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| Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7341 - 5 Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors; Fisher CE et al.; We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly) . The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly . Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase . We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor . We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment . To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7305 - 9 Formation of stable cationic lipid/DNA complexes for gene transfer; Hofland HE et al.; Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent . Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity . Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition . However, expression was completely lost 24 hr after mixing . The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum . A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency . Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid . Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant . Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7300 - 4 Molecular cloning of a rat chromosome putative recombinogenic sequence homologous to the hepatitis B virus encapsidation signal; Aoki H et al.; Previously, we reported that a 61-bp subgenomic HBV DNA sequence (designated as 15AB, nt 1855-1915) is a hot spot for genomic recombination and that a cellular protein binding to 15AB may be the putative recombinogenic protein . In the present study, we established the existence of a 15AB-like sequence in human and rat chromosomal DNA by Southern blot analysis . The 15AB-like sequence isolated from the rat chromosome demonstrated a 80.9% identity with 5'-CCAAGCTGTGCCTTGGGTGGC-3', at 1872-1892 of the hepatitis B virus genome, thought to be the essential region for recombination . Interestingly, this 15AB-like sequence also contained the pentanucleotide motifs GCTGG and CCAGC as an inverted repeat, part of the chi known hot spot for recombination in Escherichia coli . Importantly, a portion of the 15AB-like sequence is homologous (82.1%, 23/28 bp) to break point clusters of the human promyelocytic leukemia (PML) gene, characterized by a translocation {t(15;17)}, and to rearranged mouse DNA for the immunoglobulin kappa light chain . Moreover, 15AB and 15AB-like sequences have striking homologies (12/15 = 80.0% and 13/15 = 86.7%, respectively) to the consensus sequence for topoisomerase II . Our present results suggest that this 15AB-like sequence in the rat genome might be a recombinogenic candidate triggering genomic instability in carcinogenesis. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7258 - 62 Cloning and expression of human deoxyguanosine kinase cDNA; Johansson M et al.; A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base . The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level . The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113) . The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa . Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA . dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues . Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK . To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases . Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7178 - 83 Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule; Fan QR et al.; Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules . To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies . The refolded NK receptor is a monomer in solution . It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells . The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide . Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis . Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+ . The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7120 - 4 Stabilization of diverged tandem repeats by mismatch repair: evidence for deletion formation via a misaligned replication intermediate; Lovett ST et al.; A functional methyl-directed mismatch repair pathway in Escherichia coli prevents the formation of deletions between 101-bp tandem repeats with 4% sequence divergence . Deletions between perfectly homologous repeats are unaffected . Deletion in both cases occurs independently of the homologous recombination gene, recA . Because the methyl-directed mismatch repair pathway detects and excises one strand of a mispaired duplex, an intermediate for RecA-independent deletion of tandem repeats must therefore be a heteroduplex formed between strands of each repeat . We find that MutH endonuclease, which in vivo incises specifically the newly replicated strand of DNA, and the Dam methylase, the source of this strand-discrimination, are required absolutely for the exclusion of "homeologous" (imperfectly homologous) tandem deletion . This supports the idea that the heteroduplex intermediate for deletion occurs during or shortly after DNA replication in the context of hemi-methylation . Our findings confirm a "replication slippage" model for deletion formation whereby the displacement and misalignment of the nascent strand relative to the repeated sequence in the template strand accomplishes the deletion. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6953 - 8 Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme; Ibba M et al.; Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates . Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition . Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS . Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon . Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding . Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end . The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition . Furthermore, we show that E . coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA . The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6935 - 40 Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest; Gu W et al.; RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation . For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII . Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation . Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site . Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes . As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length . After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex . These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation . Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site . Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6892 - 7 Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element; Postel EH et al.; NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity . While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known . We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence . Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding . Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids . All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding . In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer . These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer . Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2. Biochemistry, 1996 Jul 9, 35(27), 8942 - 7 Essential cysteines in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase from Escherichia coli: analysis by chemical modification and site-directed mutagenesis; Salleh HM et al.; The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (EC 4.1 . 2.16) (KDO 8-P synthase) that catalyzes the condensation of D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) and inorganic phosphate (Pi) was inactivated by the thiol-modifying reagents 5,5-dithiobis (2-nitrobenzoate) (DTNB) and methyl methanethiosulfonate (MMTS) . Reaction of cloned native KDO 8-P synthase with DTNB correlated with modification of two of the four cysteine sulfhydryls per monomer of enzyme and total loss of enzymatic activity which could be partially restored by treatment with dithiothreitol (DTT) . Cyanolysis of the DTNB-inactivated enzyme with KCN led to the elimination of 2 equiv of 5-thio-2-nitrobenzoate and partial recovery of activity . The presence of either substrate(s) or product(s) provided no protection against inactivation nor affected the number of cysteines modified, indicating that the cysteines modified are most likely not at the active site of KDO 8-P synthase . Titration of denatured enzyme with DTNB resulted in the modification of all four cysteines . After treatment of native enzyme with MMTS, no cysteines could be titrated with DTNB and no enzymatic activity could be detected . Treatment of the MMTS-inactivated KDO 8-P synthase with DTT resulted in restoration of enzymatic activity and the presence of two DTNB-titratable cysteine residues . Based on these observations and a report that KDO 8-P synthase is inactivated in a time-dependent manner with 3-bromopyruvate and that the substrate PEP protects against this inactivation, all four cysteines (38, 166, 206, and 249) were individually mutated to alanines via a modified PCR methodology . The C206A and C249A mutants were both enzymatically active with K(m) and Vmax values approximately identical to those of wild-type KDO 8-P synthase, and both native mutants reacted with DTNB to modify only one of the three remaining cysteine sulfhydryls per monomer of enzyme . Titration of denatured C206A and C249A mutants resulted in the modification of three cysteines . The C38A and C166A mutants were both for the most part enzymatically inactive . Titration of native C38A and C166A with DTNB resulted in modification of two cysteines while titration of the denatured mutant protein resulted in modification of the three remaining cysteines . Circular dichroism measurements of wild-type KDO 8-P synthase and the four C --> A mutants indicate modest but significant changes in the structure of the mutants . These results indicate that C206 and C249 in native KDO 8-P synthase are readily accessible to the modification reagent DTNB and therefore inactivation may result from structural changes in the DTNB-modified KDO 8-P synthase or blockage of access of substrates to the active site . The C38 and C166 in native KDO 8-P synthase are inaccessible to the modification reagent DTNB, indicating that they are located in the interior of KDO 8-P synthase, and loss of activity in the C38A and C166A mutants suggests their essentiality in the KDO 8-P synthase reaction. Biochemistry, 1996 Jul 9, 35(27), 8934 - 41 Active site of 5-aminolevulinate synthase resides at the subunit interface . Evidence from in vivo heterodimer formation; Tan D et al.; 5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway of animals, fungi and some bacteria . It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor . In mouse erythroid 5-aminolevulinate synthase, lysine 313 has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate {Ferreira, G . C., et al . (1993) Protein Sci . 2, 1959-1965}, while arginine 149, a conserved residue among all known 5-aminolevulinate synthase sequences, is essential for function {Gong & Ferreira (1995) Biochemistry 34, 1678-1685} . To determine whether each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interface (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically inactive mouse 5-aminolevulinate synthase mutants . When R149A and K313A mutants were co-expressed in a hem A- Escherichia coli strain, which can only grow in the presence of 5-aminolevulinate or when it is transformed with an active 5-aminolevulinate synthase expression plasmid, the hem A- E . coli strain acquired heme prototrophy . The purified K313A/R149A heterodimer mixture exhibited K(m) values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system . In addition, DNA sequencing of four Saccharomyces cerevisiae 5-aminolevulinate synthase mutants, which lack ALAS activity but exhibit enzymatic complementation, revealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C . Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthase active site is located at the subunit interface and contains catalytically essential residues from the two subunits. Biochemistry, 1996 Jul 9, 35(27), 8855 - 62 A rapid screen of active site mutants in glycinamide ribonucleotide transformylase; Warren MS et al.; Specific and saturation site-directed mutageneses have been used to alter each polar residue within 6 A of the catalytic center of glycinamide ribonucleotide transformylase (EC 2.1.2.2) . These mutants were rapidly screened for catalytic activity using functional complementation of auxotrophic cells . This screen allows a rapid qualitative estimate of enzyme activity for each of these mutants . These results have shown that none of the polar residues close to the catalytic center of the enzyme are irreplaceable, although several are important for full catalytic activity, namely, Asn106, His108, Ser135, and Asp144 . A mechanism is proposed in which a fixed water molecule mediates the required proton transfers between substrate and cofactor, while the formyl group is transferred from 10-formyltetrahydrofolate by direct nucleophilic attack by the amine of glycinamide ribonucleotide . The active site polar residues may act to alter the pKa values of the attacking and leaving amino groups within a putative tetrahedral intermediate in order to facilitate the transfer of the formyl group. Biochemistry, 1996 Jul 9, 35(27), 8805 - 14 Structural influence of cation binding to recombinant human brain S100b: evidence for calcium-induced exposure of a hydrophobic surface; Smith SP et al.; The dimeric calcium-binding protein S100b is proposed to undergo a calcium-induced structural change allowing it to interact, via a hydrophobic surface, with other proteins . Previously it has been suggested that calcium binding to S100b leads to the exposure of at least one phenylalanine residue (Mani et al., 1982, 1983) . This effect appears to be "reversed" at higher ionic strength, leading to a possible reburying of phenylalanine residues (Mani et al., 1982, 1983) . To study these effects, we monitored calcium binding to recombinant human S100b by NMR spectroscopy under different salt (KCI) conditions . 15N-Labeled glycine residues in S100b showed calcium-induced chemical shift changes similar to those reported for the related monomeric protein calbindin D9k, suggesting similar conformational changes are occurring in the calcium-binding loops of these two proteins . Calcium binding to S100b also resulted in a shifting and broadening of several 1H resonances from the Ca-S100b form only including those from the side chains of residues F14, F70, and F73 but not those of residue Y17 . This broadening was enhanced with increased ionic strength (KCI) . However, small additions ( < 15% v/v) of the hydrophobic solvent trifluoroethanol relieved this phenomenon, leading to narrower line widths . These observations are consistent with the calcium-induced exposure of at least one of these hydrophobic residues, resulting in self-association of the S100b dimer . Trifluoroethanol serves to dissociate these complexes back to the dimeric calcium species . We propose that this cluster of hydrophobic residues which include F14, F73, and F88 may be important for interactions with a target protein. FEBS Lett, 1996 Jul 8, 389(3), 297 - 303 Characterization of human presenilin 1 using N-terminal specific monoclonal antibodies: Evidence that Alzheimer mutations affect proteolytic processing; Mercken M et al.; The majority of cases of early-onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS1) gene, located on chromosome 14 . PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane-spanning domain . We produced 7 monoclonal antibodies that react with 3 non-overlapping epitopes on the N-terminal hydrophilic tail of PS1 . The monoclonal antibodies can detect the full-size PS1 at Mr 47000 and a more abundant Mr 28000 product in membrane extracts from human brain and human cell lines . PC12 cells transiently transfected with PS1 constructs containing two different Alzheimer mutations fail to generate the 28 kDa degradation product in contrast to PC12 cells transfected with wild-type PS1 . Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1. Biochem Biophys Res Commun, 1996 Jul 5, 224(1), 164 - 8 Iron metabolism-related genes and mitochondrial genes are induced during involution of mouse mammary gland; Lee M et al.; To understand molecular mechanisms of mammary gland involution, several clones were isolated after the primary differential screening of a total 40,000 pfu of involution-specific cDNA library, and further characterized . The partial sequences and Northern analysis revealed that iron metabolism-related genes and mitochondrial genes were induced during mammary gland involution . The expression of the lactoferrin gene was induced at involution days 1, 2, and 3 . The expression of ferritin heavy chain gene was induced at involution days 1, 2, 3 and 4 . Cytochrome oxidase subunit 1 and cytochrome oxidase subunit 2 genes were induced at involution days 4 and 7 . The expression of cytochrome b gene was induced at involution day 7 . These results imply that iron metabolism and mitochondrial function may be altered during mammary gland involution. Biochem Biophys Res Commun, 1996 Jul 5, 224(1), 103 - 7 Association of a 14-3-3 protein with CMP-NeuAc:GM1 alpha 2,3-sialyltransferase; Gao L et al.; CMP-NeuAc:GM1 alpha 2,3-sialyltransferase (ST-IV) was purified to homogeneity from rat brain . Microsequencing of the tryptic peptides derived from the purified enzyme revealed two amino acid sequences homologous to the 14-3-3 proteins . A polyclonal antibody was raised against purified ST-IV . A 33 kDa protein was co-immunoprecipitated from rat brain extracts with the anti-(ST-IV) antibody as detected by Western blot analysis . This protein was identified as a subtype of 14-3-3 family by an anti-(14-3-3) antibody . Screening of a rat brain lambda gt11 library using the anti-(ST-IV) antibody resulted in the identification of a cDNA clone coding for the subtype of 14-3-3 protein . These results indicate an association of the 14-3-3 protein with the sialyltransferase . Since the 14-3-3 protein has PKC inhibitor activities and the activity of sialyltransferases is, at least in part, regulated by PKC, the association of the 14-3-3 protein with ST-IV may indicate a role for this protein in the post-translational regulation of the sialyltransferase activity through the processes of phosphorylation and dephosphorylation. Mutat Res, 1996 Jul 5, 354(1), 95 - 101 Specificity of spontaneous and t-butyl hydroperoxide-induced mutations in delta oxyR strains of Escherichia coli differing with respect to the SOS mutagenesis proficiency and to the MutY and MutM functions; Urios A et al.; Mutations induced by oxidative DNA damage appear to occur by two pathways, differing in their dependence on SOS mutagenesis . We have analysed the specificity of mutations produced by each pathway . Base substitutions generating extragenic suppressors were characterized in Trp+ revertants of Escherichia coli strains carrying the trpE65 ochre mutation, which were hypersensitive to oxidative mutagenesis due to a deletion of the oxyR gene . In strain IC3821, containing MucA/B proteins and therefore proficient for SOS mutagenesis, the more frequently scored base substitutions, either spontaneous or induced by t-butyl hydroperoxide (BuOOH), were T:A-A:T transversions, followed by G:C-A:T transitions, while the frequency of G:C-T:A transversions was lower . This SOS-dependent mutability could be promoted by abasic sites . In strains IC3894 (mutY) and IC3981 (mutY mutM), lacking mutagenesis proteins, SOS-independent revertants arose almost exclusively via G:C-T:A transversions probably derived from oxidatively damaged 8-oxoguanine/adenine mispairs . Formation of these mispairs in IC3894 and IC3981 would be enhanced by BuOOH treatment since it caused a significant increase in the revertant number . Strains IC3894 and IC3981 could have a complementary role to that of IC3821 to analyse the mutagenicity and the mutational specificity of oxidants. J Biol Chem, 1996 Jul 5, 271(27), 16000 - 7 Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis; Prasad R et al.; Base excision repair (BER) is a cellular defense mechanism repairing modified bases in DNA . Recently, a G:U repair reaction has been reconstituted with several purified enzymes from Escherichia coli (Dianov, G., and Lindahl, T.(1994) Curr . Biol . 4, 1069-1076) . Using bovine testis crude nuclear extract, we have shown that G:U is repaired efficiently in vitro, and DNA polymerase beta (beta-pol) is responsible for the single nucleotide gap-filling synthesis (Singhal, R . K., Prasad, R., and Wilson, S . H.(1995) J . Biol . Chem . 270, 949-957) . To investigate potential interaction of beta-pol with other BER protein(s), we developed affinity chromatography matrices by cross-linking purified rat beta-pol or antibody against beta-pol to solid supports . Crude nuclear extract from bovine testis was applied to these affinity columns, which were then extensively washed . Proteins that bound specifically to the affinity columns were co-eluted in a complex with beta-pol . This complex had a molecular mass of approximately 180 kDa and was able to conduct the complete uracil-initiated BER reaction . The BER complex contained both beta-pol and DNA ligase I . An antibody to beta-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both beta-pol and DNA ligase I . Furthermore, DNA ligase I and beta-pol were co-immunoprecipitated from the testis nuclear extract with anti beta-pol IgG . Thus, we conclude that beta-pol and DNA ligase I are components of a multiprotein complex that performs BER. J Biol Chem, 1996 Jul 5, 271(27), 16180 - 6 Fluorescence detection of symmetric GroEL14(GroES7)2 heterooligomers involved in protein release during the chaperonin cycle; Torok Z et al.; The GroEL14 chaperonin from Escherichia coli was labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (I-AEDANS), a hydrophobic probe whose fluorescent emission is sensitive to structural changes within the protein . Increasing concentrations of ATP or adenylyl imidodiphosphate but not ADP caused two successive GroES7-dependent changes in the fluorescence intensity of AEDANS-GroEL14, corresponding to the sequential binding of two GroES7 heptamers and the formation of two types of chaperonin heterooligomers, GroEL14GroES7 and GroEL14(GroES7)2 . The binding of thermally denatured malate dehydrogenase (MDH) caused a specific increase in fluorescence intensity of AEDANS-GroEL14 that allowed the direct measurement in solution at equilibrium of ATP- and GroES7-dependent protein release from the chaperonin . Structure/function analysis during the generation of ATP from ADP indicated the following sequence of events: 1) ADP-stabilized MDH-GroEL14GroES7 particles bind newly formed ATP . 2) MDH-GroEL14GroES7 particles bind a second GroES7 . 3) MDH-GroEL14(GroES7)2 particles productively release MDH . 