditioned taste aversion paradigm . To investigate this phenomenon, lipopolysaccharide (LPS) (100 micrograms/kg) was used as the unconditioned stimulus, and was paired with a novel 1% saccharin solution (conditioned stimulus) . Upon conditioned stimulus representation, the anorectic effects of LPS were observed . These data are consistent with recent literature showing acute phase events to be conditionable. Arzneimittelforschung, 1995 Feb, 45(2), 205 - 10 Conventional statistics and useful statistics; Rahlfs VW; Differences between conventional statistical methods and more useful, modern methods are demonstrated using a statistical analysis of data from therapeutic research in rheumatology . The conventional methods, t-test and graphs of mean values and the boxplot, detect almost no differences between treatment groups . A more recent procedure for analysing group differences is the Wilcoxon-Mann-Whitney test . The associated graphs are based on the cumulative distribution function of the two treatment groups and the synthetic Receiver Operating Characteristic (ROC) . Special differences, namely baseline dependencies, can be visualized in this way. Trends Biochem Sci, 1995 Feb, 20(2), 65 - 9 High selectivity with low specificity: how SecB has solved the paradox of chaperone binding; Randall LL et al.; Fundamental to the function of all molecular chaperones is their amazing ability to selectively and rapidly bind proteins in non-native states . Chaperones modulate a kinetic partitioning among the alternative pathways open to polypeptides within a cell, so that the proper pathway is taken . Here we review studies of SecB, a chaperone in Escherichia coli dedicated to facilitation of protein export, and emphasize the features that enable it to bind rapidly with high affinity and selectivity in the absence of consensus in sequence . The concepts discussed are likely to be generally applicable to chaperones. J Appl Bacteriol, 1995 Feb, 78(2), 189 - 93 Transfer of plasmids between strains of Escherichia coli under rumen conditions; Scott KP et al.; Strains of Escherichia coli originally isolated from the rumen of sheep were shown to be capable of exchanging a 60kb plasmid, conferring resistance to tetracycline and ampicillin, at low frequencies (below 10(-6) per recipient) under anaerobic conditions in the presence of (a) autoclaved and clarified rumen fluid, (b) raw clarified rumen fluid, or (c) whole rumen fluid . Under anaerobic conditions the two rumen strains showed no inhibition of growth rate when 50 mmol l-1 volatile fatty acids were added to LB medium at pH 7, although significant inhibition resulted with 100 mmol l-1 VFA . The two rumen strains, and four strains from the pig gut, showed less inhibition of anaerobic growth by volatile fatty acids than did three laboratory strains examined for comparison . These findings indicate that plasmid transfer between certain E . coli strains can occur under conditions that closely simulate an anaerobic but environment. Biochem Mol Biol Int, 1995 Feb, 35(2), 375 - 85 Expression and DNA-binding properties of the 14K carboxyl terminal fragment of Escherichia coli DNA topoisomerase I; Zhu CX et al.; DNA sequence coding for the last 121 amino acids of Escherichia coli topoisomerase I was synthesized by PCR and cloned into a plasmid under the control of the T7 promoter . Induction of T7 RNA polymerase in E . coli carrying the plasmid clone resulted in over-expression of this C-terminal domain fragment previously shown to confer higher DNA binding affinity to the enzyme . Purification to homogeneity was achieved by phosphocellulose and single-stranded DNA agaraose chromatography . Direct interaction between this 14K domain and poly(dA) was demonstrated by UV spectroscopy . Noncovalent complexes formed between this fragment and oligo(dT) 8 and oligo(dT) 16 can also be trapped by photo-crosslinking. Biochem Mol Biol Int, 1995 Feb, 35(2), 297 - 306 Evidence for the presence of two pyridine nucleotide-binding sites on the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase; Glavas NA et al.; The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta . Trypsin digestion of the purified enzyme generates fragments of the alpha subunit . The beta subunit is uncleaved unless NADP(H) is present (Tong, R.C.W., Glavas, N.A . and Bragg . P.D . (1991) Biochim . Biophys . Acta 1080, 19-28) . Purified transhydrogenase bound to either NAD- or NADP-agarose was treated with trypsin . The alpha subunit was cleaved to 16, 29 and 43 kDa fragments in both cases . The beta subunit remained bound to NAD-agarose but was released as two cleavage fragments (25 and 30 kDa) from NADP-agarose . The beta subunit of the transhydrogenase bound to NAD-agarose was cleaved by trypsin in the presence of NADP(H) to yield 25 and 30 kDa fragments of the beta subunit . These results suggest that the beta subunit contains two pyridine nucleotide-binding sites. Biochem Mol Biol Int, 1995 Feb, 35(2), 291 - 6 Variation in the modulation of superoxide-induced single-strand breaks in plasmid pBR322 DNA by biological antioxidants; Sah NK et al.; Superoxide radical (O2-.), generated by the xanthine-xanthine oxidase system, induces significant amount (20%) of single-strand breaks in plasmid pBR322 DNA . This is almost completely inhibited by its specific scavenger, superoxide dismutase . The biological antioxidants, at near physiological concentrations show great variation in their modulation of DNA damage induced by O2-. . The thiols glutathione, cysteine and dithiothreitol do not protect DNA, instead they greatly enhance the strand-breaking activity of this free radical . However, the lipid soluble antioxidants tannic acid, butein, canthaxanthin, beta-carotene and lipoate offered significant protection to plasmid DNA against O2-. . Since O2- . is the most abundant reactive oxygen species generated, the above mentioned modulating abilities of biological antioxidants may have significant biological implications. Biochem Mol Biol Int, 1995 Feb, 35(2), 283 - 90 Tissue expression of rat ciliary neurotrophic factor (CNTF) mRNA and production of the recombinant CNTF; Ohta K et al.; We used the polymerase chain reaction to show that CNTF mRNA is widely expressed in the brain, heart, lung, liver, kidney and testis of the rat, in addition to preferential expression in the sciatic nerve . We produced the recombinant CNTF in E . coli using rat sciatic nerve cDNA and obtained high yields of pure CNTF . The biological activity of the recombinant protein was comparable to that of native rat CNTF in its support of the survival of cultured embryonic day-8 chick ciliary ganglions . Western Blot analysis using antibody against the recombinant CNTF clearly showed the presence of CNTF protein (25kD) in rabbit sciatic nerve. Mol Carcinog, 1995 Feb, 12(2), 110 - 7 ogt alkyltransferase enhances dibromoalkane mutagenicity in excision repair-deficient Escherichia coli K-12; Abril N et al.; We examined the role of the O6-alkylguanine-DNA alkyltransferase encoded by ogt gene in the sensitivity of Escherichia coli to the mutagenic effects of the dibromoalkanes, dibromoethane and dibromomethane, by comparing responses in ogt- bacteria to those in their isogenic ogt+ parental counterparts . The effects of the uvrABC excision-repair system, the adaptive response, mucAB and umuDC mutagenic processing, and glutathione bioactivation on the differential responses of ogt- and ogt+ bacteria were also studied . Mutation induction was monitored by measuring the frequency of forward mutations to L-arabinose resistance . Induced mutations occurred only in excision repair-defective strains and were totally (with dibromomethane) or substantially (with dibromoethane) dependent on the alkyltransferase (ATase) encoded by the ogt gene . An increased mutagenic response to both dibromoalkan