4) Released MDH completes folding . Therefore, the symmetrical GroEL14(GroES7)2 heterooligomer is an intermediate after the formation of which the protein substrate is productively released during the chaperonin-mediated protein folding cycle. J Biol Chem, 1996 Jul 5, 271(27), 16409 - 15 Structural differences in the minimal catalytic domains of the GTPase-activating proteins p120GAP and neurofibromin; Ahmadian MR et al.; The kinetic properties for the enzymatic stimulation of the GTPase reaction of p21(ras) by the two GTPase-activating proteins (GAPs) p120(GAP) and neurofibromin are different . In order to understand these differences and since crystallization attempts have only been successful with truncated fragments, structure/function requirements of the catalytic core of these proteins were investigated . Differences in size of the minimal catalytic domains of these two proteins were found as determined by limited proteolysis . The minimal catalytic domain has a molecular mass of 30 kDa in the case of p120(GAP) and of 26 kDa in the case of neurofibromin . Both catalytic domains contain the homology boxes as well as the residues perfectly conserved among all Ras GAPs . The C termini of these fragments are identical, whereas the N-terminal part of the minimal p120(GAP) domain is 47 amino acids longer . These newly identified minimal catalytic fragments were as active in stimulating GTPase activity toward p21(ras) as the corresponding larger fragments GAP-334 and NF1-333 from which they had been generated via proteolytic digestion . Recently it was postulated that a fragment of 91 amino acids from neurofibromin located outside the conserved domain contains catalytic activity . In our hands this protein is unstable and has no catalytic activity . Thus, we believe that we have defined the true minimal domains of p120(GAP) (GAP-273, residues Met714-His986) and neurofibromin (NF1-230, residues Asp1248-Phe1477), which can be expressed via LMM fusion vectors in Escherichia coli and isolated in high purity. J Biol Chem, 1996 Jul 5, 271(27), 16288 - 93 Cloning of a cDNA encoding an aldehyde dehydrogenase and its expression in Escherichia coli . Recognition of retinal as substrate; Wang X et al.; The biosynthesis of the hormone retinoic acid from retinol (vitamin A) involves two sequential steps, catalyzed by retinol dehydrogenases and retinal dehydrogenases, respectively . This report describes the cloning of a cDNA encoding a heretofore unknown aldehyde dehydrogenase from a rat testis library and its expression in Escherichia coli . This enzyme has been designated retinal dehydrogenase, type II, RalDH(II) . The deduced amino acid sequence of RalDH(II) had the highest identity with mammalian aldehyde dehydrogenases that feature low Km values (microM) for retinal: human ALDH1 (72.2%), rat retinal dehydrogenase, type I (71.5%), bovine retina (72.7%), and mouse AHD-2 (71.5%) . RalDH(II) expressed in E . coli recognizes as substrates free retinal, with a Km of approximately 0.7 microM, and cellular retinol-binding protein-bound retinal, with a Km of approximately 0.2 microM . RalDH(II) also can utilize as substrate retinal generated in situ by microsomal retinol dehydrogenases, from the physiologically most abundant substrate: retinol bound to cellular retinol-binding protein . Rat testis expresses RalDH(II) mRNA most abundantly, followed by (relative to testis): lung (6.7%), brain (6.3%), heart (5.2%), liver (4.4%), and kidney (2.7%) . RalDH(II) does not recognize citral, benzaldehyde, acetaldehyde, and propanal efficiently as substrates, but does metabolize octanal and decanal efficiently . These data support a function for RalDH(II) in the pathway of retinoic acid biogenesis. J Biol Chem, 1996 Jul 5, 271(27), 15905 - 10 An isoleucine to valine substitution in Escherichia coli acyl carrier protein results in a functional protein of decreased molecular radius at elevated pH; Keating DH et al.; Escherichia coli acyl carrier protein (ACP) has been reported to exist in at least two distinct conformers in solution . A novel form of ACP having an increased electrophoretic mobility on polyacrylamide gel electrophoresis was noted previously during work on beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants of E . coli (Jackowski, S., and Rock, C . O.(1987) J . Bacteriol . 169, 1469-1473) . These workers reported that the increased electrophoretic mobility of the ACP from fabF strains occurred irrespective of prosthetic group attachment or the state of acylation of the prosthetic group . Since these workers were unable to detect a difference between the amino acid sequence of the ACP from the fabF mutants and that of wild type ACP, they suggested that the increased electrophoretic mobility was due to an unknown post-translational modification of the polypeptide chain . We have reinvestigated these mutants and report that the increased electrophoretic mobility is due to a mutation within the gene (acpP) that encodes ACP . This mutation results in substitution of isoleucine for valine 43 of ACP . Site-directed mutagenesis of a synthetic ACP gene demonstrated that the amino acid substitution at residue 43 is the cause of the increased electrophoretic mobility . Gel filtration experiments indicated that the increased electrophoretic mobility results from the more compact structure of V43I ACP at high pH . The altered residue lies within the ACP region of greatest conformational lability, and thus the V43I substitution may shift the equilibrium toward the more compact conformation(s) . The disulfide-linked dimer of V43I ACP was readily formed and had an electrophoretic migration greater than the dimer of wild type ACP, suggesting that formation of ACP.ACP dimers does not require structural deformation of the protein. J Biol Chem, 1996 Jul 5, 271(27), 16218 - 26 Identification of the structural and functional domains of MutY, an Escherichia coli DNA mismatch repair enzyme; Manuel RC et al.; The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates . In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization . Proteolysis by trypsin cleaved the enzyme into 26- and 13-kDa fragments . NH2-terminal sequencing showed that the p13 domain begins at Gln226, indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain . The large p26 domain is almost equivalent to the size of endonuclease III . Binding activity of the p26 domain to a DNA substrate containing an A.G mismatch was comparable with that of the intact enzyme . In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2'-deoxyguanine . Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain . The structure predicted by molecular modeling indicates that the region of MutY (Met1-Trp216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin. J Biol Chem, 1996 Jul 5, 271(27), 15942 - 9 Biochemical characterization of p16INK4- and p18-containing complexes in human cell lines; Ragione FD et al.; The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G1 cell cycle phase . One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs . We have evaluated the amount of two such inhibitors, namely p16(INK4) and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins . The results obtained indicated that (i) p18 gene expression, unlike p16(INK4) gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16(INK4) and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase . Moreover, employing the human native partially purified p16(INK4)or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16(INK4) bimolecular complex . Our findings suggest that during the cell division cycle the members of the p16(INK4) protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G1 --> S transition. J Biol Chem, 1996 Jul 5, 271(27), 16294 - 9 Molecular cloning and identification of N-acyl-D-glucosamine 2-epimerase from porcine kidney as a renin-binding protein; Maru I et al.; N-Acetylneuraminic acid (NeuAc) is an important molecule in biological recognition systems . NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) . However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated . To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis, the enzyme and its gene were isolated from porcine kidney cortex . Escherichia coli cells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney . Sequence analysis indicated that the gene was capable of synthesizing a 46.5-kDa protein (402 amino acids) with a conserved leucine zipper motif . Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein (RnBP) in porcine kidney (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y.(1990) J . Biol . Chem . 265, 6556-6561) (identity: 99.6% in nucleotide sequence, 99.0% in amino acid sequence) . That GlcNAc 2-epimerase is a RnBP was confirmed by its ability to bind porcine kidney renin and mask its protease activity . These findings provide unequivocal evidence that the enzyme GlcNAc 2-epimerase is a RnBP. J Biol Chem, 1996 Jul 5, 271(27), 16068 - 74 Escherichia coli contains a protein that is homologous in function and N-terminal sequence to the protein encoded by the nifS gene of Azotobacter vinelandii and that can participate in the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase; Flint DH; In this paper, I report the purification of a protein from Escherichia coli that is very similar in sequence, molecular weight, and the reactions it can catalyze to the protein encoded by the Azotobacter vinelandii nifS gene . This E . coli protein contains pyridoxal phosphate as a cofactor and catalyzes the removal of sulfur from cysteine to form alanine and S0 . When dithiothreitol is present along with cysteine, the S0 formed is reduced to S2- . This protein has a reactive sulfhydryl group that is essential for activity . As isolated, this sulfhydryl group appears to be in a disulfide linkage with the sulfhydryl group from the phosphopantetheine moiety of the acyl carrier protein . The purified E . coli protein can mobilize the sulfur from cysteine and contribute it to the formation of a {4Fe-4S} cluster on the apoprotein of E . coli dihydroxy-acid dehydratase . A mechanism is proposed for the early stages of the synthesis of Fe-S clusters using this protein and sulfur in the S0 oxidation state. J Biol Chem, 1996 Jul 5, 271(27), 16053 - 67 Studies on the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase in escherichia coli crude extract . Isolation of O-acetylserine sulfhydrylases A and B and beta-cystathionase based on their ability to mobilize sulfur from cysteine and to participate in Fe-S cluster synthesis; Flint DH et al.; The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential {4Fe-4S} cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis . The inactive apoprotein could be reactivated in vitro by factors present in the crude extract of E . coli and to a much smaller extent in the presence of Fe3+, S2-, and dithiothreitol . This reactivation occurs as a result of Fe-S cluster synthesis . It is anticipated that the Fe-S cluster synthesis observed in crude extracts in vitro may involve some of the components that participate in Fe-S cluster synthesis in vivo . The origin of the sulfur used to form Fe-S clusters was investigated . Four enzymatic activities in the crude extract of E . coli were found that can provide sulfur for Fe-S cluster synthesis in vitro by mobilizing the sulfur from cysteine . The purification of the proteins responsible for three of these activities is reported in this paper . The three proteins have been identified as O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B, and beta-cystathionase . The rate and extent of sulfide mobilization from cysteine in the reaction catalyzed by O-acetylserine sulfhydrylases A and B depend on the presence of nucleophiles that can add to the aminoacrylate formed on the enzyme following the removal of sulfide from cysteine . A new amino acid is formed when the nucleophiles add to the aminoacrylate . Sulfur mobilization by beta-cystathionase does not require a nucleophile, and the reaction is a minor variation on the cleavage of beta-cystathionine, with pyruvate, ammonia, and sulfide being the products . Once sulfur is mobilized by these enzymes, its efficient use in Fe-S cluster synthesis seems to be affected by the presence of yet unidentified factors present in crude extract . In crude extract and partially purified preparations from E . coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions. Science, 1996 Jul 5, 273(5271), 107 - 9 Mapping of catalytic residues in the RNA polymerase active center; Zaychikov E et al.; When the Mg2+ ion in the catalytic center of Escherichia coli RNA polymerase (RNAP) is replaced with Fe2+, hydroxyl radicals are generated . In the promoter complex, such radicals cleave template DNA near the transcription start site, whereas the beta' subunit is cleaved at a conserved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp) . Substitution of the three aspartate residues with alanine creates a dominant lethal mutation . The mutant RNAP is catalytically inactive but can bind promoters and form an open complex . The mutant fails to support Fe2+-induced cleavage of DNA or protein . Thus, the NAD-FDGD motif is involved in chelation of the active center Mg2+. Biochemistry, 1996 Jul 2, 35(26), 8786 - 93 Thermodynamic stabilization of nucleotide binding to thymidylate synthase by a potent benzoquinazoline folate analogue inhibitor; Chen CH et al.; The stabilization of dUMP, FdUMP, and dGMP binding to Escherichia coli thymidylate synthase (TS) in the presence and absence of a folate analogue inhibitor of TS, 1843U, was determined by differential scanning calorimetry . When the enzyme is thermally unfolded in the presence of dUMP, two separate temperature transitions are evident, although only one binding site/dimer was detected in equilibrium dialysis experiments . In the absence of dUMP, TS shows a major peak of unfolding at 45 degrees C with a shoulder at 47 degrees C . In the presence of increasing amounts of dUMP progressive changes in the size of each peak occur, each associated with a higher temperature of unfolding . At a ratio of dUMP/TS of 100, a major peak predominates with an unfolding temperature (Td) of 60 degrees C . FdUMP shows a similar profile, while dGMP does not alter the Td of the enzyme since dGMP alone does not bind to TS . Despite the fact that 1843U binds tightly to TS in the absence of nucleotide ligands {Dev, I . K., Dallas, W.S., Ferone, R., Hanlon, H., McKee, D.D., & Yates, B . B . (1994) J.Biol . Chem . 269, 1873-1882}, it exhibits only a small effect on the Td profile of TS . However, when 1843U is present, in addition to the nucleotides (dUMP, FdUMP, or dGMP), a Td of 72 degrees C is achieved and the enthalpy of unfolding is increased by one-third . The stabilizing effect of substrate binding to TS by 1843U examined by thermodynamic parameters can be attributed to the considerable extra amount of free energy released on formation of the ternary complex of TS-1843U-nucleotide . The tightness of this complex is due to the stacking energy that results from Van der Waals contacts between the nucleotide purine or pyrimidine ring and the benzoquinazoline ring of 1843U {Weichsel, A., Montfort, W . R., Ciesla, J., & Maley, F . (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 3493-3497}, which induces a local conformational change in the protein . This conformational change is associated with a significant positive entropy change, which suggests that water is expelled from the active site region. Biochemistry, 1996 Jul 2, 35(26), 8610 - 8 Escherichia coli diacylglycerol kinase is an alpha-helical polytopic membrane protein and can spontaneously insert into preformed lipid vesicles; Sanders CR 2nd et al.; Escherichia coli diacylglycerol kinase (DAGK) is a 13.2 kDa enzyme which spans the cytoplasmic membrane three times . Functional DAGK was purified to homogeneity using a polyhistidine tag and Ni(II)-chelate chromatography . Transmission Fourier transform infrared spectroscopy (FT-IR) of DAGK in phosphatidylcholine multilayers led to the conclusion that > or = 90 of DAGK's native 121 residues are alpha-helical, consistent with a model in which DAGK consists of two amphipathic alpha-helices and three transmembrane helices . Polarized attenuated total reflection FT-IR studies of DAGK in oriented multilamellae yielded data consistent with a topological arrangement in which the three transmembrane helices are well-aligned with the bilayer normal while the two amphipathic helices are approximately parallel with the membrane plane . The ability of DAGK to spontaneously insert into preformed lipid vesicles was examined using a novel assay system involving DAGK-catalyzed phosphorylation of a fluorescently tagged diacylglycerol . When micellar DAGK is diluted into L alpha-phase vesicles spontaneous insertion of the enzyme is fairly efficient (ca . 30%) . DAGK refolding and insertion from delipidated urea-solubilized DAGK into lipid vesicles is also modestly efficient (3.8 +/- 2.1%) above the gel to liquid crystalline phase transition temperature . The insertion studies indicate that the difference in energy barriers (delta delta G++) between pathways leading to catalytically productive folding and insertion of DAGK relative to unproductive pathways is < 4 kcal/mol . However, additional studies carried out with mutant forms of DAGK indicated that the differences between refolding/insertion pathways for DAGK in vivo and in vitro can be significant. Biochemistry, 1996 Jul 2, 35(26), 8595 - 602 Inactivation of Escherichia coli ribonucleotide reductase by 2'-deoxy-2'-mercaptouridine 5'-diphosphate . Electron paramagnetic resonance evidence for a transient protein perthiyl radical; Coves J et al.; Ribonucleotide reductase catalyzes a key step in DNA biosynthesis and repair, supplying the cell with the four common deoxyribonucleotides . It is thus the target of antiproliferative agents . The enzyme consists of two subunits named protein R1 and protein R2 . R1 provides the sites for the nucleotide substrates and redox-active cysteines required for catalysis . R2 harbors a tyrosyl radical essential for activity . We show here that 2'-deoxy-2'-mercaptouridine 5'-diphosphate, a substrate analog, is a very efficient inactivator of ribonucleotide reductase (Ki = 35 microM, Kinact = 0.18 s-1) . Inactivation is due to specific scavenging of the protein R2 tyrosyl radical . This unique feature sets this compound apart from other mechanism-based inhibitors such as 2'-azido-or 2'-chloro-2'-deoxyribonucleotide which induce partial or total protein R1 inactivation . During reaction, a transient organic radical was detected by EPR spectroscopy . Its g anisotropy (gz = 2.0620, gy = 2.0265, and gx = 2.0019) and its hyperfine structure are consistent with a perthiyl RSS . radical . The loss of the hyperfine structure by deuterium labeling of the beta protons of R1 cysteines unambiguously shows that the perthiyl radical is located on protein R1 . We thus conclude that inactivation of ribonucleotide reductase by 2'-deoxy-2'-mercaptouridine 5'-diphosphate is due to an irreversible transfer of the radical located on protein R2 to a cysteine residue of protein R1. Biochemistry, 1996 Jul 2, 35(26), 8587 - 94 Functional properties of the histidine-aspartate ion pair of flavocytochrome b2 (L-lactate dehydrogenase): substitution of Asp282 with asparagine; Gondry M et al.; The FMN prosthetic group of flavocytochrome b2 or L-lactate dehydrogenase oxidizes lactate to pyruvate . The reducing equivalents are then transferred one by one, intramolecularly, to heme b2 and then to external acceptors . Substrate oxidation is thought to begin with abstraction of the substrate alpha-hydrogen as a proton by an enzyme base . It has been proposed that this role is played by His373, which lies close to the flavin in the crystal structure and interacts with Asp282 . It has also been shown before, using hydrogen exchange measurements, that the pKa of His373 is substantially increased in the wild-type reduced enzyme compared to that in the oxidized state . We report here the enzymatic properties of the D282N mutant flavocytochrome b2 . Steady-state rate measurements with {2-1H}lactate and {2-2H}-lactate indicate that, as predicted, the Michaelis complex stability is hardly affected, whereas the transition state for proton abstraction increases in energy by 2.8 kcal/mol . Steady-state inhibition studies were conducted with a number of active-site ligands: sulfite, D-lactate, pyruvate, and oxalate . Binding was found to be most affected for oxalate, but kinetic patterns indicated oxalate and pyruvate were still capable of binding to the enzyme both at the oxidized and semiquinone stages, whereas inhibition by excess substrate, due to lactate binding at the semiquinone stage, was lost . Finally, analysis of the intermolecular hydrogen transfer catalyzed by the enzyme between {2-3H}lactate and fluoropyruvate indicated that the substitution with asparagine facilitates exchange of the histidine-bound proton and hence induces a decrease in the pKa value of H373 in the reduced enzyme of about 1.4 pH units . Nevertheless, the rate constant value for exchange with the solvent of the enzyme-bound substrate alpha-proton indicates that H373 is still protonated in the reduced mutant enzyme at neutral pH . Thus, the D282N mutation destabilizes the transition state for proton abstraction and decreases the pKa of H373 in the reduced enzyme but is insufficient to bring it back to a normal value. Vopr Virusol, 1996 Jul-Aug, 41(4), 150 - 3 {Immunochemical properties and specificity of monoclonal antibodies against N- and C-terminal sites of the recombinant protein of the nucleocapsid of hepatitis C virus (HCcAg)}; Masalova OV et al.; Highly affine murine monoclonal antibodies (MAB) to recombinant nucleocapsid (core) protein of hepatitis C virus (rHCcAg) expressed in E . coli were obtained . The MABs were analyzed by solid-phase enzyme immunoassay (EIA), immunodot, immunoblotting, and competitive immunochemical analysis . For estimating the epitope specificity of MAB, several immunoreactive fragments of different length were cloned from the HCcAg region overlapping 160 N-terminal amino acid (a . a.) residues . Use of these fragments and the competitive EIA demonstrated that MAB recognize 4 non-overlapping epitopes, 2 of which are localized in the 1-80 a . a . and 2 other in the 80-150 a . a . regions . A protocol of EIA for detecting HCcAg using MABs to two nonoverlapping HCcAg epitopes has been designed . The sensitivity of double-site sandwich is 1 ng/ml for the recombinant protein. Mol Gen Mikrobiol Virusol, 1996 Jul-Sep, (3), 36 - 40 {Synthesis, cloning, and expression in Escherichia coli cells of human alpha1-antitrypsin cDNA}; Tikunova NV et al.; cDNA coding for the full-length human alpha 1-antitrypsin (AAT) and its leader sequence has been cloned and sequenced . DNA sequences encoding the deletion variants and a full-length copy of AAT were cloned and expressed under trp-promoter control . It has been shown that guanine replacing right upstream and downstream the ATG of deletion variant increases the expression to ten-fold . The synthesis of deletion AAT in E . coli was evaluated immunologically and the level of the synthesis was shown to be 4-5% of total cellular protein. Mol Gen Mikrobiol Virusol, 1996 Jul-Sep, (3), 23 - 6 {Biosynthesis of alpha2-interferon and tumor necrosis factor at different stages of culturing}; Andreeva IS et al.; Study of the biosynthesis of human alpha 2-interferon and tumor necrosis factor in derivatives of E . coli SG200-50 recipient strain containing pIF16, pTNF311, and pLT21 plasmids demonstrated elimination of the plasmids from the cells without degradation thereof in the course of culturing . Methods increasing the content of the end products in the biomass are proposed. Bioorg Khim, 1996 Jul, 22(7), 489 - 502 {Biosynthesis and conformational state of 17-kDa and 27-kDa N-terminal fragments of elongation factor EF-2 in solution}; Plotnikov AN et al.; N-Terminal fragments of the rat liver elongation factor EF-2 containing 162 (17 kDa) and 244 (27 kDa) amino acid residues of 857 (95 kDa) residues of the native protein were synthesized in E . coli cells and in a wheat germ cell-free translation system, and their conformations were studied . Both fragments were synthesized as inclusion bodies (nonspecific molecular aggregates) . The conformations of the fragments in a solution were studied at neutral pH values by CD, fluorescence spectroscopy, scanning microcalorimetry, viscosimetry, gel-filtration, limited proteolysis, and interaction with monospecific anti-EF-2 antibodies and GroEL/ES molecular chaperone . Under nondenaturing conditions, both fragments existed in a solution as associates within a broad range of molecular masses, contained a considerable amount of elements of the intramolecular secondary structure, and represented globules without rigid tertiary structure (molten globules) . A rigid tertiary structure was not formed even after the interaction of the fragments with the GroEL/ES molecular chaperone, thus indicating that the C-terminal fragment is essential for the formation of the rigid tertiary structure . Both fragments contained conformational antigenic determinants similar to those in the whole protein; i.e., despite the absence of the rigid tertiary structure, the fragments contained elements whose structure was similar to that of the corresponding regions in the whole protein. Mikrobiologiia, 1996 Jul-Aug, 65(4), 481 - 7 {Catabolism of methylphosphonic acid and its physiological regulation in Escherichia coli}; Matys SV et al.; It was found that methyl phosphonic acid (Pn) was degraded by different Escherichia coli strains, which utilized it as the sole phosphorus source with resulting methane formation . This ability was influenced by mutations in the regulatory genes of the pho regulon . Thus, Pn was not degraded by an E . coli mutant defective in the regulatory phoB gene, responsible for the induction of pho-regulon proteins during phosphorus starvation . The intensity of Pn degradation depended on the age and concentration of the inoculum . Preincubation of bacteria in the presence of Pn accelerated subsequent degradation of both methyl phosphonic acid and its esters . Cultures developing from a small amount of inoculum degraded Pn more efficiently than heavily inoculated cultures that underwent only one cell division . However, cultures heavily inoculated with adapted cells degraded Pn as efficiently as cultures developing from a small amount of inoculum . Aeration was an important factor regulating Pn degradation: Pn was degraded more efficiently under anaerobic conditions regardless of the amount of inoculum. J Ind Microbiol, 1996 Jul, 17(1), 47 - 52 A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli; Gosset G et al.; Different approaches to increasing carbon commitment to aromatic amino acid biosynthesis were compared in isogenic strains of Escherichia coli . In a strain having a wild-type PEP:glucose phosphotransferase (PTS) system, inactivation of the genes encoding pyruvate kinase (pykA and pykF) resulted in a 3.4 fold increase in carbon flow to aromatic biosynthesis . In a strain already having increased carbon flow to aromatics by virtue of overexpression of the tktA gene (encoding transketolase), the pykA and/or pykf mutations had no effect . A PTS- glucose+ mutant showed a 1.6-fold increase in carbon flow to aromatics compared to the PTS+ control strain . In the PTS- glucose+ host background, overexpression of tktA caused a further 3.7-fold increase in carbon flow, while inactivation of pykA and pykF caused a 5.8-fold increase . When all of the variables tested (PTS-glucose+, pykA, pykF, and overexpressed tktA) were combined in a single strain, a 19.9-fold increase in carbon commitment to aromatic biosynthesis was achieved. Biotechnol Prog, 1996 Jul-Aug, 12(4), 572 - 4 A quantitative immunoassay utilizing Escherichia coli cells possessing surface-expressed single chain Fv molecules; Chen G et al.; A facile, quantitative immunoassay is described that utilizes Escherichia coli (E . coli) bacteria expressing single chain Fv (scFv) antibody fragments attached to the cell surface . A Scatchard analysis demonstrated that the antibodies on the surface of the cells retained full binding activity (Kd = 2.2 x 10(-9) M) and that there are 60,000 scFv molecules per cell . The cells are used as the antibody reagent in the assay, and, following incubation with analyte, simple centrifugation is used to separate the antibody-bound from unbound analyte . The immunoassay is rapid and accurate down to the nanomolar level . In addition, a variety of detection strategies can be used, and the immunoassay is not adversely affected by the presence of animal serum . A key advantage of the new immunoassay is that the antibody reagent can be inexpensively produced in a "ready to use" form by simply growing cultures of the bacteria. Appl Microbiol Biotechnol, 1996 Jul, 45(6), 755 - 63 The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori; van Gemeren IA et al.; A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A . awamori endoxylanase II (exlA) promoter and terminator . The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus . Transformants containing a construct encoding a direct, inframe fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide . The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-pro-sequence . The multicopy strains showed a 6-to 12-fold increased production of extracellular cutinase relative to the single-copy strains . No linear dose response relation to the number of expression cassettes present in the strains was observed . The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion. Exp Eye Res, 1996 Jul, 63(1), 35 - 50 Morphological and physiological consequences of the selective elimination of rod photoreceptors in transgenic mice; McCall MA et al.; We have produced transgenic mice (rdta mice) that express the gene for an attenuated diphtheria toxin under the control of a portion of the rhodopsin promotor . Morphologically, expression of this transgene results in the elimination of the majority of cell bodies in the outer nuclear layer (ONL) of the retina . This cell loss is evident as early as postnatal day 7 (P7), which corresponds closely to the onset of expression of rhodopsin in the mouse retina that occurs about P5 . Reverse transcription-PCR (RT-PCR) analysis of mRNA from the retinae of rdta mice shows that the level of rhodopsin mRNA is reduced by 50% as early as P14 and by P28, has declined to approximately 15% of that in the retinae of control mice . Electroretinographic recordings from the dark-adapted rdta mice at P17 reveal that their retinae do not generate any rod-mediated signals . The majority of the cell bodies that persist in the ONL of the rdta retinae have the morphological features of cone photoreceptors, although these cells never develop normal inner and outer segments . To confirm that the surviving cells are cones we crossed the rdta mice to a different line of transgenic mice that express the E . coli beta-galactosidase (lacZ positive) reporter gene in all cone photoreceptors . In retinae from mice that inherit both transgenes, nearly every cell that remains in the ONL expresses lacZ and, thus, is a cone . This finding also is consistent with RT-PCR analyses, which show that cone opsin mRNAs persist in the retinae of our rdta mice at ages when rhodopsin mRNA is significantly reduced . Electroretinograms can be obtained from the rdta mice under conditions that saturate the rod response and, thus, providing evidence that the cones that remain are functional, even though they lack inner and outer segments . Finally, we have examined the inner nuclear layer for changes that result from rod photorecptors ablation . We show that, while the elimination of the rod photoreceptors has little or no effect on the morphology of the post-synaptic neurons, this deletion does alter their laminar position. Genetika, 1996 Jul, 32(7), 1013 - 6 {Attenuation of type I restriction in Escherichia coli: effect of the ard gene in UV-irradiated cells}; Zavil'gel'skii GB et al.; The effect of conjugative plasmids ColIb-P9 (incI1) and pKM 101 (incN), containing the active ard gene, on the efficiency of EcoK restriction of nonmodified phage lambda .0 in UV-irradiated Escherichia coli cells was studied . ard-Dependent antirestriction enzyme activity was shown to decrease in UV-irradiated cells . The efficiency of action of the ard plasmid gene on lambda .0 was also shown not to depend on cell helicases RecBCD and UvrD, in contrast to the UV-induced alleviation of EcoK restriction (SOS alleviation). Gac Med Mex, 1996 Jul-Aug, 132(4), 367 - 75 {Importance of questioning and physical examination in pediatric clinical diagnosis}; Rendon-Macias ME et al.; In order to determine the certainty of the nosologic as well as the etiologic diagnosis of a complication that a group of physicians can achieve with the use of the interrogatory and physical examination in solving pediatric cases, a comparative survey was carried out with 36 pediatricians . Three clinical cases were chosen (A, B and C) in three different versions (1 = interrogatory, 2 = interrogatory plus physical examination, and 3 = the same plus clinical laboratory tests) . Randomly, the physicians reached a diagnosis in all three cases in any one of its versions . Generally, through interrogatory, 66.6% of the physicians (24/36) reached a nosologic diagnosis, 8.3% (3/26) diagnosed the complication and 41% (15/36) the etiological . Together with the physical examination, the percentages increased to 67.5% (25/ 37), 18.9% (7/37) and 43.2% (16/37), respectively (p < 0.05) . In Case A, the main nosologic diagnosis was reached by 71, 90 and 91% of the physicians, V = 1, 2 and 3 (p = ns) . In Case B, the main nosologic diagnosis was reached by all of the physicians, and in C, 10, 21 and 90% (p < 0.05) of the physicians, respectively . No differences were found in determining the etiologic diagnosis in versions one and two of the three cases . Differences were found in V3 (p < 0.001) . More physicians reached the diagnosis of the complications in Cases A and C . Having previous experience in similar cases allowed for a greater percentage of physicians to reach the main nosologic diagnosis (75%, 18/24 vs . 50%, 6/ 112, p = 0.03) . The interrogatory and the physical examinations in pediatrics continue to be a useful tool, allowing for a certain diagnosis in 70% to 100% of the cases of common ambulatory pathology . Previous experience in similar cases is a determining factor in reaching a correct diagnosis. Plasmid, 1996 Jul, 36(1), 26 - 35 Molecular analysis of the replication elements of the broad-host-range RepA/C replicon; Llanes C et al.; RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1 . The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number . The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa protein (ORF2) (Llanes et al., 1994b) . In this work, using an in vitro transcription/translation system, we detected a polypeptide whose size corresponded well to that of the deduced product of ORF1 . Deletion and insertion mutation analysis showed that ORF1 is essential for replication; it encodes an initiator protein (called RepA) . ORF2 was not essential for replication in Escherichia coli and its function remains to be determined . Using complementation experiments, the replication origin (ori) of RepA/C was defined . The ori was located in a 600-bp fragment downstream from repA, containing 10 direct repeats . To study the control of repA expression, a transcriptional fusion PrepA::lacZ was constructed . Its analysis showed that repA is transcriptionally autoregulated as are most repA genes of replicons controlled by iterons. J Endod, 1996 Jul, 22(7), 358 - 61 Regulation of pulp cell matrix metalloproteinase production by cytokines and lipopolysaccharides; Panagakos FS et al.; Little information is currently known regarding the effects of cytokines and lipopolysaccharides (LPS's) on matrix metalloproteinase (MMP) production by pulp cells in vitro . In this study, human pulp cells (HPC's) and clonal rat pulp cells RPC-C2A were treated with interleukin (IL)-1 alpha, IL-1 beta, tumor necrosis factor (TNF)-alpha, and LPS for 24 h . Conditioned medium and cell lysates were collected and analyzed by gelatin zymography . RPC-C2A cells treated with IL-1 beta and TNF-alpha displayed elevated levels of MMP's in conditioned medium fractions . LPS's at increasing concentrations had a similar effect . HPC's treated with either cytokines or LPS's had no change in the pattern of MMP's produced or secreted in either cellular or conditioned medium fractions . These studies indicate that the effects of cytokines and LPS's on pulp cells are not identical for cells from different species and requires further investigation to clarify these variations. Chirurg, 1996 Jul, 67(7), 754 - 6 {Paranephritic abscess . Local late complication after laparoscopic cholecystectomy}; Sichardt G et al.; Spilled gallstones abandoned intraperitoneally may cause serious complications . The following case history describes a patient who had undergone a laparoscopic cholecystectomy elsewhere 2 years previously, and who had to have a laparotomy because of a pararenal abscess caused by spilled gallstones . fatal sepsis ensued and the patient died on the 6th postoperative day. Biol Chem, 1996 Jul-Aug, 377(7-8), 535 - 8 Characterization of the preprotein translocase of the outer mitochondrial membrane by blue native electrophoresis; Dekker PJ et al.; The mitochondrial outer membrane contains import receptors for nuclear-encoded preproteins and a general import pore responsible for membrane translocation of preproteins . Receptors and the general import pore have been suggested to assemble into a loose complex . However, biochemical characterization of the complex has been limited so far . We report that blue native electrophoresis separates two complexes . One complex of approximately 400 kDa contains the receptor Tom22 and the general import pore component Tom40, the other complex of approximately 120 kDa contains the receptor Tom70 . A preprotein accumulated at the general import pore apparently co-migrates with the larger complex, suggesting the functionality of the complex . We conclude that the translocase of the outer membrane consists of at least two subcomplexes and that blue native electrophoresis will be a powerful tool for biochemical analysis of the complexes. Biol Chem, 1996 Jul-Aug, 377(7-8), 505 - 12 Three extremely thermostable proteins from Sulfolobus and a reappraisal of the 'traffic rules'; Schafer T et al.; Three cytosolic enzymes from the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius (DSM 639) have been investigated: adenylate kinase, pyrophosphatase and superoxide dismutase . The latter was isolated from S . acidocaldarius cells, the others were heterologically overproduced in Escherichia coli . Long-term thermostability, flexibility, catalytic activity, and thermal denaturation were investigated by biochemical and physical methods . Superoxide dismutase is hyperthermostable over several days . The other enzymes have Tm values between 87 degrees C - 98 degrees C depending on conditions and reveal long-term stability in the range of hours . On the basis of sequence alignments, core structures were defined and compared to mesophilic homologues selected by growth temperature of organisms from 25 degrees C to 88 degrees C . The data set confirms none of the simple sequence based 'traffic rules' previously proposed by others . Some aspects of thermostability based on molecular modeling studies are discussed which remain to be proved by the 3D structures . All three enzymes could be crystallized. Ultramicroscopy, 1996 Jul, 63(3-4), 205 - 18 A common-lines based method for determining orientations for N > 3 particle projections simultaneously; Penczek PA et al.; A method is proposed for determining the directions of projections . An arbitrary number of projections of unknown three-dimensional structure are simultaneously used as input . The method is based on common lines and uses a new discrepancy measure accounting for the uneven distribution of common lines in angular space . An application to the 70S Escherichia coli ribosome data obtained from an energy-filtering electron microscope is described. J Protein Chem, 1996 Jul, 15(5), 491 - 9 The carboxy-terminal region of human interleukin-5 is essential for maintenance of tertiary structure but not for dimerization; Proudfoot AE et al.; The C-terminal region of interleukin-5 has previously been suggested to be important for biological activity {Mackenzie et al., (1991), Mol . Immunol . 28, 155-158; Kodama et al . (1991), Biochem . Biophys . Res . Commun . 178, 514-519} . We have investigated this region by making a series of truncation mutants . The proteins were expressed in Escherichia coli, purified from inclusion bodies, and were able to refold with the disulfide homodimeric topology typical of interleukin-5 . Analysis of the truncated carboxy-terminal proteins in an interleukin-5-dependent proliferation assay on TF-1 cells showed a rapid loss of activity as the C-terminal was shortened by more than two amino acids . This loss of biological activity correlated with a drop in binding affinity to both the alpha chain of the receptor and the high-affinity complex consisting of the alpha and beta subunits . Analysis of the proteins by 1H-NMR showed that the truncated mutants have higher exchange rates with solvent, indicating a less rigid structure . The carboxy-terminal region is therefore necessary to maintain the stability of the four-helix bundle and to orient correctly the important residues of the fourth helix . Inspection of the structure determined by X-ray crystallography shows that Trp-110 acts as the major residue in anchoring the fourth helix. J Protein Chem, 1996 Jul, 15(5), 481 - 9 Splase: a new class IIS zinc-finger restriction endonuclease with specificity for Sp1 binding sites; Huang B et al.; A new restriction endonuclease, named Splase, was constructed by genetically fusing the DNA-cleavage domain of the restriction endonuclease Fok1 with the zinc-finger DNA-binding domain of the transcription factor Sp1 . The resulting protein was expressed in Escherichia coli., partially purified, and shown to selectively digest plasmid DNA harboring consensus Sp1 sites . Splase was also shown to selectively digest the long terminal repeat of the HIV-1 DNA at Sp1 sites . Splase recognizes a 10-bp DNA sequence and hydrolyzes phosphodiester bonds upstream of the binding sequence . The binding specificity of Splase makes this a "rare cutter" restriction enzyme which could be valuable in creating large DNA fragments for genome sequencing projects . The result also presents the opportunity to create other restriction enzymes by altering the binding specificity of the zinc-finger recognition helix. J Invest Surg, 1996 Jul-Aug, 9(4), 313 - 9 Secretory phospholipase A2 levels in rat small bowel; Sonnino RE et al.; Preliminary studies on ischemia/reperfusion injury in transplanted small bowel grafts showed that secretory phospholipase A2 (sPLA2) may play a substantial role by breaking down membrane phospholipids . This study sought to determine the normal values of sPLA2 in the rat small bowel as a function of site and length as a baseline for future studies . The entire small bowel of male Lewis rats (200 g) was flushed with normal saline to eliminate solid contents . In group 1, the entire small bowel was divided into 5-cm segments (numbered 1-9), which were snap frozen and processed the same day for sPLA2 . In group 2, a 25-cm segment of bowel (corresponding to segments 2-6 in group 1) was harvested from each animal, snap frozen, and immediately processed for sPLA2 . To assess the effect of bowel storage on enzyme content, group 3 and group 4 grafts were stored for 7 and 14 days, respectively, at -85 degrees C prior to processing . All samples were homogenized in buffer, extracted with H2SO4 and assayed for sPLA2 activity using {1-14C}oleate-labeled autoclaved Escherichia coli as substrate . Results were analyzed statistically by ANOVA . sPLA2 activity rose from 85.46 +/- 14.46% hydrolysis/min fraction-1 in segment 1, to 476.38 +/- 176.75% hydrolysis/min fraction-1 in segment 9 . The increase was linear and statistically significant (p < .0001) . There was no significant difference in enzymatic activity between groups 2, 3, and 4 . Group 2 activity was 263.02 +/- 43.74% hydrolysis/min fraction-1 . This value was not statistically different from the mathematically calculated mean of segments 2-6 in group 1 (237.75) . The results show that (1) sPLA2 activity increases predictably with distance from the ligament of Treitz (2) storage at -85 degrees C does not affect sPLA2, activity, and (3) 25-cm grafts may be evaluated in toto with reproducible baseline enzyme activity . Given the variability of enzyme activity along the course of the rat small bowel, it is imperative that exact location be identified in any studies evaluating sPLA2 activity. Hepatogastroenterology, 1996 Jul-Aug, 43(10), 914 - 8 Influence of endotoxemia on hepatic energy metabolism in rats with obstructive jaundice; Takada T et al.; BACKGROUND/AIMS: A secondary insult in patients with obstructive jaundice can lead to multiple system organ failure . We evaluated the influence of endotoxin on hepatic energy metabolism and hepatic tissue blood flow in obstructive jaundiced rats . MATERIAL AND METHODS: Male Sprague-Dawley rats were divided into a control group, an endotoxin administration group, an obstructive jaundice group, and an obstructive jaundice with endotoxin administration group . To evaluate hepatic energy metabolism, we have measured arterial blood ketone body ratio, and arterial blood total ketone body concentration . Hepatic tissue blood flow was determined by laser Doppler velocimetry . RESULTS: In the endotoxin administration group, no change was observed in hepatic energy metabolism . However, the obstructive jaundice group was associated with decreased hepatic tissue blood flow shortly after the outset of jaundice, while no change was observed in hepatic energy metabolism until 3 weeks later . In the obstructive jaundice with endotoxin administration, a significant decrease in hepatic tissue blood flow and an increase in hepatic energy metabolism were measured . CONCLUSION: Endotoxin administration alone had no influence on hepatic energy metabolism, while endotoxin administration in the presence of obstructive jaundice results in a rapid decrease in hepatic energy metabolism . This occurred as a result of the secondary insult of endotoxin in the setting of decreased hepatic tissue blood flow caused by obstructive jaundice. Avian Dis, 1996 Jul-Sep, 40(3), 690 - 8 Description of cellulitis lesions and associations between cellulitis and other categories of condemnation; Elfadil AA et al.; A total of 110 broiler flocks processed in a single processing plant in southern Ontario were studied for purposes of describing the cellulitis lesions and investigating possible associations between cellulitis and other categories of condemnation at the processing plant . Two hundred and ninety-five carcasses condemned for cellulitis were examined . They came from 65 of the 110 flocks . The lesions tended to be unilateral with most carcasses (87%) having one lesion . The majority of the lesions (92%) were located on the abdomen . Almost 65% of the lesions were large (> or = 8.1 cm2), and 27% were medium (2.1-8.0 cm2) . On the basis of gross appearance, 69% of the lesions were classified as severe, 26% moderate, and 5% mild . Of 149 lesions examined histologically, 74% were classified as chronic, 21% ongoing, and 5% mild-acute . Condemnation data from the 110 broiler flocks were analyzed using Poisson regression . Simple relationships were examined between a count outcome (number of cellulitis-condemned carcasses per flock) and other categories of condemnation and average bird weight . Cellulitis was significantly associated with average bird weight (P = 0.0018), Escherichia coli-related conditions (SEROSITIS; P < or = 0.0001), ascites (P = 0.0004), cyanosis (P < or = 0.0001), valgus varus deformity (P < or = 0.0001), REJECT (combined carcass condemnations for bruising, mutilation, and contamination; P = 0.0003), and the interaction terms "average bird weight and ascites" (AVWT*ASCIT; P < or = 0.0001) and "average bird weight and cyanosis" (AVWT*CYAN; P < or = 0.0001) . Average bird weight, SEROSITIS, ascites, cyanosis, valgus varus deformity, and AVWT*ASCIT were the only significant factors after adjusting for clustering . No association was observed between cellulitis and emaciation and dead on arrival . Variables significantly associated with cellulitis in the multivariate analysis could be considered as potential predictors . These predictors may share common risk factors predisposing broiler chickens to cellulitis. Avian Dis, 1996 Jul-Sep, 40(3), 677 - 89 A prospective study of cellulitis in broiler chickens in southern Ontario; Elfadil AA et al.; This study investigated the associations among cellulitis and hatchery, farm, and abattoir factors . Forty-four broiler flocks from 24 farms located in southern Ontario were followed from hatching to processing . Poisson regression was used to analyze the data . Cellulitis as a count outcome (CELLCOUNT) was significantly associated (P < or = 0.05) with the hatchery of origin, strain of birds, farm size, type of litter, lighting system, total down time, prevalence of abdominal scratches, Escherichia coli-related conditions (SEROSITIS), ascites, and valgus varus deformity . However, only farm size, abdominal scratches, SEROSITIS, ascites, and valgus varus deformity were significant (P < or = 0.05) after adjusting for clustering . No significant associations were found between cellulitis and source of eggs, sex, average bird weight, feed company, growth promoter, or stocking density . Factors significantly associated with cellulitis in this study could be considered as potential risk factors for cellulitis in broiler chickens in southern Ontario. Avian Dis, 1996 Jul-Sep, 40(3), 634 - 44 Divergent antibody responses to vaccines and divergent body weights of chicken lines selected for high and low humoral responsiveness to sheep red blood cells; Parmentier HK et al.; Primary and secondary antibody responses to intramuscularly administered proteins of Eschericia coli (F11), Newcastle disease virus (NCD), infectious bronchitis virus (IB), and infectious bursal disease virus (IBD), respectively, were measured at weekly intervals in two chicken lines . The latter had been divergently selected for high and low antibody responses to sheep red blood cells (SRBC), and in a random-bred control line . An oil-based adjuvant was required to induce primary and secondary antibody responses to NCD, IB, and IBD . With respect to F11, elevated antibody responses were found in birds sensitized and boosted to F11 with and without adjuvant . The humoral response to F11 and to all viral antigens was significantly higher in the high (H) line than in the low (L) line, whereas the control (C) line showed intermediate titers . At 5 and 17 weeks of age, L line birds were significantly heavier than birds of the H and the C lines . A negative phenotypic correlation within lines between body weight at 17 weeks of age and antibody titers at 1 week after sensitization was found, but no further correlations between humoral responses and body weight or growth could be established . The present results suggest that selection for enhanced humoral responsiveness to SRBC resulted in enhanced responsiveness to components of several vaccines . Mechanisms underlying the relationship between divergent selection for immune responsiveness and body weight are discussed. Avian Dis, 1996 Jul-Sep, 40(3), 540 - 5 Lethality, hemagglutination and adhesion of Escherichia coli strains (serotype 01) isolated in Morocco from chickens with colibacillosis; Amara A et al.; Six strains of Escherichia coli, isolated from chickens with colibacillosis and belonging to serotype 01, were studied . All of them were lethal for 1-day-old chicks and were strongly adherent in vitro . Hemagglutination tests showed a strong reaction to horse and chicken erythrocytes . This reaction was inhibited by the addition of 5% D-mannose and also by overnight incubation at 18 C . When the strains were heated at 65 C or at 85 C for 1 hr, agglutination of horse and chicken erythrocytes was conserved only at 65 C . Purification of the subunit of fimbriae by several cycles of solubilization-crystallization using MgCl2 gave good results . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight for one strain was about 18.4 kD. Genet Anal, 1996 Jul, 13(2), 45 - 7 Isolation of genes encoding tRNA binding proteins by probing an expression library with unmodified tRNA; Gustafsson C; A rapid method for isolating Escherichia coli genes encoding proteins which bind unmodified, but not modified, tRNA is described . The method is generally applicable, and can be used to clone many RNA binding proteins where a structured RNA ligand is known. Eur J Surg, 1996 Jul, 162(7), 561 - 5 Quantification of intestinal blood flow by ultrasonic transit time flowmetry in fed and endotoxaemic rats; Haque SM et al.; OBJECTIVE: To compare the dye dilution method with ultrasonic transit time flowmetry (UTTF) for quantifying intestinal blood flow in the same experimental animals . DESIGN: Open experimental study . SETTING: University hospital, Osaka, Japan . MATERIAL: 11 (experiment 1) and 25 (experiment 2) adult male Sprague-Dawley rats . INTERVENTIONS: In experiment 1, the rats were fasted overnight . In experiment 2, endotoxin, Escherichia coli lipopolysaccharide (endotoxin) or saline (sham) was injected intraperitoneally . MAIN OUTCOME MEASURES: Superior mesenteric venous and abdominal aortic blood flow . RESULTS: Experiment 1: intestinal blood flow measured by UTTF was significantly decreased by 17% (p < 0.05) and 56% (p < 0.05) after 30 minutes infusion through the tertiary branch of the mesenteric vein and during simultaneous drawing of blood samples from both the carotid artery and the superior mesenteric vein over a 1.5 minute period, respectively . The intestinal blood flow measurements obtained by UTTF at different intervals before simultaneous drawing of blood from the carotid artery and the superior mesenteric vein differed significantly (p < 0.05) from those obtained by the dye dilution method . Experiment 2: intestinal blood flow was also decreased by 21%-34% (p < 0.05) during similar simultaneous drawing of blood . Aortic blood flow in the endotoxin group was reduced by 66% (p < 0.05) compared with fed animals and 63% (p < 0.05) compared with sham animals . Simultaneously, intestinal blood flow in the endotoxin group was also reduced by 57% (p < 0.05) compared with fed or 48% (p < 0.05) compared with sham treated animals . CONCLUSION: Real intestinal blood flow might not be measured by the procedure of simultaneously drawing blood from the carotid artery and the superior mesenteric vein in rats as is usually done in the dye dilution method . Intraperitoneal endotoxin reduced aortic as well as intestinal blood flow . We propose that UTTF is an alternative method for quantifying intestinal blood flow in fed and endotoxaemic animals. Vaccine, 1996 Jul, 14(10), 949 - 58 Construction, purification and immunogenicity of antigen-antibody-LTB complexes; Green EA et al.; An oligonucleotide, encoding a short epitope peptide tag, termed Pk, was inserted at the 3'-end of the gene coding B-subunit of Escherichia coli heat-labile enterotoxin (LTB) . The presence of the Pk epitope on LTB-Pk was used to construct novel macromolecular assemblies comprising LTB-Pk, an anti-Pk mAb, (mAb SV5-P-k) and Pk-linked recombinant SIV proteins . The 1:1:1 stoichiometry of such complexes was ensured by binding LTB-Pk to one arm of mAb SV5-P-k and an SIV-Pk antigen to the other arm of the antibody . Such SIV-mAb-LTB macromolecular complexes bound to GM1-ganglioside in vitro, and when immunized systemically into mice were highly immunogenic, inducing both humoral and cell-mediated responses to the recombinant SIV antigens. Exp Lung Res, 1996 Jul-Aug, 22(4), 449 - 65 Neutrophil activation and lung injury associated with chronic endotoxemia in rabbits; Klut ME et al.; Chronic endotoxemia produces emphysematous lung destruction in several animal models . The present study was designed to examine changes in the polymorphonuclear leukocytes (PMN) and the lung parenchyma of rabbits that received either saline (control, n = 6) or Escherichia coli endotoxin (LPS, n = 6) 2-3 times weekly for 15 to 28 weeks . Peripheral blood was collected just before and after each intravenous injection and lung tissue was processed at the end of the experiment . PMN myeloperoxidase was stained with diaminobenzidine tetrahydrochloride (DAB)-H2O2, and CD11/CD18 was detected with immunogold . The changes in the PMN and the lung parenchyma were quantitatively analyzed . The results show that each dose of LPS produced an initial fall, followed by a rise in the circulating mature and immature PMN cell counts . Repeated doses of LPS induced PMN activation, degranulation, and an increase in the mean thickness of the alveolar wall (control, 4.1 +/- 0.2; LPS, 5.1 +/- 0.5; p < .05) at 28 weeks without evidence of alveolar septa destruction . Morphometric analysis of intravascular PMN showed an increase in the volume (V) of myeloperoxidase-containing azurophil granules (control, 6.1 +/- 1.3 microns3; LPS, 13.1 +/- 2.8 microns3; p < .05); a trend for a decrease in the V of specific granules (control, 15.8 +/- 3.4 microns3; LPS, 10.2 +/- 1.5 microns3; p = .09); an increase in the V of the cytoplasm (control, 37.3 +/- 6.4 microns3; LPS, 54.5 +/- 7.1 microns3; p < .05); and an increase in CD11/CD18 expressed as the number of gold particles per micrometer of cell surface membrane (G/micron) (control, 7.1 +/- 1.4 G/micron; LPS, 18.1 +/- 7.8 G/micron; p < .05) . The results indicate that chronic endoxemia in rabbits, accelerates the release of PMN from the bone marrow, enhances the retention of both mature and immature activated PMN in the pulmonary microvessels, and causes alveolar wall thickening rather than emphysematous lung destruction. J Perinatol, 1996 Jul-Aug, 16(4), 281 - 4 Pentoxifylline does not delay bacterially induced preterm delivery in rabbits; Walton DL et al.; OBJECTIVE: Our purpose was to determine whether pentoxifylline, a potent cytokine inhibitor, would delay bacterially induced preterm delivery in rabbits . STUDY DESIGN: The study was a randomized, blinded, prospective trial . Twenty-seven rabbits underwent laparotomy . Of these, five (shams) received an intrauterine injection of endotoxin-free water, and the remaining 22 received an injection of 10(5) Escherichia coli into the lower uterine segment . Postoperatively the animals that received the Escherichia coli were divided into two groups . The placebo group (n = 11) received subcutaneous injections of saline solution three times a day and the treatment group (n = 11) received pentoxifylline 20 mg/kg/day in three divided doses . The following parameters were evaluated: (1) time until delivery, (2) time until death, and (3) intrauterine pathologic features . RESULTS: All five of the sham rabbits were delivered at term without any evidence of infection . There were no differences in the preterm delivery rates between the placebo group (eight of 11) and the pentoxifylline-treated group (seven to 11) . However, there was a trend toward prolonging time until death in the treatment group . There were no differences in intrauterine pathologic feature between the placebo and treatment groups . CONCLUSION: Pentoxifylline does not delay Escherichia coli induced preterm delivery in rabbits. J Biochem (Tokyo), 1996 Jul, 120(1), 153 - 9 Human homolog of Drosophila heterochromatin-associated protein 1 (HP1) is a DNA-binding protein which possesses a DNA-binding motif with weak similarity to that of human centromere protein C (CENP-C); Sugimoto K et al.; Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal component tightly associated with the pericentromeric heterochromatic region of fruit fly, mouse, and human throughout the cell cycle . Drosophila HP1 has been shown to be involved in position effect variegation and to be required for the correct chromosome segregation in vivo, while the biological activity of human homolog (HP1Hsa) has not yet been characterized . We previously reported that human CENP-B and CENP-C, two major centromere heterochromatin autoantigens often recognized by autosera in scleroderma patients, possess DNA-binding activity in vitro . Here, we show that human HP1, which is also an autoantigen targeted by some types of anticentromere autosera, is a DNA-binding protein . Human HP1 was expressed as a GST-fusion in Escherichia coli and purified with glutathione-Sepharose . The DNA-binding activity of the recombinant HP1 was demonstrated by gel mobility shift assay and South-Western-type blotting . The minimum DNA-binding region was further limited to the internal 64-amino acid stretch that is less-conserved between human and fruit fly but retains a helix-enriched motif with weak similarity to CENP-C . This suggests that HP1 is involved in the pericentromeric heterochromatin formation by directly associating with genomic DNA. J Biochem (Tokyo), 1996 Jul, 120(1), 14 - 7 Three-dimensional structure of porcine kidney D-amino acid oxidase at 3.0 A resolution; Mizutani H et al.; The X-ray crystallographic structure of porcine kidney D-amino acid oxidase, which had been expressed in Escherichia coli transformed with a vector containing DAO cDNA, was determined by the isomorphous replacement method for the complex form with benzoate . The known amino acid sequence, FAD and benzoate were fitted to an electron density map of 3.0 A resolution with an R-factor of 21.0% . The overall dimeric structure exhibits an elongated ellipsoidal framework . The prosthetic group, FAD, was found to be in an extended conformation, the isoalloxazine ring being buried in the protein core . The ADP moiety of FAD was located in the typical beta alpha beta dinucleotide binding motif, with the alpha-helix dipole stabilizing the pyrophosphate negative charge . The substrate analog, benzoate, is located on the re-face of the isoalloxazine ring, while the si-face is blocked by hydrophobic residues . The carboxylate group of benzoate is ion-paired with the Arg283 side chain and is within interacting distance with the hydroxy moiety of Tyr228 . The phenol ring of Tyr224 is located just above the benzene ring of benzoate, implying the importance of this residue for catalysis . There is no positive charge or alpha-helix dipole near N(1) of flavin . Hydrogen bonds were observed at C(2) = O, N(3)-H, C(4) = O, and N(5) of the flavin ring. Exp Nephrol, 1996 Jul-Aug, 4(4), 222 - 30 Lipopolysaccharide-induced glomerulonephritis develops in the absence of interferon-gamma signaling; Haas C et al.; IFN gamma is a costimulator of macrophage activation and it plays an important role as a proinflammatory cytokine by upregulation of adhesion molecules and MHC antigens . In this study we tested the role of IFN gamma in a model of endotoxin-induced glomerulonephritis . A systemic lupus-like disease was induced by injection of 50 micrograms bacterial LPS twice a week for 4 weeks in wild-type and in IFN gamma receptor-deficient (IFN gamma R-/-) mice . The renal cortex was examined by immunofluorescence and by light microscopy . LPS treatment induced an increase in serum levels of IgG and anti-dsDNA antibodies . A mild glomerulonephritis was characterized morphologically, but proteinuria was not observed . The main histological features of glomerulonephritis were an increase in ICAM-1 expression, deposition of immune complexes and of complement in the glomeruli, increased mesangial matrix and mesangial hypercellularity . The number of intraglomerular leukocytes, detected by MHC class-II and LFA-1 expression increased roughly 4-fold . All those alterations took place in a similar manner in wild-type and in IFN gamma R-/-mice . Therefore it is concluded that IFN gamma does not play an important role in the development of endotoxic glomerulonephritis. Cell Calcium, 1996 Jul, 20(1), 63 - 72 The calcium-binding protein calretinin-22k, an alternative splicing product of the calretinin gene is expressed in several colon adeno carcinoma cell lines; Gander JC et al.; An alternatively spliced mRNA for the calcium-binding protein calretinin (CR) is present in the colon adenocarcinoma cell line WiDr . As a consequence of a frame shift, the resulting protein, calretinin-22k (CR-22k), consists of the first 178 amino acids of calretinin followed by a carboxy-terminal peptide of 14 amino acids that is not present in full-length calretinin . Antibodies specific for this C-terminal region have been generated by 2 different methods . A peptide corresponding to the specific C-terminal region of CR-22k was either chemically synthesized and coupled to a carrier protein or was expressed in Escherichia coli as a carboxyterminal fusion to a carrier protein applying recombinant techniques . Both antisera produced in rabbits were tested in Western blots and immuno-histochemical experiments . The antisera recognized human recombinant CR-22k overexpressed in E . coli, but not fulllength calretinin and stained fixed WiDr cells . The presence of CR-22k was also confirmed in the colon cell lines CO115/3 in which mRNA coding for CR-22k mRNA coding for CR-22k mRNA is present as well as in the lines COLO205 and LS-180, all of which also express full-length calretinin . Although the intracellular distribution of CR-22k and CR are similar as evidenced by immunohistochemical stainings, CR-22k is preferentially localized in the nucleus in the cell lines LS-180 and Co115/3 suggesting potentially different roles for the two proteins. Mol Microbiol, 1996 Jul, 21(2), 409 - 19 Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolK, nodZ as well as noeC and/or downstream genes; Mergaert P et al.; The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon . The A . caulinodans wild-type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D-arabinosyl and/or an L-fucosyl branch . Nod factors produced by Tn5-insertion mutants in nodZ, noeC, and the separate nolK locus, were analysed by thin-layer chromatography and mass spectrometry . Fucosylation of Nod factors depended on both nodZ and nolK . Arabinosylation depended on noeC and/or downstream genes . Protein extracts of A . caulinodans contained an enzymatic activity for fucose transfer from GDP-fucose to chitooligosaccharides and to Nod factors . By mutant analysis and expression of nodZ in Escherichia coli, the fucosyltransferase activity was ascribed to the protein encoded by nodZ . In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A . caulinodans . Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP-fucose. Mol Microbiol, 1996 Jul, 21(2), 361 - 72 The coupling between ftsZ transcription and initiation of DNA replication is not mediated by the DnaA-boxes upstream of ftsZ or by DnaA; Smith RW et al.; The DnaA protein of Escherichia coli is a multi-functional protein which, In addition to promoting initiation of replication, can regulate the initiation or termination of transcription of a variety of genes . It acts by binding to DNA at a defined sequence, termed a DnaA-box . Three candidate DnaA-boxes which occur within the essential cell-division genes, ftsQ and ftsA, have been hypothesized to mediate the response of the downstream ftsZ gene to intracellular levels of DnaA, and thus to couple the processes of initiation and cell division . We show here that, although transcription from promoters upstream of ftsZ is increased when initiation of chromosome replication is blocked by DnaA inactivation, this response is not mediated by the DnaA-boxes near these promoters, nor is it specific to DnaA . We show, furthermore, that mutational inactivation of the putative DnaA-binding sites in the fts region of the chromosome does not lead to impaired growth or reduced survival of cells. Mol Microbiol, 1996 Jul, 21(2), 347 - 60 Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo; Sussman JK et al.; In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene (tnp), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3) . Several lines of evidence led us to conclude that transposase expression, per se, was not increased . Rather, these mutations appear to increase expression of the tnp'-'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons . To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons . Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide . These potential start codons fall into distinct classes with regard to translation efficiency in vivo: Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1-3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification, importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3-5-fold, and this same effect was seen in other gene contexts . Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo, consistent with earlier in vitro observations . We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function. Mol Microbiol, 1996 Jul, 21(2), 331 - 46 The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli; Sacerdot C et al.; The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e . the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type . The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo . In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl-tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background . The same result was obtained with the rpsO gene encoding ribosomal protein S15 . We also show that derepression of infC, thrS, and rpsO is obtained with other 'abnormal' initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency . Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other 'abnormal' initiation codons . Under the same conditions and with the same set of 'abnormal' initiation codons, the repression of thrS and rpsO expression is weaker . This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3 . We also studied another peculiarity of the infC message, namely the role of a GC-rich sequence located immediately downstream of the initiation codon and conserved through evolution . This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation . Unexpectedly, mutating this GC-rich sequence increases infC expression, indicating that this sequence has no enhancing role . Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC-rich sequence might pair with another region of the mRNA . On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between 'normal' and 'abnormal' initiation codons. Mol Microbiol, 1996 Jul, 21(2), 301 - 11 A comprehensive set of DnaA-box mutations in the replication origin, oriC, of Escherichia coli; Langer U et al.; We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1-R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes . For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype . This demonstrates that all DnaA boxes in oriC have a function in the initiation process . Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency . Inversion or scrambling of DnaA boxes R1 or M inactivated oriC-dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites . In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations . We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC . Mutants in DnaA box R3 behaved essentially like wild-type oriC, except for those in which the low-affinity box R3 was replaced by the high-affinity box R1 . Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation . Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo. Mol Microbiol, 1996 Jul, 21(2), 281 - 92 The clp (CS31A) operon is negatively controlled by Lrp, ClpB, and L-alanine at the transcriptional level; Martin C; Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase-variation control and repressed by L-alanine . Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, revealed the presence of a regulatory gene, clpB . The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa-3 fimbriae, respectively . The clp regulatory region contained two deoxyadenosine methylase sites (GATC-I and GATC-II) . The leucine-responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC-II site . The activity of the clp promoter was monitored in a clp-lacZYA single-copy fusion . The cloned DNA used in this study did not contain a related papl gene . In these conditions, we showed, as expected, that phase variation did not occur . However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase . In the presence of AfaF, a Papl equivalent, the phase-variation control was restored . We concluded that two regulatory mechanisms were superimposed to control the clp expression . Phase variation, mediated by Lrp and a Papl equivalent, controlled the number of cells producing CS31A in a single colony . The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell . Furthermore, we showed that L-alanine reduced, by about twofold, the clp promoter activity independently of a Papl equivalent, ClpB, Lrp or Dam methylase . In addition, the presence of L-alanine prevented the phase-variation control mediated by AfaF. Mol Microbiol, 1996 Jul, 21(2), 257 - 66 Definition of a consensus DNA-binding site for the Escherichia coli pleiotropic regulatory protein, FruR; Negre D et al.; The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism . The sequence determinants of the FruR-binding site were analysed by using 6x His-tagged FruR and a series of double-stranded randomized oligonucleotides . FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction-assisted binding-site selection method (BSS) using nitrocellulose-immobilized DNA-binding protein . FruR was demonstrated to require, for binding, an 8 bp left half-site motif and a 3 bp conserved right half-site with the following sequence: 5'-GNNGAATC/GNT-3' . In this sequence, the left half-site AATC/ consensus tetranucleotide is a typical motif of the DNA-binding site of the regulators of the GalR-Lacl family . On the other hand, the high degree of degeneracy found in the right half-site of this palindrome-like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half-sites of its operator . However, potentially FruR-target sites showing a high degree of symmetry were detected in 13 genes/operons . Among these, we have focused our interest on the pfkA gene, encoding phosphofructo-kinase-1, which is negatively regulated by FruR. Mol Microbiol, 1996 Jul, 21(2), 213 - 9 Three, four or more: the translational stop signal at length; Tate WP et al.; Translational stop signals are defined in the genetic code as UAA, UAG and UGA, although the mechanism of their decoding via protein factors is clearly different from that of the other codons . There are strong biases in the upstream and downstream nucleotides surrounding stop codons . Experimental tests have shown that termination-signal strength is strongly influenced by the identity of the nucleotide immediately downstream of the codon (+4), with a correlation between the strength of this four-base signal and its occurrence at termination sites . The +4 nucleotide and other biases downstream of the stop codon may reflect sites of contact between the release factor and the mRNA, whereas upstream biases may be due to coding restrictions, with the release factor perhaps recognizing the final tRNA and the last two amino acids of the polypeptide undergoing synthesis . This means that the translational stop signal is probably larger than the triplet codon, but its exact length will be clearer when it is known which nucleotides are in direct contact with the release factor . Ultimately it will be defined exactly when a crystal structure of the release factor with its recognition substrate becomes available. In Vitro Cell Dev Biol Anim, 1996 Jul-Aug, 32(7), 446 - 50 Mycobacterium tuberculosis heat shock protein 10 increases both proliferation and death in mouse P19 teratocarcinoma cells; Galli G et al.; Exogenously added Mycobacterium tuberculosis Hsp10, either synthetic or recombinant, but not other related heat shock proteins (GroES from Escherichia coli or bovine Ubiquitin), increases apoptosis in serum-deprived P19 mouse teratocarcinoma cells . The effect is dose-dependent, with a bell-shaped curve and peak activity at 10(-9) M (maximal effect: 62.9 +/- 17.7% increase, mean +/- SD, n = 10) and is specifically inhibited by a polyclonal antibody raised against the synthetic protein . On the other hand, when the same cells are exponentially growing, M . tuberculosis Hsp10 increases cell proliferation with a bell-shaped dose-response curve and a moderate decrease in potency (peak-activity at 10(-8)-10(-7) M, with a 43.7 +/- 8.1% increase, mean +/- SD, n = 3) . Therefore, it appears that this bacterial protein can exert two opposite effects, behaving either as a death- or as a growth-promoting factor, depending on the conditions of the target. FEMS Immunol Med Microbiol, 1996 Jul, 14(4), 231 - 44 Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli; Garcia-Martinez J et al.; Molecular methods allow an extremely fine strain typing that can be used to establish the population structure of bacterial species . This methodology has been used to characterize a collection of 74 uropathogenic Escherichia coli obtained from three hospitals located in geographically distant towns in Spain, some representatives of the ECOR collection and other reference strains . Genomic DNA was analyzed by RAPD (Random Amplified Polymorphic DNA) that can characterize a bacterial strain to the level of defining individual clones . The 16S rDNA-23S rDNA spacers were amplified by PCR and submitted to restriction analysis . Finally, the presence or absence of G adhesins in Escherichia coli as well as the type of adhesin (three types are known) have been shown by PCR amplification followed by digestion with restriction enzymes . As expected a wide diversity was shown by RAPD and identical patterns were only found in the case of strains isolated from the same individual, an obvious case of relapse . Analysis of the spacers' restriction patterns showed the presence of two markedly differentiated clusters that we have named alpha and beta . Both RAPD and spacer restriction patterns originated similar clusters of strains showing a consistency in the evolution of the global genome with the sequence variation of the ribosomal spacers . Furthermore, most of the strains having G-adhesin, with only a few exceptions, corresponded to the alpha rRNA spacer group . The two spacer types detected were also consistent with some phenotypic markers such as sucrose and raffinose utilization . The alpha and beta clusters could be intraspecific groups produced by partial sexual isolation or other barriers that are originating a divergent evolution. J Appl Toxicol, 1996 Jul-Aug, 16(4), 309 - 15 Nitric oxide (NO) synthase activity in the lung and NO synthesis in alveolar macrophages of rats increased on exposure to asbestos; Iguchi H et al.; We examined whether the lung of rats induced nitric oxide synthase (NOS) on instillation of Union Internationale Contre le Cancer (UICC) asbestos into the trachea, and whether alveolar macrophages (M phi s) of rats increased synthesis of nitric oxide (NO) on exposure to the asbestos . Instillation of chrysotile asbestos (CH) into rat tracheas increased NOS activity in the lung up to fourfold compared with that of normal control rats . On instillation of amosite asbestos (AM), the activity did not significantly increase . In the culture of alveolar M phi s with CH, NO2-, a stable oxidation product of NO, in the conditioned medium markedly increased while in the culture with AM or crocidolite (CR) asbestos it was comparable to that in the control culture without asbestos . Thus, it was assumed that the lung as well as alveolar M phi s induced NOS on exposure to CH . Viability of M phi s was lower in the culture with CH than in the culture with AM or CR . When alveolar M phi s were cultured in the presence of lipopolysaccharide (LPS), a potent inducer of NOS, NO2- in the medium dramatically increased, indicating a possible induction of NOS, while NO2- was decreased significantly in the presence of both LPS and CH, AM or CR . On the other hand, when M phi s which had previously been stimulated with LPS were cultured in LPS-free medium, NO2- synthesis retained a significantly increased level, irrespective of the presence or absence of asbestos . Thus, the three kinds of asbestos used seemed to interfere with LPS in inducing NOS and to decrease subsequently the synthesis of NO2-, but not to affect the synthesis of NO2- by LPS-induced NOS . Taken altogether, lung or alveolar M phi s of rats induced NOS to increase NO synthesis on exposure to CH. Cancer Gene Ther, 1996 Jul-Aug, 3(4), 245 - 9 High levels of gene transduction in human lung tumors following intralesional injection of recombinant adenovirus; Cusack JC et al.; To effect gene transfer into large solid malignancies for the purpose of clinical application, new treatment strategies using intralesional administration of adenovirus were studied . Replication-deficient adenovirus Ad5LacZ, containing the Escherichia coli beta-galactosidase (beta-gal) gene (LacZ), was injected directly into 1-cm x 1-cm subcutaneous xenograph tumors of human large cell lung cancers (H460 and H1299) . Each tumor received a single injection or three injections of purified virus, diluted in 200 microL of phosphate-buffered saline . The tumors were harvested 3 days after the last injection, serially sectioned, and stained with X-gal . The cells expressing beta-gal were counted by using digital image analysis and the percentage of tumor cells transduced was calculated . After a single viral injection of 1 x 10(9) PFU, 5 x 10(9) PFU, or 1 x 10(10) PFU solid tumor transduction increased significantly with dose escalation . At a dose of 1 x 10(10) PFU, transduction of the H1299 and H460 tumors was 80.2% and 46.7%, respectively . Dividing the viral dose into three injections given on alternating days had no significant effect on viral transduction . These data demonstrate that a large portion of an established human lung cancer cell line tumor undergoes gene transduction after a single intralesional injection of recombinant adenovirus. Bioconjug Chem, 1996 Jul-Aug, 7(4), 413 - 20 Optimizing the targeted chemical nuclease activity of 1,10-phenanthroline-copper by ligand modification; Gallagher J et al.; Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins . Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1,10-phenanthroline, we have compared the activity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-OP) . Tethering MOP derivatives to the Escherichia coli Fis protein enhances DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP . However, scission efficiency is not increased at the stronger cleavage sites, or when scission is targeted to single-stranded DNA by a complementary oligonucleotide . These results are consistent with a change in the rate-determining step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant . Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neocuproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complementary DNA sequence . These results establish that the nucleolytic species in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-oxo intermediate rather than a metal-coordinated peroxy species . However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of the phenanthroline-copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for cleavage of single-stranded regions and the failure of neocuproine-DNA-binding protein chimeras and a C2-tethered chimera to cleave DNA. Proteins, 1996 Jul, 25(3), 300 - 14 Identification and analysis of long-range electrostatic effects in proteins by computer modeling:aspartate transcarbamylase; Oberoi H et al.; While ion pairs are readily identified in crystal structures, longer range electrostatic interactions cannot be identified from the three-dimensional structure alone . These interactions are likely to be important in large, multisubunit proteins that are regulated by allosteric interactions . In this paper, we show that these interactions are readily detected by electrostatic modeling, using, as an example, unliganded Escherichia coli aspartate transcarbamylase, a widely studied allosteric enzyme with 12 subunits and a molecular weight of 310 kD . The Born, dipolar, and site-site interaction terms of the free energy of protonation of the 810 titratable sites in the holoenzyme were calculated using the multigrid solution of the nonlinear Poisson-Boltzmann equation . Calculated titration curves are in good agreement with experimental titration curves, and the structural asymmetry observed in the crystal structure is readily apparent in the calculated free energies and pK1/2 values . Most of the residues with pK1/2 values that differ substantially from those of model compounds are buried in the low dielectric medium of the protein, particularly at the intersubunit interfaces . The dependence of the site-site interaction free energies on distance is complex, with a steep dependence at distances less than 5 A and a more shallow dependence at longer distances . Interactions over distances of 6 to 15 A require a bridging residue and are often not apparent in the structure . The network of interactions between ionizable groups extends across and between subunits and provides a potential mechanism for transmitting long-range structural effects and allosteric signals. Protein Eng, 1996 Jul, 9(7), 555 - 8 The influence of Glu44 and Glu56 of cytochrome b5 on the protein structure and interaction with cytochrome c; Sun YL et al.; The gene encoding trypsin-solubilized bovine liver microsomal cytochrome b5 (82 residues in length) has been mutated, in which the codons of Glu44 and Glu56 were changed to those of Ala . The mutated genes were expressed in Escherichia coli successfully and three mutant proteins (E44A, E56A and E44/56A) were obtained . The UV-visible, CD and 1H NMR spectra of proteins have been studied . The results show that the mutagenesis at surface residues does not alter the secondary and tertiary structures of cytochrome b5 significantly . The interactions between recombinant cytochrome b5 and its mutants with cytochrome c were studied by using optical difference spectra . The results demonstrated that both Glu44 and Glu56 of cytochrome b5 participate in the formation of a complex between cytochrome b5 and cytochrome c. J Vet Diagn Invest, 1996 Jul, 8(3), 345 - 50 Production of gamma-interferon by peripheral blood mononuclear cells: an important diagnostic tool for detection of subclinical paratuberculosis; Stabel JR; Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne's disease) . Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutinin-P (PHAP), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide . In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M . paratu-berculosis at 10:1 bacteria: cell ratio . After incubation, cell-free supernatants were analyzed for gamma-interferon (gamma-IFN) production . Cells from subclinical cows produced significantly higher levels of gamma-IFN than did cells from clinical animals after stimulation with mitogens ConA, PHAP, and PWM . Levels of gamma-IFN produced by noninfected control animals generally followed the pattern of those of subclinical animals . After incubation with MpS, significantly greater quantities of gamma-IFN were produced by cells isolated from subclinical animals than by cells from clinical cows and noninfected controls . Stimulation of cells with heat-killed or live M . paratuberculosis evoked a similar response . This study indicates that gamma-IFN production by peripheral blood mononuclear cells in response to M . paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinically affected animals. Mol Microbiol, 1996 Jul, 21(1), 133 - 46 An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli; van Heeswijk WC et al.; The PII protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity . Here we show that PII, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain . We describe a new operon, glnK amtB, which encodes a homologue of PII and a putative ammonia transporter . We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as PII, reacted with polyclonal PII antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli PII . Like PII, purified GlnK can activate the adenylylation of glutamine synthetase in vitro, and, in vivo, the GlnK protein is uridylylated in a glnD-dependent fashion . Unlike PII, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia . Because of a NRI and a sigma N (sigma 54) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two-component regulatory system . Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on PII . Possible regulatory implications of this conditional redundancy of PII are discussed. Mol Microbiol, 1996 Jul, 21(1), 77 - 96 A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome; Redenbach M et al.; A Supercos-1 library carrying chromosomal DNA of a plasmid-free derivative of Streptomyces coelicolor A3(2) was organized into an ordered encyclopaedia of overlapping clones by hybridization . The minimum set of overlapping clones representing the entire chromosome (with three short gaps) consists of 319 cosmids . The average insert size is 37.5 kb and the set of clones therefore divides the chromosome into 637 alternating unique and overlapping segments which have an average length of approx . 12.5 kb . More than 170 genes, gene clusters and other genetic markers were mapped to their specific segment by hybridization to the encyclopaedia . Genes could be cloned by direct transformation and complementation of S . coelicolor mutants with cosmids isolated from Escherichia coli, selecting for insertion into the chromosome by homologous recombination . As in other streptomycetes, the ends of the chromosome have long terminal inverted repeats. Mol Microbiol, 1996 Jul, 21(1), 69 - 76 Modulation of lambda integrase synthesis by rare arginine tRNA; Zahn K et al.; Lambda's int gene contains an anomalously high frequency of the rare arginine codons AGA and AGG when compared to genes of Escherichia coli or to the rest of phage lambda . These are the least frequent codons in genes of E . coli and are recognized by the rarest tRNAs . The presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms . In this study, we show that expression of the natural int gene may also be modulated by rare arginine codon usage, and we explore this mechanism. Mol Microbiol, 1996 Jul, 21(1), 55 - 68 In vitro analysis of mRNA processing by RNase E in the pap operon of Escherichia coli; Naureckiene S et al.; Differential gene expression from operons encoding fimbrial adhesins in Escherichia coli involves processing and differential decay of polycistronic transcripts . Previous analyses of mRNA processing in vivo using ribonuclease mutants of E . coli have given different results with the different fimbrial gene systems tested . For the pap operon from uropathogenic E . coli, the results suggested that the mRNA processing is dependent on ribonuclease E (RNase E), whereas in other fimbrial operons with similar genetic organisation, the processing was concluded to be RNase E independent . We have developed an in vitro system allowing us to assess the cleavage of pap mRNA, to study the mRNA processing of a fimbrial operon in more detail, and to define the enzymatic activity and target . The results of this study establish that RNase E does indeed cleave the papBA intercistronic transcript . Analysis of the cleavage products reveals that in vitro RNase E can cleave the mRNA at other positions in addition to the site preferentially cleaved in vivo . The specificity of the cleavage pattern was assessed using transcripts derived from mutants with base substitutions near, or within, the major in vivo cleavage site . Such mutants have alternative cleavage sites . A common feature of the different cleavage sites is a high A/U nucleotide content, similar to other known RNase E cleavage sites . Features of the secondary structure of the papBA intercistronic mRNA were investigated using single-strand-specific and double-strand-specific nucleases . The secondary structure model derived from stability calculations and our results from the nuclease-probing experiments indicate that the positions subject to RNase E cleavage are mainly single stranded and flanked by more stable stem-loop structures . The results are consistent with the notion that an mRNA conformation exposing A/U-rich, non-paired regions constitutes the target, i.e . a flexible determinant, for processing by RNase E in the pap transcript . The findings are discussed in relation to the existence of a potential recognition site for RNase E and the analysis of RNase E cleavages in other RNA molecules. Biochem Mol Biol Int, 1996 Jul, 39(4), 833 - 42 Cloning and characterization of cDNA for human adenylate kinase 2A; Lee Y et al.; We have isolated and characterized a cDNA clone encoding human adenylate kinase 2A (AK2A) from cDNA libraries of fetal liver origin . The complete nucleotide sequence of the cloned 889 nucleotide cDNA fragment indicated that the deduced gene product of human AK2A is composed of 239 amino acids with a molecular weight of 26 kD . Comparison of these nucleotide and amino acid sequences with the corresponding sequences of bovine AK2A and rat AK2 revealed a high degree of conservation . It showed 91% and 98% homologies in DNA and amino acid sequences, respectively, with bovine AK2A throughout the full open reading frame . RNA blot analysis revealed that three species of mRNA were present with approximate sizes of 3.4, 2.1, and 1.0 kb . This gene was expressed in E . coli cells and the recombinant protein was enzymatically active. Biochem Mol Biol Int, 1996 Jul, 39(4), 789 - 95 Development of a metallothionein based heavy metal biosorbent; Pazirandeh M; The potential utility of a recombinant E . coli expressing the Neurospora crassa metallothionein gene (NCP) as a heavy metal biosorbent was investigated . It was shown that the NCP was capable of efficiently removing low levels of several metals (including cadmium, lead, and mercury) from solutions . The reusability of the NCP was demonstrated through 5 cycles of metal binding, stripping with dilute acid, and regeneration of the binding sites with out any adverse effect on the metal binding activity . The NCP was successfully encapsulated in alginate and acrylamide without any inhibitory effect on its metal uptake activity . Furthermore, the metal uptake activity of the NCP was shown to be metabolism independent and resistant to solvents and other compounds (eg . polyaromatic hydrocarbons) which are often present along with heavy metals in waste waters thereby creating the potential for non-viable, encapsulated cells to be used. Biochem Mol Biol Int, 1996 Jul, 39(4), 657 - 64 Structure of the N-terminus of the Escherichia coli ribosomal protein L7/L12; Todorova RT; Three mutant forms of the ribosomal protein L7/L12 (S1Y, M14Y and M26Y) were constructed in order to use the Tyr residues as internal labels at the N-terminal region . The proteins were studied by the methods of 1H-NMR, Fluorescence, Circular dichroism spectroscopy and Differencial scanning calorimetry . The secondary structure of N-terminus (residues 1-36) was predicted by the programme "Globule" {1, 2} . Y1 is located in a structurally disordered region at the free end of the molecule . M14 and M26 are located in structurally ordered regions . The replacement M14 does not change the structure of the protein . The replacement M26 disturbs the helicity in the region of the mutation . The first 1-5 positions from the N-terminus of L7/L12 are disordered, the position 7-14 participate in an alpha-helical/beta-structural region and position 19-30 in a strongly alpha-helical region. New Microbiol, 1996 Jul, 19(3), 211 - 20 Simpler peptidoglycan chemical composition in the highly transformant Escherichia coli DH5 alpha strain; Signoretto C et al.; Artificial transformation of Escherichia coli is obtainable by treating the culture with CA2+ and other substances known to increase the permeability of the outer membrane . Nevertheless, particular strains of E . coli are more useful for transformation since the number of transformants obtained is far higher . We postulate that an additional layer of the envelope may play an important role comparable to that of the outer membrane . The chemical composition of the peptidoglycan of a highly efficient transformant E . coli strain (DH5 alpha) was analyzed in comparison with a normal and poorly transformant E . coli strain (KN126) revealing a simpler peptidoglycan chemical composition in the DH5 alpha strain . This may be responsible for the simpler architecture of the peptidoglycan which, in turn, may interfere less with the passage of the DNA across the bacterial envelope. Biol Pharm Bull, 1996 Jul, 19(7), 922 - 31 Influence of the linking number of template DNA on sigma 32-dependent transcription initiation in vitro of the Escherichia coli groE gene; Kurokawa K et al.; Plasmids carrying the groE promoter with a superhelical density of 0 to -0.091 were used for in vitro transcription with RNA polymerase holoenzyme containing sigma 32 . The promoter activity of the groE gene in vitro on the plasmid DNA was decreased to 100-fold when the superhelical density of the template DNA decreased from -0.059 to 0 . As the superhelical density of DNA inside the cells was estimated to be approximately -0.025, it seems likely that the increase in the linking number of template DNA caused by stress which induces heat shock response may not stimulate the groE promoter activity in vivo on the chromosomal DNA . At higher concentrations of salt, the dependence of the groE promoter activity on negative supercoiling was even more apparent . Since melting of the DNA duplex is inhibited by high salt concentrations, DNA supercoiling may facilitate melting of the groE promoter by an RNA polymerase holoenzyme containing sigma 32. Histol Histopathol, 1996 Jul, 11(3), 597 - 606 Comparative in vivo and in vitro models to approach the cellular basis of endotoxic shock . The role of sinusoidal liver cells; Pagani R et al.; During endotoxic shock, the liver exerts a lipopolysaccharide (LPS) clearance function with the participation of both parenchymal and sinusoidal cells . Liver damage could be caused by LPS direct action, hypoxia and/or inflammatory mediators released by Kupffer cells . The aim of this study is to establish an experimental model that could allow us to understand the direct E . coli 011:B4 LPS action on sinusoidal cells . A comparative study was carried out, in vivo and in vitro, using either a rat reversible endotoxic shock model or sinusoidal cell cultures . The LPS was found to induce important and similar morphological alterations both in vivo and in vitro, specially in Kupffer cells . These cells present mitochondrial damage, nuclear membrane swelling, and increased number of phagosomes, including lamellar bodies . An immunocolloidal gold technique shows, in vitro, the LPS mainly located on Kupffer cell membrane and in phagosomes . The LPS binding to membrane, as a primary step of Kupffer cell activation, increases the phagocytosis . This effect could be related to a decrease of fluidity on the external membrane portion. Res Virol, 1996 Jul-Aug, 147(4), 203 - 11 Immunological identification of Tacaribe virus proteins; Rossi C et al.; Tacaribe virus (TV), an arenavirus, is an enveloped virus with genetic information encoded in two segments of single-stranded RNA . The completed sequence of TV led to the identification of four open reading frames (ORF) . In order to establish a direct link between ORFs in the sequence of TV and proteins present in virus particles and virus-infected cells, segments of the molecularly cloned TV genome were engineered so as to be expressed in Escherichia coli to produce fusion proteins that were used to raise antisera . The antisera were in turn employed to identify the TV gene products . Serum to the putative nucleocapsid (N) protein reacted with a 68-kDa protein, both in TV particles and in the infected cells . Sera raised to the glycoprotein precursor (GPC) immunoprecipitated two proteins of 68 and 70 kDa from infected cell lysates . Analysis of GPC synthesis in the presence of tunicamycin revealed that the unglycosylated GPC appeared as two polypeptides of 43 and 46 kDa . The putative RNA polymerase gene product (L) was detected as a approximately 240-kDa protein . Serum to the small zinc-binding domain protein (p11-Z) recognized a protein of approximately 11kDa . Immunological evidence is presented that in addition to N and L, two glycoproteins (GP1 and GP2) and p11-Z are structural components of Tacaribe virions. Curr Biol, 1996 Jul 1, 6(7), 825 - 7 Receptor signaling: dimerization and beyond; Stock J; It is has been proposed that hormone receptors which have only a single transmembrane sequence mediate signaling via hormone-induced monomer-to-dimer transitions . But recent studies of analogous receptors in bacteria indicate that dimerization may be only a prerequisite for signaling. J Cell Sci, 1996 Jul, 109 ( Pt 7), 1909 - 17 Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip; Soltys BJ et al.; Giardia lamblia trophozoites contain a complex endomembrane system as demonstrated by fluorescence and cryoelectron microscopy . The endomembrane system was weakly detected in live cells using the fluorescent membrane dye 3,3'-dihexyloxacarbocyanine iodide . The definitive identification of endoplasmic reticulum required the development of a molecular label . We expressed Giardial Bip in Escherichia coli and raised a polyclonal antibody to the purified protein . In western blots, the antibody was specific for Giardial Bip and did not react with human, monkey and rodent homologs . By immunofluorescence microscopy in methanol fixed cells the antibody visualized tubular structures and other subcellular components that required characterization by electron microscopy . Using cryotechniques we directly demonstrate the presence of a complex endomembrane system at the ultrastructural level . In conjunction with Bip immunogold labeling of cryosections we identify: (1) endoplasmic reticulum cisternae and tubules; (2) stacked perinuclear membranes; and (3) Bip presence in the nuclear envelope . Both the endoplasmic reticulum and nuclear envelope were found either with or without a cleft region suggesting each may contain common specialized sub-regions . In stacked perinuclear membranes, which may represent either multilamellar endoplasmic reticulum or a Golgi apparatus, Bip labeling was restricted to peripheral layers, also suggesting specialized sub-regions . Labeled endomembrane systems could be observed associated with microtubule structures, including axonemes and the adhesive disk . The presence of an extensive endomembrane system in Giardia lamblia, which represents one of the earliest diverging eukaryotic species, supports the view that both the nucleus and endomembrane system co-evolved in a common ancestor of eukaryotic cells. Cell Mol Biol (Noisy-le-grand), 1996 Jul, 42(5), 703 - 10 Medium scale production and purification to homogeneity of a recombinant trans-sialidase from Trypanosoma cruzi; Buschiazzo A et al.; Trypanosoma cruzi, the agent of Chagas' disease, presents an enzyme that catalyzes the transfer of sialic acid among glycoproteins and glycolipids known as trans-sialidase (TS), displaying some interesting features: 1) It differs from all other eucaryotic sialyltransferases, both kinetically and in substrate specificity and 2) it is involved in the parasite's mechanism of mammalian host cell invasion . We report here the production and purification to homogeneity of an enzymatically active recombinant TS (rTS) lacking the C-terminal amino acid repeats, using iminodiacetic metal affinity chromatography . Initial ratios of non-fusion recombinant versus total protein were very low in several expression systems tested, mainly due to high degradation rate . However, after purifying 1,330 times, we were able to obtain an essentially homogeneous preparation of rTS with a final yield of 29% . After minor changes, a modified protocol for a medium scale production was designed obtaining 0.5 mg of homogeneous rTS per liter of bacterial culture . The purified rTS behaved as a homogeneous protein in silver-stained denaturing gels, isoelectrofocusing and N-terminal sequencing having identical pH and temperature optima as the natural enzyme . Conditions to keep the rTS for long periods without a significant loss of activity were identified. Cell Mol Biol (Noisy-le-grand), 1996 Jul, 42(5), 673 - 82 Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase; Hagelin K et al.; The primary structure of several chloroplast fructose-1,6-bisphosphatase (CFBPase) was deduced from DNA sequences, but only spinach, pea and rapeseed enzymes have been characterized structurally . We analyzed whether CFBPases from different phylogenetic origin contain a common epitope . To this end a DNA fragment of 1200 base pairs encoding 338 amino acid residues of wheat CFBPase (38 kDa) was cloned in the expression plasmid pGEX-1 in frame with the gene coding for glutathione S-transferase (GT) of Schistosoma japonicun (26.5 kDa) . Upon transformation of Escherichia coli and induction with isopropyl-beta-D-thiogalactopyranoside, centrifugation of the lysate partitioned 10% of the fusion protein in the supernatant fraction and the remaining 90% in the precipitate . The expected 65 kDa protein was purified from both the soluble and the particulate fraction by affinity chromatography on columns of glutathione-agarose . This fusion protein was successfully used to produce a monoclonal antibody that specifically recognized the CFBPase region of the fusion protein but not the GT moiety . Moreover, the monoclonal antibody immunoreacted not only with polypeptides (ca . 40 kDa) present in leaf crude extracts of other varieties of wheat (Triticum spelta, T . aestivum and T . durum), but also with homogeneous preparations of the spinach (Spinacia oleracea) and rapeseed (Brassica napus) enzymes . Thus, the cross reaction of this monoclonal antibody with counterparts from different plant species indicates the persistency of a common epitope through biological evolution. Br J Pharmacol, 1996 Jul, 118(6), 1335 - 40 Modulation by nitric oxide of spontaneous motility of the rat isolated duodenum: role of tachykinins; Martinez-Cuesta MA et al.; 1 . Incubation of proximal segments of the rat isolated duodenum with NG-nitro-L-arginine (L-NOARG; 3-100 microM) produced a concentration-dependent increase in both resting tone and the amplitude of the spontaneous contractions . These effects were attenuated by concurrent incubation with L-arginine (1 mM) but not D-arginine (1 mM) . 2 . These changes in resting tone and motility induced by L-NOARG (30 microM) were substantially reduced by concurrent incubation with tetrodotoxin (1 microM) or hexamethonium (10 microM), implicating the involvement of a local neuronal response . 3 . The L-NOARG-induced increase in duodenal motility was not, however, inhibited by atropine (1 microM), guanethidine (6.4 microM) phentolamine (1 microM), or indomethacin (10 microM), indicating a non-cholinergic, non-adrenergic and non-prostanoid-mediated contractile response . 4 . The NK1/NK2 tachykinin receptor antagonist, (D-Pro2, D-Trp7.9 substance P, 1-10 microM), and the NK2-receptor antagonists, MEN 10,207 and MEN 10,376 (1-5 microM), concentration-dependently reduced the effect of L-NOARG (30 microM) on spontaneous duodenal motility . 5 . The resting tone and amplitude of the spontaneous contractions was likewise increased by incubation with NG-monomethyl-L-arginine (L-NMMA; 100-1000 microM) . However, incubation with L-NMMA (100 microM) attenuated the actions of more potent L-NOARG (30 microM) on resting motility . 6 . Administration of E.coli endotoxin (3 mg kg-1, i.v.) to the rat 5 h prior to tissue removal, at a time of known induction of NO synthase, reduced the amplitude of spontaneous contractions of the isolated duodenum, an effect inhibited by pretreatment of the rats with dexamethasone (1 mg kg-1) 2 h prior to endotoxin challenge . 7 . These findings indicate a role of endogenous NO in the modulation of spontaneous tone and motility in the rat duodenum . Induction of NO synthase may result in a reduction in spontaneous motility of the tissue . By contrast, inhibition of constitutive NO biosynthesis unmasks a contractile response that is neuronally mediated and involves tachykinin NK2 receptors. Am J Reprod Immunol, 1996 Jul, 36(1), 49 - 57 Characterization of a sperm nuclear protein; Batova IN et al.; PROBLEM: The molecular identity of sperm DNA-binding structural proteins contributing to the integrity of a sperm residual high salt/nuclease resistant nuclear structure is studied by cDNA cloning and monoclonal antibodies to the recombinant polypeptide . This structure, which is likely to be transferred unimpaired in the oocyte and is anticipated as a molecular correlate of the nuclear scaffold (nuclear matrix/envelope) in somatic cells, may be essential with respect to its DNA organization for the recovery and assembly of somatic-type chromatin in the zygote . Recently, a cDNA encoding one of these proteins has been cloned and the recombinant polypeptide expressed in E . coli as a beta-galactosidase fusion protein . The main objective of the present study is the identification of the native sperm antigen by monoclonal antibodies raised against the recombinant molecule . METHODS: We evaluated the possibility of immunizing by direct intrasplenic deposition in BALB/c mice of the recombinant fusion protein available as transblotted on nitrocellulose membrane carriers or as nitrocellulose protein-bearing particles . Isolated sperm DNA/tight binding protein complexes were used in ELISA and Western blotting for selection of monoclonal antibodies specific to self epitopes of the nuclear antigen, as well as immunofluorescence of swollen human spermatozoa subjected to in situ extraction with high salt/beta-mercaptoethanol/DNase I and proteolysis, and of a cultured fibroblast cell line L-929 . RESULTS: A monoclonal antibody, Mab 2C4, was selected which recognized a 52 kDa protein in the fraction of sperm high salt/urea resistant proteins . The target polypeptide was detected on swollen spermatozoa primarily to the post-acrosomal and/or equatorial regions whereas in nonextracted sperm cells the epitope was exceedingly unavailable . The somatic cell location of the cognate epitope was confined to the nuclear envelope displaying a cap-like pattern of staining, and also in a juxtanuclear cytoplasmic randomly coiled filamentous network and in compact bodies . CONCLUSIONS: A nuclear protein salt-stably bound to the sperm residual structure has been identified . The antigen appears localized in sperm exclusively to perinuclear subacrosomal sites that may be anchored at the male nuclear envelope, given the occurrence of the target epitope in somatic cells as well in nuclear and cytoplasmic sites adjacent to the nuclear membrane. Chem Res Toxicol, 1996 Jul-Aug, 9(5), 891 - 6 Deamination of single-stranded DNA cytosine residues in aerobic nitric oxide solution at micromolar total NO exposures; Merchant K et al.; Deamination of cytosine to uracil is a potential source of mutations in DNA . Here we examine the deaminating ability of aerobic nitric oxide (NO) toward single-stranded DNA at very low (micromolar and below) total exposures, using a sensitive genetic method that allows us to study a single deamination event at a specific site in a 7200-nucleotide DNA molecule within a pool of ca . 100,000 other identical DNA molecules . We incubated gapped C141 M13mp2 DNA with the NO-generating compound, Et2N{N(O)NO}Na (DEA/NO), in aerobic buffer for 16 h to ensure complete autoxidation at pH 7.4 and 37 degrees C . After ultrafiltration to remove small molecules, the DNA was transformed into isogenic Escherichia coli cultures that were either deficient (NR9404, ung-) or proficient (MC1061, ung+) in uracil-DNA glycosylase activity . The gapped DNA was constructed such that the target (CCC) codon was contained in a short single-stranded segment of otherwise double-stranded circular DNA, and the incubation was performed in a closed system to prevent loss of NO to the atmosphere before the reaction was complete . An increase in the reversion frequency in the ung- strain was noted between 0 and 1 microM DEA/NO, and the reversion frequency leveled out between 3 and 30 microM . However, 30 microM "spent" DEA/NO (i.e., that which was similarly incubated for 4 h to complete the autoxidation of NO before the DNA was added) did not increase reversion frequency relative to control . Nearly all (42/43) of the mutations identified after 1 microM DEA/NO treatment were C-->T transitions, and reversion frequency in the isogenic ung+ strain was lower than in the ung- strain . The data are consistent with the hypothesis that total NO exposures in the mumol/L range can lead to C-->T mutations via a mechanism most probably involving deamination of DNA cytosine residues. Microb Pathog, 1996 Jul, 21(1), 35 - 45 Monoclonal antibodies against fimbrial subunits of colonization factor antigen I (CFA/I) inhibit binding to human enterocytes and protect against enterotoxigenic Escherichia coli expressing heterologous colonization factors; Rudin A et al.; Enterotoxigenic Escherichia coli (ETEC) bind to enterocytes in the small intestine by means of antigenically distinct colonization factors (CFs) . By immunizing with isolated subunits of CFA/I fimbriae we have previously produced monoclonal antibodies (MAbs) that cross-react immunologically in vitro with several CFs . Two of these MAbs {S(subunit)-CFA/ I 17:8 and S-CFA/I 5:6} were found to significantly inhibit the binding of ETEC strains expressing either homologous or heterologous CFs, i.e . CFA/I and CS4, to isolated human jejunal enterocytes . The two MAbs also conferred passive protection against fluid accumulation in rabbit ileal loops caused by CFA/I-as well as CS4-expressing ETEC strains . Immunoelectron microscopy studies showed that both MAbs bound specifically to CFA/I as well as to CS4 fimbriae expressed on bacteria . These results indicate the possibility to induce anti-CF antibodies that can protect against ETEC infection caused by bacteria expressing not only homologous but also heterologous CFs, by immunizing with fimbrial subunits. J Lipid Res, 1996 Jul, 37(7), 1519 - 28 High yield overexpression and characterization of human recombinant proapolipoprotein A-I; McGuire KA et al.; Human apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) where it defines the particle structure and stability and functions as the main activator of the enzyme lecithin:cholesterol acyltransferase (LCAT) . ApoA-I is expressed in the liver as a preproprotein that is targeted to the endoplasmic reticulum for secretion; in plasma, an unknown protease removes the six amino acid long propeptide . In this study, the cDNA coding the human proapoA-I was cloned into an Escherichia coli vector; the overexpressed protein was purified to 99% homogeneity and was extensively characterized together with mature apoA-I purified from plasma . SDS-PAGE, mass spectrometry, and Edman sequence analysis showed that the initial Met residue needed for translation in E . coli is posttranslationally removed from the N-terminal sequence of the proapoA-I . The structural and functional analyses were carried out on the lipid-free and the lipid-bound proteins . ProapoA-I self associated, interacted with dimyristoyl phosphatidylcholine vesicles, and formed secondary structures very similar to the lipid-free apoA-I . Reconstituted HDL particles made with two initial molar ratios of palmitoyloleoyl phosphatidylcholine/cholesterol/apolipoprotein/Na-cholate had identical particle sizes and distributions when apoA-I or proapoA-I were used . Particles having diameters of 79 A and 98 A, containing two apoA-I or proapoA-I molecules per particle, were isolated and characterized . The particles contained the same amounts of alpha-helical structure, had very similar fluorescence properties, and activated LCAT equally well . We conclude that proapoA-I expressed and purified from E . coli is functionally and structurally indistinguishable from mature apoA-I purified from plasma when analyzed in vitro . Therefore, this recombinant proapoA-I and mutants derived from it will be important sources of protein for analyzing apoA-I structure and function, as well as for studies of proapoA-I processing. Res Vet Sci, 1996 Jul, 61(1), 72 - 7 Effect of supplementary feeding during the sucking period on net absorption from the small intestine of weaned pigs; Nabuurs MJ et al.; An intestinal perfusion technique was used to measure the effects of supplementary feeding (experiment 1) and temporary weaning (experiment 2) during the sucking period on the net absorption of fluid, sodium, chloride and potassium from the small intestine of pigs after weaning . The technique was also applied to control pigs which did not receive supplementary feed and were not temporarily weaned during the sucking period . Paired intestinal segments were prepared at five sites along the small intestine in each of 80 pigs in experiment 1 and 36 pigs in experiment 2 . The cranial segment of each pair was infected with enterotoxigenic Escherichia coli (ETEC) . In both experiments, four days after weaning, the net absorption of fluid, sodium and chloride in the uninfected segments was in general significantly less in all the pigs than at weaning or seven, 11 and 14 days after weaning and it was significantly greater in the pigs given supplementary feed than in the other pigs . Net absorption values in the infected segments of the control pigs were less four days after weaning than on the day of weaning . No differences were found between pigs that were temporarily weaned or not and those that were not fed during the sucking period . Supplementary feeding during the sucking period partially prevented the decrease of net absorption usually observed after weaning in uninfected and in ETEC-infected segments of intestine. Protein Sci, 1996 Jul, 5(7), 1316 - 24 Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit; Grant BD et al.; The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family . Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition . Glu 230, the most buried of these acidic residues, was mutated to Ala (rC{E230A}) and Gln (rC{E230Q}) and overexpressed in Escherichia coli . In contrast to the mostly insoluble and destabilized rC{E230A}, rC{E230Q} is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability . The mutation in rC{E230Q} causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide . In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway . Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed . Electrostatic alterations in the rC{E230Q} active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally . These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis . Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site. Protein Sci, 1996 Jul, 5(7), 1290 - 300 In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: implications for protein folding and assembly; Zhang P et al.; Because the N- and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild-type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain . These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR . Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants . Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers . The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16-21 mumol carbamoylaspartate per microgram per h, compared with 15 for wild-type ATCase, and the values for K0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM . Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild-type enzyme . As observed with wild-type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH) . In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild-type ATCase . Thus, the enzyme containing the permuted chains with widely diverse N- and C-termini exhibited the homotropic and heterotropic effects characteristic of wild-type ATCase . The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand-promoted allosteric transition from the taut (T) state to the relaxed (R) conformation . These ATCase molecules with new N- and C-termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains. Protein Sci, 1996 Jul, 5(7), 1250 - 60 The lipocalin Xlcpl1 expressed in the neural plate of Xenopus laevis embryos is a secreted retinaldehyde binding protein; Lepperdinger G et al.; The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate of Xenopus laevis embryos and the adult choroid plexus have been investigated . It was found that this lipocalin, termed Xlcpl1, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol . Furthermore, this protein also binds D/L thyroxine . The Xlcpl1 cDNA was expressed in cell culture using the vaccinia virus expression system . In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway . We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids . Therefore, cpl1-expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers . These retinoids may enter the cytosol by diffusion or receptor-controlled mechanisms, as has been shown for exogenously applied retinoids . Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development. J Pharm Sci, 1996 Jul, 85(7), 680 - 4 Influence of Escherichia coli endotoxin on the pharmacokinetics and respiratory depressant effect of alfentanil in rabbits; Bjorkman S et al.; Postoperative fever occurs in many surgical patients, due to release of pyrogens into the circulation . Pyrogens and fever may influence the pharmacokinetics and effects of drugs . The aim of this study was to investigate the interactions of a pyrogen with the opioid analgesic alfentanil in an animal model . Alfentanil was infused over 10 min into 17 conscious rabbits and over 120 min in another 12 animals . Some of them had been randomized to pretreatment with Escherichia coli endotoxin . Arterial plasma concentrations of alfentanil were determined repeatedly, and arterial blood gas values were recorded . Endotoxin antagonized the respiratory depressant effect of the 10-min infusion of alfentanil by increasing drug distribution clearance during the infusion . In addition, endotoxin raised the total volume of distribution of alfentanil from 0.70 to 1.4 L/kg . Plasma protein binding and elimination clearance were not affected . The increase in volume of distribution and the lack of effect of endotoxin on elimination clearance were confirmed in the 120-min infusion experiments . Apparent concentration-effect relationships for the actions of alfentanil on arterial CO2, O2, and pH were not influenced by the endotoxin . Circulating pyrogens can thus influence the pharmacokinetics and, indirectly, the effects of alfentanil. Gene Ther, 1996 Jul, 3(7), 615 - 23 Long-term persistence of defective HSV-1 vectors in the rat brain is demonstrated by reactivation of vector gene expression; Starr PA et al.; Wild-type HSV-1 is known to persist indefinitely in neurons in the latent state; however, defective HSV-1 vectors, or amplicons, contain only approximately 1% of the HSV-1 genome and persistence of these HSV-1 vectors has not been studied even semiquantitatively in the adult rat brain . Defective HSV-1 vectors contain both an HSV-1 origin of replication and a packaging site, and in the presence of helper virus can undergo DNA replication and packaging into HSV-1 particles . Our prototype defective HSV-1 vector, pHSVlac, uses the HSV-1 immediate-early (IE) promoter to regulate expression of the Escherichia coli lacZ gene . Using cultured neuronal cells, we have previously shown that expression from pHSVlac can be augmented by superinfection with a helper virus . In this study, pHSVlac was delivered into the adult rat striatum or hippocampus, and 2-3 months after gene transfer we utilized superinfection with several replication-incompetent HSV-1 mutants to reactivate expression from pHSVlac in approximately 30% of the number of cells observed at 4 days after gene transfer . Thus, HSV-1 plasmid vectors can persist for at least 2-3 months in at least approximately 30% of the cells which are initially infected. Equine Vet J, 1996 Jul, 28(4), 269 - 74 Induction of early-phase endotoxin tolerance in horses; Allen GK et al.; Six, clinically healthy horses, of mixed age and sex, were infused via a jugular venous catheter with 100 ml of pyrogenfree sterile saline (PFSS; 0.9% NaCl) . Animals were infused with Escherichia coli O55:B5 endotoxin (total dose = 50 ng/kg bwt), 24 (LPS-1) and 48 h (LPS-2) after PFSS infusion . Blood was collected before, and every 15 min after, each infusion for the first 8 h and then every 2 h for the following 14 h . Clinical responses (rectal temperature, heart rate, respiration rate and blood pressure) were determined before and every 4 h after each infusion for 20 h . Geometric mean anti-endotoxin antibody titres in serum samples, harvested just before each infusion, were unchanged over the course of the experiment . Serum tumour necrosis factor-alpha (TNF alpha) activity was estimated using a cytotoxic bioassay and WEHI 164 clone 13 murine fibrosarcoma cells as targets . Mean clinical parameter values and geometric mean serum TNF alpha activity at given time points were compared across the 3 infusions . Both LPS-1 and LPS-2 resulted in elevated mean rectal temperature at 4 h after infusion . However, duration of mean rectal temperature elevation was greater (P < 0.05) after LPS-1 (through 12 h) than after LPS-2 (through 8 h) . More substantial increases in systolic and diastolic blood pressure were observed after LPS-1 than LPS-2 and mean systolic blood pressure after LPS-1 was elevated at 4 h when compared to PFSS (P < 0.05) . Decreased systolic and diastolic blood pressures were observed at 16 h after both LPS infusions, when compared to PFSS infusion . Heart rate was increased, compared to PFSS, after both LPS-1 (8-12 h) and LPS-2 (4-12 h) (P < 0.05) . No significant elevations in mean respiratory rate were observed after either LPS-1 or LPS-2 when compared to PFSS . However, at 4 h post infusion, mean respiratory rate after LPS-2 was greater (P < 0.05) than that after LPS-1 . Serum TNF alpha activity was not detected after infusion of PFSS, but was detected after both LPS-1 and LPS-2 . Serum TNF alpha activity was elevated earlier, was present in higher concentrations and persisted longer after LPS-1 than after LPS-2 (P < 0.05) . The decreased duration of fever and attenuated serum TNF alpha response subsequent to successive sublethal LPS challenge observed in this study support the conclusion that these horses developed early-phase endotoxin tolerance (EPET) and, therefore, contributes to the understanding of the role of endotoxaemia in a number of clinical conditions in horses. Br J Pharmacol, 1996 Jul, 118(5), 1223 - 31 Effects of UR-12633, a new antagonist of platelet-activating factor, in rodent models of endotoxic shock; Giral M et al.; 1 . The effects of the selective and potent novel platelet-activating factor (PAF) antagonist, UR-12633 (1-(3,3-diphenylpropionyl)-4-(3-pyridylcyanomethyl)piperidin e) on several markers of endotoxic shock syndrome were evaluated in rats and mice . 2 . UR-12633, administered 60 min after E . coli lipopolysaccharide (LPS), reversed the LPS-induced sustained hypotension in rats at doses of 0.01 to 1 mg kg-1, i.v . The reference compound WEB-2086 (1 mg kg-1) also reversed the LPS-induced hypotension . UR-12633 (1 mg kg-1), administered 10 min before LPS, almost fully inhibited sustained hypotension . The immediate hypotension (within 1 min) caused by LPS was not prevented by either UR-12633 or WEB-2086 . 3 . Pretreatment with 10 mg kg-1, i.v . of either UR-12633 or WEB-2086 inhibited the increase in disseminated intravascular coagulation markers, such as activated partial thromboplastin time (55 and 74% inhibition, respectively), and prothrombin time (22 and 72% inhibition) and prevented the decrease in plasma fibrinogen content (100 and 29% inhibition) . 4 . Increases in acid phosphatase (ACP) plasma activity, a marker of lysosomal activation, and in lactate dehydrogenase (LDH), a marker of tissue damage, were inhibited by pretreatment with 10 mg kg-1, i.v . of either UR-12633 or WEB-2086 (100% and 69% inhibition, ACP; 62 and 48% inhibition, LDH) . Hyperglycaemia (71 and 46%) and hyperlactacidaemia (92 and 56%) were also inhibited . 5 . UR-12633, but not WEB-2086, inhibited the LPS-induced increase in vascular permeability in rats, as shown by prevention of haemoconcentration and, to a lesser degree, the increase in Evans blue dye extravasation . 6 . In a series of nine reference compounds and UR-12633, we found a high correlation (P < 0.001) between PAF antagonist activity, measured as the inhibition of PAF-induced rabbit platelet aggregation or PAF-induced mortality in mice and the inhibition of LPS-induced mortality . 7 . In spite of the multifactorial nature of endotoxic shock, in which many mediators may be involved, the new potent PAF antagonist, UR-12633, proved effective in protecting against changes in most shock markers . These data strongly suggest a key role for PAF in the pathogenesis of endotoxic shock in rodents. Br J Pharmacol, 1996 Jul, 118(5), 1218 - 22 Alteration by lipopolysaccharide of the relationship between intracellular calcium levels and contraction in rat mesenteric artery; Martinez MC et al.; 1 . The aim of this work was to investigate the effect of lipopolysaccharide (LPS) treatment on the relationship between the cytosolic Ca2+ ion concentration ({Ca2+}i) and contraction in rat resistance arteries, and the involvement of the L-arginine-nitric oxide (NO)-guanosine 3'-5' cyclic monophosphate (cyclic GMP) pathway in these effects . 2 . {Ca2+}i and tension were simultaneously recorded in small mesenteric arteries removed from rats 4 h after intraperitoneal injection of E . coli LPS (30 mg kg-1) or solvent . Cyclic GMP was assayed in vessels submitted to identical treatments . 3 . Basal {Ca2+}i was higher in vessels from LPS-treated rats compared to controls . LPS did not modify the concentration-contraction curve of noradrenaline . However, the increase in basal {Ca2+}i produced by LPS resulted in a shift of the noradrenaline {Ca2+}i-contraction curve to higher {Ca2+}i concentrations . 4 . L-Arginine (300 microM) relaxed noradrenaline (10 microM) pre-contracted arteries from LPS-treated but not from control rats . This effect of L-arginine was reversed by two inhibitors of NO synthase: N omega-nitro-L-arginine-methyl-ester (L-NAME, 1 mM) and S-methyl-isothiourea (SMT, 0.1 mM) . Both the relaxing effect of L-arginine and its reversal by L-NAME or SMT occurred without any change in {Ca2+}i . 5 . LPS treatment did not modify the cyclic GMP content of the small mesenteric arteries . In arteries removed from LPS-treated rats but not from controls, addition of L-arginine (300 microM) was associated with a significant increase in cyclic GMP content, an effect which was prevented by both L-NAME (1 mM) and SMT (0.1 mM) . 6 . L-NAME (1 mM) produced a greater reduction in cyclic GMP content than SMT (0.1 mM) in control vessels exposed to L-arginine (300 microM) . Under the same conditions, SMT produced a larger decrease in cyclic GMP level than L-NAME in arteries taken from LPS-treated rats, consistent with selective inhibition by SMT of the inducible NO-synthase after LPS . 7 . These results show that LPS produced two effects in small mesenteric arteries: (i) alterations in Ca2+ handling and a decreased sensitivity of myofilaments to Ca2+, (ii) induction of NO-synthase activity resulting in exogenous L-arginine-dependent production of NO and cyclic GMP accumulation . Both effects are likely to be involved in the hyporeactivity induced by LPS in resistance arteries. Hum Mol Genet, 1996 Jul, 5(7), 1023 - 8 Recessively inherited L-DOPA-responsive parkinsonism in infancy caused by a point mutation (L205P) in the tyrosine hydroxylase gene; Ludecke B et al.; Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in the biosynthesis of dopamine . This report describes a missense point mutation in the human TH (hTH) gene in a girl presenting parkinsonian symptoms in early infancy and a very low level of the dopamine metabolite homovanillic acid in the CSF . DNA sequencing revealed a T614-to-C transition in exon 5 (L205P) . Both parents and the patient's brother are heterozygous for the mutation . Site-directed mutagenesis and expression in different systems revealed that the recombinant mutant enzyme had a low homospecific activity, i.e . approximately 1.5% of wt-hTH in E . coli and approximately 16% in a cell-free in vitro transcription-translation system . When transiently expressed in human embryonic kidney (A293) cells a very low specific activity (approximately 0.3% of wt-hTH) and immunoreactive hTH (< 2%) was obtained . The expression studies are compatible with the severe clinical phenotype of the L205P homozygous patient carrying this recessively inherited mutation . Treatment with L-DOPA resulted in normalisation of the CSF homovanillic acid concentration and a sustained improvement in parkinsonian symptoms. Hum Mol Genet, 1996 Jul, 5(7), 1011 - 6 Clustering of mutations in the biotin-binding region of holocarboxylase synthetase in biotin-responsive multiple carboxylase deficiency; Dupuis L et al.; Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans . A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency (MCD) . We have identified six different point mutations in the HCS gene in nine patients with MCD . Two of the mutations are frequent among the MCD patients analyzed . Four of the mutations cluster in the putative biotin-binding domain as deduced from the corresponding Escherichia coli enzyme and consistent with an explanation for biotin-responsiveness based on altered affinity for biotin . The two others may define an additional domain involved in biotin-binding or biotin-mediated stabilization of the protein. Biotechniques, 1996 Jul, 21(1), 92 - 4, 96-9 Comparison of plasmid DNA preparation methods for direct gene transfer and genetic immunization; Davis HL et al.; Plasmid DNA is widely used for direct gene transfer in animals to study gene therapy, gene regulation, drug delivery and genetic immunization . Here we compare cesium chloride and anion-exchange purified plasmid DNA for direct gene transfer in mouse muscle and show no differences in efficiency of transfection with reporter genes or in humoral response to DNA-based immunization. Z Naturforsch {C}, 1996 Jul-Aug, 51(7-8), 534 - 8 A new non-radioactive assay of phytoene desaturase to evaluate bleaching herbicides; Sandmann G et al.; A non-radioactive cell-free assay was developed to quantitatively determine inhibition of plant-type phytoene desaturase by bleaching herbicides . An active desaturase was prepared from an appropriately cloned E . coli transformant . Another E . coli transformant was used to produce the required phytoene . Phytofluene and zeta-carotene, the products of the desaturase reaction, were either determined by HPLC or optical absorption spectra . Enzyme kinetics and inhibition data for the bleaching tetrazole herbicide WL110547 are presented as an example. Hokkaido Igaku Zasshi, 1996 Jul, 71(4), 463 - 73 {Study on the specificity of a monoclonal antibody against recombinant envelope proteins of a human endogenous retrovirus, ERV3}; Shibata M; Human endogenous retroviruses (HERVs) and its related sequences have been known to occupy about 1% on human genome, and the expression of messenger RNA has been demonstrated by many investigators including our laboratory . However, only few papers have been indicated they are translated in proteins . Either physiological roles in man or pathogenic roles in diseases are still unknown at present . In this study, to investigate the protein expression of one copy type HERV, ERV3 and its pathogenicity, a recombinant protein to ERV3 env region was produced using E . coli expression system, and a monoclonal antibody to the recombinant protein was prepared . The monoclonal antibody evidenced the translation of ERV3, and 170 and 180 kilodaltons of proteins were detected in normal placentas and adrenal glands by Western blot . Natural antibodies to the products of ERV3 env region with high reactivities were detected in sera from some patients with systemic lupus erythematosus (SLE) compared with normal population and patients with rheumatoid arthritis (RA) . The result suggests that ERV3 products may play as one of autoantigens in patients with SLE . The recombinant protein and monoclonal antibody described here may be useful to study the role of ERV3 in vivo and the etiologic relation of HERVs to autoimmune diseases. Chem Biol, 1996 Jul, 3(7), 579 - 89 The mismatch-repair protein hMSH2 binds selectively to DNA adducts of the anticancer drug cisplatin; Mello JA et al.; BACKGROUND: The antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) exerts its cytotoxic effects through the formation of covalent DNA adducts . A family of proteins possessing a common HMG box motif that binds specifically to cisplatin DNA adducts has been previously suggested to be important in the clinical efficacy of the drug . RESULTS: We have shown that the human mismatch-repair protein, hMSH2, also binds specifically to DNA containing cisplatin adducts and displays selectivity for the DNA adducts of therapeutically active platinum complexes . Moreover, hMSH2 is overexpressed in testicular and ovarian tissue; tumors in these tissues are most effectively treated by cisplatin . CONCLUSIONS: Our results suggest a role for hMSH2 in mediating cisplatin toxicity . Supporting this view, previous studies in Escherichia coli dam- strains demonstrate that mutations in mismatch-repair proteins confer resistance to cisplatin toxicity . Mismatch-repair deficiency is also correlated with tolerance to O6-methylguanine, a cytotoxic DNA lesion formed by methylating agents . A current model ascribes O6-methylguanine toxicity to unsuccessful attempts at repair of this lesion by mismatch-repair proteins, resulting in a futile cycle of incision and synthesis, leading ultimately to lethal DNA-strand breaks . We propose that mismatch repair may contribute to cisplatin toxicity by a similar mechanism . Alternatively, hMSH2 may shield cisplatin adducts from repair, allowing adducts to persist, thus enhancing lethality. Genetics, 1996 Jul, 143(3), 1127 - 35 Mismatch repair mutants in yeast are not defective in transcription-coupled DNA repair of UV-induced DNA damage; Sweder KS et al.; Transcription-coupled repair, the targeted repair of the transcribed strands of active genes, is defective in bacteria, yeast, and human cells carrying mutations in mfd, RAD26 and ERCC6, respectively . Other factors probably are also uniquely involved in transcription-repair coupling . Recently, a defect was described in transcription-coupled repair for Escherichia coli mismatch repair mutants and human tumor cell lines with mutations in mismatch repair genes . We examined removal of UV-induced DNA damage in yeast strains mutated in mismatch repair genes in an effort to confirm a defect in transcription-coupled repair in this system . In addition, we determined the contribution of the mismatch repair gene MSH2 to transcription-coupled repair in the absence of global genomic repair using rad7 delta mutants . We also determined whether the Rad26-independent transcription-coupled repair observed in rad26 delta and rad7 delta rad26 delta mutants depends on MSH2 by examining repair deficiencies of rad26 delta msh2 delta and rad7 delta rad26 delta msh2 delta mutants . We found no defects in transcription-coupled repair caused by mutations in the mismatch repair genes MSH2, MLH1, PMS1, and MSH3 . Yeast appears to differ from bacteria and human cells in the capacity for transcription-coupled repair in a mismatch repair mutant background. Genetics, 1996 Jul, 143(3), 1101 - 14 Recombination-dependent growth in exonuclease-depleted recBC sbcBC strains of Escherichia coli K-12; Ryder L et al.; Analysis of the aroLM-sbcCD interval of the Escherichia coli K-12 chromosome revealed a new gene (rdgC) encoding a function required for growth in recombination-deficient recBC sbcBC strains . Deletion of rdgC does not reduce viability, conjugational recombination, or DNA repair in rec+, recA, recB, recF, or recJ mutants . However, it makes the growth of recBC sbcBC strains reliant on the RecA, RecF, and RuvC proteins and, to a large extent, on RuvAB . The recBC sbcBC delta rdgC ruvAB construct forms colonies, but cell viability is reduced to < 5% . A recBC sbcBC delta rdgC derivative carrying the temperature-sensitive recA200 allele grows at 32 degrees but not 42 degrees . Multicopy rdgC+ plasmids reduce the growth rate of recBC sbcBC strains, while multicopy sbcC+ plasmids that reactivate SbcCD nuclease cannot be maintained without RdgC protein . The data presented are interpreted to suggest that exonuclease-depleted recBC sbcBC strains have difficulty removing the displaced arm of a collapsed replication fork and that this problem is compounded in the absence of RdgC . Recombination then becomes necessary to repair the fork and allow chromosome duplication to be completed . The possibility that RdgC is an exonuclease is discussed. Genetics, 1996 Jul, 143(3), 1091 - 100 Mosaic structure of plasmids from natural populations of Escherichia coli; Boyd EF et al.; The distribution of plasmids related to the fertility factor F was examined in the ECOR reference collection of Escherichia coli . Probes specific for four F-related genes were isolated and used to survey the collection by DNA hybridization . To estimate the genetic diversity of genes in F-like plasmids, DNA sequences were obtained for four plasmid genes . The phylogenetic relationships among the plasmids in the ECOR strains is very different from that of the strains themselves . This finding supports the view that plasmid transfer has been frequent within and between the major groups of ECOR . Furthermore, the sequences indicate that recombination between genes in plasmids takes place at a considerably higher frequency than that observed for chromosomal genes . The plasmid genes, and by inference the plasmids themselves, are mosaic in structure with different regions acquired from different sources . Comparison of gene sequences from a variety of naturally occurring plasmids suggested a plausible donor of some of the recombinant regions as well as implicating a chi site in the mechanism of genetic exchange . The relatively high rate of recombination in F-plasmid genes suggests that conjugational gene transfer may play a greater role in bacterial population structure than previously appreciated. Antimicrob Agents Chemother, 1996 Jul, 40(7), 1751 - 3 Novel method for assessing postantibiotic effect by using the Coulter counter; Li RC et al.; To study postantibiotic effect, viable counts are routinely acquired by the pour plate technique . However, this technique is laborious and time-consuming; the required sample dilutions also cause excessive errors and material wastage . A new total cell counting technique in which a Coulter counter is used to obtain more efficient postantibiotic effect measurements has been developed. Am J Vet Res, 1996 Jul, 57(7), 1063 - 6 Use of detergent to prevent initial responses to endotoxin in horses; Longworth KE et al.; OBJECTIVE: To determine whether a detergent can prevent most of the early effects of i.v . infusion with Escherichia coli endotoxin (< 100 ng/kg of body weight) in horses: marked pulmonary hypertension, acute leukopenia, and fever . ANIMALS: 8 healthy adult horses (4 male, 4 female), 415 to 615 kg . DESIGN AND PROCEDURE: Control and detergent experiments were performed in each horse while it was awake but sedated . In control experiments, 10 to 100 ng of E coli endotoxin/kg was given . In detergent experiments, 100 mg of detergent/kg was given 1 hour before injecting endotoxin, similar to the control experiments . RESULTS: In control experiments, pulmonary arterial pressure increased transiently over 40 minutes by 33 +/- 8 mm of Hg (mean +/- SD; P < 0.001), then returned to baseline . Circulating leukocytes decreased to 47 +/- 19% (P < 0.02) of baseline by 1 hour after endotoxin, then increased above baseline by 6 hours . Rectal temperature increased by 0.7 +/- 0.4 C (P < 0.01) . In detergent experiments, the increase in pulmonary arterial pressure was much less than that in the control experiments (8 +/- 7 mm of Hg; P < 0.001) . Circulating leukocytes did not decrease, and the increase in rectal temperature after endotoxic was blocked . CONCLUSIONS: This attenuation of te response to endotoxin may occur because the normal steps in the response of pulmonary intravascular macrophages (ie, endocytosis of endotoxin and subsequent release of inflammatory mediators) are altered by the detergent . This low-technology, inexpensive, and safe treatment could be an important new clinical tool for veterinarians in combating endotoxemia. Plant Mol Biol, 1996 Jul, 31(4), 917 - 22 Isolation of a cDNA from tomato coding for an unregulated, cytosolic chorismate mutase; Eberhard J et al.; A cDNA coding for chorismate mutase was isolated from tomato by complementing a chorismate mutase-deficient Escherichia coli strain with a cDNA library . Southern blot analysis suggests the existence of a single gene of this chorismate mutase type per haploid tomato genome . The abundance of the corresponding transcripts was highest in roots, lower in stems and cotyledons, and even lower in flowers and leaves . The activity of the protein expressed in E . coli was not regulated by the three aromatic amino acids . Characteristics of the sequence and of the enzymatic activity suggest that the identified cDNA encodes a cytosolic, unregulated CM-2 type chorismate mutase. Poult Sci, 1996 Jul, 75(7), 821 - 7 Feeding regimen by sire family interactions on growth, immunocompetence, and disease resistance in chickens; Praharaj NK et al.; Progeny from matings of 12 sires from a White Plymouth Rock line selected for high juvenile BW and 96 dams from a White Leghorn line selected for low antibody production to SRBC were reared under alternate-day (AD) or ad libitum (AL) feeding regimens . Within a feeding regimen males were heavier than females, and within a sex, chicks fed AL were heavier than those fed AD . Feeding regimen by sire family interactions were significant for BW at 21 d of age for both male and female progeny . The interaction was due to differences among sires in the magnitude of the AD: AL relationship . Product moment correlation coefficients between feeding regimens for male and female progeny of sire families for 21-d BW were essentially zero, which was consistent with the sire family by feeding regimen interactions observed at this age . At 41 d of age, relative to BW, weights of empty esophagus plus crop and of crop contents were greater for AD than AL chicks . There were differences among sire families for crop content and breast weights relative to BW . Lesion scores to Escherichia coli challenge were lower and antibody titers to SRBC antigen were higher in AD than in AL chicks . Sire families differed in antibody titers to SRBC antigen . Feeding regimen by sire family interactions were significant for percentage change in BW 144 h after E . coli challenge and lesion scores were greater for AL than AD chicks. Pediatr Res, 1996 Jul, 40(1), 142 - 7 Surfactant protein A-producing cells in human fetal lung are good targets for recombinant adenovirus-mediated gene transfer; Messina E et al.; Local delivery of Escherichia coli beta-galactosidase gene (beta-gal) to surfactant protein-A (SP-A)-producing cells by a replication-defective recombinant adenovirus (AdCMV.beta-gal) was tested in human 8-12-wk-old fetal lung explants cultured in Waymouth's medium . In contrast to uninfected explants, direct addition of 0.8-1.6 x 10(6) plaque-forming units of AdCMV.beta-gal resulted in beta-galactosidase (beta-Gal)-specific staining of the pulmonary epithelium . SP-A localization by indirect immunofluorescence showed positive specific staining of the beta-Gal+ lung epithelial cells, demonstrating that recombinant-defective adenoviruses efficiently transfer reporter genes to fetal lung SP-A+ cells . The reporter gene expression in SPA+ cells persisted for more than 1 mo . No apparent alteration of morphology, phenotype, and growth was observed . The in vitro human lung model described may be useful for testing DNA constructs for vector-mediated gene therapy, as an approach to the treatment of congenital defects and neonatal disorders, such as respiratory distress syndrome and bronchopulmonary dysplasia. J Cardiovasc Pharmacol, 1996 Jul, 28(1), 75 - 81 Reversal by L- and D-enantiomers of NG-nitro-arginine of endotoxin-induced hypotension and vascular hyporesponsiveness; Cheng X et al.; We examined the effects of D-NNA (NG-nitro-D-arginine) and L-NNA (NG-nitro-L-arginine) on suppression of Escherichia coli lipopolysaccharide (LPS)-induced vascular hyporeactivity in pentobarbital-anesthetized rats . Mean arterial pressure (MAP) and pressor response to norepinephrine (NE) were reduced at 40 min (early phase) and 3.5-4 h (late phase) after i.v . injection of LPS (10 mg/kg) . Pretreatment with either D-NNA (16 mg/kg) or L-NNA (8 mg/kg) abolished LPS-induced reduction in MAP and hyporesponsiveness to NE during the early phase but not the late phase of endotoxemia and increased mortality . In contrast, posttreatment with D-NNA and L-NNA at 3 h after the injection of LPS prevented further decreases of MAP and pressor response to NE during the late phase of endotoxemia . The restoration of vascular response by pretreatment with either D-NNA or L-NNA during the early phases or posttreatment with either of these two agents during the late phase of endotoxemia was abolished by i.v . infusion (10 mg/kg/min) of L-arginine (L-Arg), but not D-arginine (D-Arg), suggesting involvement of the L-Arg/ nitric oxide pathway. Photochem Photobiol, 1996 Jul, 64(1), 205 - 10 Effect of glucose on photodynamic action of methylene blue in Escherichia coli cells; Capella M et al.; The results of this work show that the resistance of Escherichia coli cells to the photodynamic action of methylene blue is increased by the addition of glucose to the media in which they are grown . It is postulated that the increased resistance may be due to lowered retention of the dye by cells grown in the presence of glucose, leading to the diminution in DNA damage revealed in the alkaline sucrose gradients . The role of cyclic adenosine-monophosphate in the protective action of glucose is discussed. Appl Environ Microbiol, 1996 Jul, 62(7), 2303 - 10 Cloning of the aldehyde reductase gene from a red yeast, Sporobolomyces salmonicolor, and characterization of the gene and its product; Kita K et al.; An NADPH-dependent aldehyde reductase (ALR) isolated from a red yeast, Sporobolomyces salmonicolor, catalyzes the reduction of a variety of carbonyl compounds . To investigate its primary structure, we cloned and sequenced the cDNA coding for ALR . The aldehyde reductase gene (ALR) comprises 969 bp and encodes a polypeptide of 35,232 Da . The deduced amino acid sequence showed a high degree of similarity to other members of the aldo-keto reductase superfamily . Analysis of the genomic DNA sequence indicated that the ALR gene was interrupted by six introns (two in the 5' noncoding region and four in the coding region) . Southern hybridization analysis of the genomic DNA from S . salmonicolor indicated that there was one copy of the gene . The ALR gene was expressed in Escherichia coli under the control of the tac promoter . The enzyme expressed in E . coli was purified to homogeneity and showed the same catalytic properties as did the enzyme from S . salmonicolor. Endocrinology, 1996 Jul, 137(7), 2947 - 53 Hypothalamic and pituitary leukemia inhibitory factor gene expression in vivo: a novel endotoxin-inducible neuro-endocrine interface; Wang Z et al.; We have recently shown expression of leukemia inhibitory factor (LIF) in human fetal pituitary tissue and its in vitro induction of POMC transcription . We now use qualitative and semiquantitative RT-PCR to demonstrate that LIF and LIF-receptor (LIF-R) are constitutively expressed in the normal mouse hypothalamus and pituitary . Hypothalamic and pituitary LIF and LIF-R are significantly induced (up to 6- and 4-fold, respectively) in vivo in response to lipopolysaccharide endotoxin (LPS) administered to B6D2F1 and C57BL/6 mice . In contrast to the nearly exclusive expression of matrix-associated LIF messenger RNA (mRNA) in control hypothalamus and pituitary, both diffusible and matrix-associated LIF mRNA alternate transcripts are induced by LPS . Furthermore, the time course of peripheral ACTH-response to LPS peaks at 60 min, whereas hypothalamic LIF mRNA increase occurs at 30 min and pituitary LIF induction occurs at 60 min . These results show that mLIF is a novel LPS-inducible proinflammatory neuroendocrine cytokine and the alternatively spliced diffusible LIF may play a paracrine role in activating pituitary ACTH secretion in synergy with hypothalamic CRH, implying a mechanism for central nervous system cytokine responses to immune signals. Rev Soc Bras Med Trop, 1996 Jul-Aug, 29(4), 359 - 62 {Pyogenic liver abscess and schistosomiasis mansoni in the state of EspÃrito Santo}; Musso C et al.; A possible association of the acute toxemic form of schistosomiasis and pyogenic liver abscess (PLA) has been recently suggested . As in the west of the Espirito Santo state schistosomiasis is endemic and PLA are frequently diagnosed in the Children's Hospital of Vitoria we reviewed the records of the Hospital during the period from May 1991 to April 1993 to: a) identify all cases of PLA in which Schistosoma mansoni infection was present and b) annotate the procedence of each case to verify if there is an association of the two diseases . 65 cases of PLA were recorded and 39 had the result of a stool examination, being three positive for Schistosoma mansoni (7.6%) and 26 for other helminth (mainly Ascaris and Trichocephalus) . The procedence of the patients showed that only 7 (10.7%) came from endemic areas . These results show that an association of Schistosoma mansoni infection and PLA was not significative in the country, where the acute toxemic form is not frequent . The great majority of PLA in this study came from the urban periphery of Vitoria, where transmission of schistosomiasis does not occur but intestinal helminth infections are extremely frequent . The great majority of PLA in this study came from the urban periphery of Vitoria, where transmission of schistosomiasis does not occur but intestinal helminth infections are extremely frequent . As 40% of these PLA were cryptogenetic it is possible that the immunomodulation induced by intestinal parasites and the liver granulomas produced by the larvae of these helminths would be predisposing factors for pyogenic liver abscess. Cell Tissue Res, 1996 Jul, 285(1), 39 - 49 Tumor necrosis factor-alpha in macrophages of heart, liver, kidney, and in the pituitary gland; Arras M et al.; Tumor necrosis factor-alpha (TNFalpha) is an important mediator in bacterial lipopolysaccharide (LPS)-induced fever and shock . New data on TNFalpha-producing macrophages in heart, pituitary gland, kidneys and liver in correlation with TNFalpha plasma levels are reported here . In adult rabbits, core temperature and TNFalpha plasma levels are significantly increased at 3 and 24 h after treatment with LPS . After a delay of 6-12 h, the number of TNFalpha-containing macrophages, determined by immunohistochemistry, increases more than fivefold in all organs investigated . With the exception of the pituitary gland, the increase in cell number is correlated with the degree of cellular injury, indicating the involvement of TNFalpha in LPS-induced organ damage that is accompanied by the synthesis of the cytokine . Cortisol levels also increase for at least 24 h after LPS treatment, show peak values 6 h after interleukin-1 treatment, and are unchanged after TNFalpha treatment, indicating the different effects of these factors on the hypothalamo-hypophyseal-adrenocortical axis . This study provides evidence that macrophageal TNFalpha of multi-organ origin is involved in LPS-induced tissue injury and supports the concept of a systemic inflammatory response syndrome . We also show for the first time that in the anterior lobe of the pituitary gland TNFalpha is a normal constituent in cells producing growth hormone but not ACTH . Moreover, most cells of the intermediate lobe are positive for TNFalpha. FEBS Lett, 1996 Jul 1, 389(2), 145 - 8 Crystal structure of macrophage migration inhibitory factor from human lymphocyte at 2.1 A resolution; Sugimoto H et al.; The three-dimensional structure of the macrophage migration inhibitory factor (MIF) from human lymphocytes has been determined by X-ray crystallography at 2.1 A resolution . The structure was solved by a molecular replacement technique using the coordinates of rat MIF . The molecule forms a trimer structure similar to the rat MIF . However, unlike the rat MIF whose C-terminal tail (residues 104-114) is disordered in the crystal, human MIF has a definite main-chain conformation up to the C-terminal end . These eleven residues create two more beta-strands and join to the inter-subunit beta-sheet, which contribute to forming a trimer structure . Thus, the trimer structure consists of three seven-stranded beta-sheets surrounded by six alpha-helices . Each beta-sheet is comprised of beta-strands from each of the three monomers . This architecture is almost identical to 5-carboxymethyl-2-hydroxymuconate isomerase (CHMI) and is related to the E . coli signal transducing protein PII. Pflugers Arch, 1996 Jul, 432(3), 574 - 7 Effects of endotoxin on tone and pressure-responsiveness of preglomerular juxtamedullary vessels; van Lambalgen AA et al.; Endotoxin might affect renal vasoreactivity, but in vivo this is difficult to assess (systemic influences) . Therefore, we used the in vitro blood-perfused juxtamedullary nephron preparation to study early changes in preglomerular vascular reactivity induced by exposure to endotoxin . Pressure-evoked vasomotor responses were determined videometrically by measuring steady-state inside vessel diameters at a perfusion pressure of 60 or 120 mmHg . Intraluminal application of endotoxin (primary contact with endothelium) for 120 min elicited an early (within 30 min) and sustained approximately 25% vasoconstriction from arcuate artery to the distal portions of the afferent arterioles; autoregulatory responses, indicated by pressure-induced vasoconstriction, were unchanged . When topically applied, endotoxin (primary contact with smooth muscle cells) had no vasomotor effects . Significant constrictions, and increases in autoregulatory responses were obtained when the preparation was taken from kidneys from endotoxin-treated rats . Endotoxin had no effect on efferent arteriolar dimensions . Such preferential preglomerular early vasoconstriction is consistent with the early increase in renal resistance and parallel decrease in renal blood flow and glomerular filtration observed during endotoxin shock in vivo . Our results support the concept of local, endothelium-mediated effects of endotoxin on renal vessels. J Allergy Clin Immunol, 1996 Jul, 98(1), 172 - 80 Isolation and characterization of a clone encoding a major allergen (Bla g Bd90K) involved in IgE-mediated cockroach hypersensitivity; Helm R et al.; Previous studies have established that atopic individuals living in cockroach-infested housing become sensitized to cockroach aeroallergens and produce IgE antibodies to a variety of proteins . We describe the isolation of a complementary DNA clone from an expression library, constructed with messenger RNA from German (Blattella germanica) cockroaches, which encodes a major allergen involved in mediating cockroach hypersensitivity . Approximately 0.2% of the clones from a lambda ZAP XR cDNA library bound IgE from a patient with cockroach sensitivity . A randomly selected subset of these clones revealed that they were either different isolates of the same gene or members of a closely related gene family . One of the largest clones (a 4 kb insert) from this subset, Bla g Bd90K hybridized to a single mRNA of approximately the same size . DNA sequence analysis showed that this gene consisted of seven 576 bp tandem repeats with a short unique region at either end . No significant sequence homologies were found between the cockroach clone and any other gene reported in the GenBank database . Serum from 17 of 22 (77%) patients with cockroach hypersensitivity identified IgE-binding recombinant protein expressed from clone Bla g Bd90K in Escherichia coli XL-Blue cells as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis/immunoblot analysis . This recombinant protein migrates with a molecular weight (90 kd) apparently similar to one identified in whole body extracts . We have identified and isolated a cDNA that encodes a major cockroach allergen (Bla g Bd90K) present in German cockroaches. Genetica, 1996 Jul, 98(1), 33 - 41 Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase; Elick TA et al.; The piggyBac Lepidopteran transposable element moves from the cellular genome into infecting baculovirus genomes during passage of the virus in cultured TN-368 cells . We have constructed genetically tagged piggyBac elements that permit analysis of excision when transiently introduced on plasmids into the piggyBac-deficient Spodoptera frugiperda IPLB-SF21AE cell line . Precise excision of the element from these plasmids occurs at a higher frequency in the presence of a helper plasmid that presumably supplies the piggyBac transposase . The results suggest that the piggyBac transposon encodes a protein that functions to facilitate not only insertion, but precise excision as well . This is the first demonstration of piggyBac mobility from plasmid sources in uninfected Lepidopteran cells. J Orthop Res, 1996 Jul, 14(4), 513 - 7 Adenovirus-mediated gene transfer into tendon and tendon sheath; Lou J et al.; In this study, we successfully transferred the Escherichia coli beta-galactosidase gene . LacZ, into the chicken tendon and tendon sheath by a recombinant adenovirus . The recombinant adenovirus Adv-beta gal that carried the E . coli LacZ gene was constructed by homologous recombination in 293 cells (human transformed embryonic kidney) between the expressing vector and the ClaI large fragment of adenovirus 5 genome . Each chicken received a 10 microliters injection containing 10(5) plaque-forming units of recombinant virus Adv-beta gal . into the tendon sheath of the long toe Samples of tendon and tendon sheath were harvested at 3.30, and 75 days after the injection . The LacZ gene transfer was detected for its coding product beta-galactosidase by staining with X-gal solution . The results showed that all tendon and tendon sheath samples from the three harvest times stained positive (blue) . The tendon sheath samples were more extensively stained; staining of the tendon was limited to the epitenon layer . These data suggest that a functional exogenous gene can potentially be transferred into the tendon and tendon sheath by similar techniques; such techniques may be used to improve healing and reduce adhesion formation. FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 159 - 63 A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene; Daruwala R et al.; A new gene (menF) encoding an isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis has been cloned and sequenced . Overexpression of the encoded polypeptide under the influence of a T7 promoter showed an increase in specific activity of 2200-fold . Treatment with protamine sulfate resulted in another 3.5-fold increase in specific activity (7700-fold compared to the parent strain) . The relative molecular mass of the overexpressed protein was M(r) 49 000, which is in full agreement with the DNA sequence predicted molecular mass of 48 777 Da . Purified enzyme converted chorismate to isochorismate with the product of the reaction shown to be isochorismate by its thermal conversion to salicylic acid . The fluorescence spectrum generated by the formed salicylic acid was identical to that of authentic salicylic acid . The 5' end of the flanking menD gene has also be redefined. FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 139 - 44 Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep; Cloeckaert A et al.; We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and which in addition made possible the serological differentiation between infected and B . melitensis Rev . 1 vaccinated sheep . Monoclonal antibodies against CP28 were used to screen a B . melitensis 16M genomic library and to clone the corresponding gene . DNA sequencing of the gene encoding CP28 of B . melitensis 16M revealed that it was nearly identical to that of the recently published bp26 gene of Brucella abortus vaccine strain S19 coding for a periplasmic protein . The differences between the B . melitensis 16M gene and that of B . abortus S19 consisted of single nucleotide substitutions, one or two codon deletions, one codon addition, and most importantly a 21-bp deletion . The corresponding region of B . abortus S19 contains two 10-bp direct repeats which could have been involved in the genesis of the deletion . Expression of the B . melitensis 16M bp26 gene in Escherichia coli studied by the use of the monoclonal antibodies showed the same characteristics as reported for the B . abortus S19 bp26 gene, i.e . the presence of a higher molecular mass preprotein and a lower molecular mass band which probably corresponds to the mature protein exported to the periplasm . Immunoblotting performed with sera from either naturally infected or B . melitensis H38 experimentally infected sheep confirmed the importance of the B . melitensis CP28/BP26 protein as diagnostic antigen. J Bacteriol, 1996 Jul, 178(14), 4310 - 2 Molybdenum cofactor biosynthesis in Escherichia coli mod and mog mutants; Joshi MS et al.; The molybdopterin content of Escherichia coli mod and mog mutants was estimated by conversion to the form A derivative . The results are in accord with complete phenotypic repair of mod, and incomplete repair of mog, by culture in high concentrations of molybdate . A possible role for Mog as a molybdochelatase is discussed. J Bacteriol, 1996 Jul, 178(14), 4294 - 6 Overexpression of vsr in Escherichia coli is mutagenic; Doiron KM et al.; Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations . The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair. J Bacteriol, 1996 Jul, 178(14), 4289 - 93 Chloramphenicol causes fusion of separated nucleoids in Escherichia coli K-12 cells and filaments; van Helvoort JM et al.; Chloramphenicol is frequently used for better visualization of the Escherichia coli nucleoid . Here, we show that chloramphenicol causes not only rounding off of the nucleoid but also fusion of as many as four separated nucleoids . Nucleoid fusion occurred in fast-growing cells and in filaments obtained by dicF antisense RNA induction or in ftsZ84(Ts) and pbpB(Ts) mutants . Thus, treatment with chloramphenicol erroneously suggests that DNA segregation is inhibited. J Bacteriol, 1996 Jul, 178(14), 4208 - 15 Tyrosine 106 of CheY plays an important role in chemotaxis signal transduction in Escherichia coli; Zhu X et al.; CheY is the response regulator in the signal transduction pathway of bacterial chemotaxis . Position 106 of CheY is occupied by a conserved aromatic residue (tyrosine or phenylalanine) in the response regulator superfamily . A number of substitutions at position 106 have been made and characterized by both behavioral and biochemical studies . On the basis of the behavioral studies, the phenotypes of the mutants at position 106 can be divided into three categories: (i) hyperactivity, with a tyrosine-to-tryptophan mutation (Y106W) causing increased tumble signaling but impairing chemotaxis; (ii) low-level activity, with a tyrosine-to-phenylalanine change (Y106F) resulting in decreased tumble signaling and chemotaxis; and (iii) no activity, with substitutions such as Y106L, Y106I, Y106V, Y106G, and Y106C resulting in no chemotaxis and a smooth-swimming phenotype . All three types of mutants can be phosphorylated by CheA-phosphate in vitro to a level similar to that of wild-type CheY . Autodephosphorylation rates are similar for all categories of mutants . All mutant proteins displayed less than twofold increased rates compared with wild-type CheY . Binding of the mutant proteins to FliM was similar to that of the wild-type CheY in the CheY-FliM binding assays . The combined results from in vivo behavioral and in vitro biochemical studies suggest that the diverse phenotypes of the Y106 mutants are not due to a variation in phosphorylation or dephosphorylation ability nor in affinity for the switch . With reference to the structures of wild-type CheY and the T871 CheY mutant, our results suggest that rearrangements of the orientation of the tyrosine side chain at position 106 are involved in the signal transduction of CheY . These data also suggest that the binding of phosphoryl-CheY to the flagellar motor is a necessary, but not sufficient, event for signal transduction. J Bacteriol, 1996 Jul, 178(14), 4176 - 81 Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli; Rajkumari K et al.; We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the sigma(s) and sigma70-bearing RNA polymerase holoenzymes, respectively . Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of sigma(s) selectivity at the P1 promoter . Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate . This study validates the existence of dual promoters with dual specificities for proU transcription . Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS. J Bacteriol, 1996 Jul, 178(14), 4105 - 14 Roles of SpoT and FNR in NH4+ assimilation and osmoregulation in GOGAT (glutamate synthase)-deficient mutants of Escherichia coli; Saroja GN et al.; An osmosensitive mutant of Escherichia coli was isolated and shown to harbor two mutations that were together necessary for osmosensitivity . One (ossB) was an insertion mutation in the gltBD operon, which encodes the enzyme glutamate synthase (GOGAT), involved in ammonia assimilation and L-glutamate biosynthesis . The other (ossA) was in the fnr gene, encoding the regulator protein FNR for anaerobic gene expression . Several missense or deletion mutations in fnr and gltBD behaved like ossA and ossB, respectively, in conferring osmosensitivity . A mutation affecting the DNA-binding domain of FNR was recessive to fnr+ with respect to the osmotolerance phenotype but was dominant-negative for its effect on expression of genes in anaerobic respiration . Our results may most simply be interpreted as suggesting the requirement for monomeric FNR during aerobic growth of E . coli in high-osmolarity media, presumably for L-glutamate accumulation via the GOGAT-independent pathway (catalyzed by glutamate dehydrogenase {GDH}), but the mechanism of FNR action is not known . We also found that the spoT gene (encoding guanosine 3',5'-bispyrophosphate {ppGpp} synthetase II/ppGpp-3' pyrophosphohydrolase), in multiple copies, overcomes the defect in NH4+ assimilation associated with GOGAT deficiency and thereby suppresses osmosensitivity in gltBD fnr strains . Enhancement of GDH activity in these derivatives appears to be responsible for the observed suppression . Its likely physiological relevance was established by the demonstration that growth of gltBD mutants (that are haploid for spoT+) on moderately low {NH4+} was restored with the use of C sources poorer than glucose in the medium . Our results raise the possibility that SpoT-mediated accumulation of ppGpp during C-limited growth leads to GDH activation and that the latter enzyme plays an important role in N assimilation in situ hitherto unrecognized from studies on laboratory-grown cultures. J Bacteriol, 1996 Jul, 178(14), 4012 - 9 Transcript analysis of the c-vac region and differential synthesis of the two regulatory gas vesicle proteins GvpD and GvpE in Halobacterium salinarium PHH4; Kruger K et al.; Halobacterium salinarium PHH4 synthesizes gas vesicles in the stationary growth phase by the expression of 14 gyp genes arranged in two clusters . The chromosomal gvpACNO (c-gvpACNO) gene cluster (encoding the major structural gas vesicle protein GvpA and the minor structural protein GvpC was transcribed as three mRNA species starting at one promoter during the stationary phase of growth . The second gene cluster, c-gvpDEFGHIKLM), was transcribed during all stages of growth as a relatively unstable, single mRNA with a maximal length of 6 kb . In addition, a 1.7-kb c-gvpD transcript was synthesized during stationary growth starting at the same promotor as that of the cgvpDEFGHIJKLM mRNA . The expression of the first two genes located in this unit (c-gvpD and c-gvpE) was also monitored by Western blot (immunoblot) analyses using antisera raised against these proteins synthesized in Escherichia coli . While the cGvpD protein was present only during early exponential growth and disappeared during gas vesicle formation, the cGvpE protein was present during cGvpA and gas vesicle synthesis in the early stationary phase of growth . Previous data indicated that cGvpD is involved in repression of gas vesicle formation, whereas cGvpE is a transcriptional activator for the c-gvpA promoter . The appearance of both proteins during the growth cycle is in line with the functions of these proteins in gas vesicle synthesis . The mechanism of the differential translation of cGvpD and cGvpE from the c-gvpDEFGHIJKLM rnRNA still has to be elucidated, but antisense RNAs complementary to the 5' terminus as well as the 3' portion of the c-gvpD mRNA might be involved in this regulation . Such RNAs occurred during early stationary growth when the cGvpD protein level decreased and may possibly inhibit the translation of the c-gvpD mRNA. Comp Biochem Physiol B Biochem Mol Biol, 1996 Jul, 114(3), 303 - 11 Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution; Hakansson K et al.; Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized . The mouse and rat inhibitors were both fully active in titrations of papain . Determination of equilibrium constants for dissociation (Ki) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (Ki 0.07-0.13 nM) . Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors . Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay . The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates . All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues . For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content . In the mouse, a notable high cystatin C content in parotid gland tissue was observed . The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy. Am J Physiol, 1996 Jul, 271(1 Pt 2), R244 - 53 First and second phases of biphasic fever: two sequential stages of the sickness syndrome? Romanovsky AA, Kulchitsky VA, Akulich NV, Koulchitsky SV, Simons CT, Sessler DI, Gourine VN. We hypothesized that the systemic inflammatory response undergoes two consecutive stages, each characterized by different nonspecific sickness patterns . To test this hypothesis, we studied thermal, nociceptive, and motor responses to lipopolysaccharide (LPS) in 43 unanesthetized, habituated, and lightly restrained male Wistar rats previously implanted with a catheter in the jugular vein . Escherichia coli LPS was injected intravenously in a dose of 0, 0.1, 1, 10, 100, or 1,000 micrograms/kg . Colonic temperature (Tc) was measured with a thermocouple . Changes in nociception were assessed by tail flick latency (TFL) to a noxious heat stimulus . Motor activity was evaluated using an observation-based activity score (AS) . The two lowest doses were apyrogenic . The next dose induced a monophasic fever with a maximal Tc rise of 0.9 +/- 0.2 degrees C at 108 +/- 11 min post-LPS . The next two higher doses caused biphasic fevers with the first and second peaks of 0.7 +/- 0.1 and 1.4 +/- 0.1 degrees C (10 micrograms/kg) and 0.7 +/- 0.1 and 1.4 +/- 0.2 degrees C (100 micrograms/kg) occurring at 60 +/- 6 and 165 +/- 17 min and at 45 +/- 3 and 141 +/- 6 min, respectively . The highest dose of LPS resulted in a Tc fall (nadir, -0.6 +/- 0.1 degree C at 83 +/- 6 min) . Two different sickness patterns were exhibited . The first (high Tc, low TFL and high AS) occurred during the monophasic fever and the first (early) phase of the biphasic fevers, and it was termed the early phase syndrome . The second pattern (high or low Tc, high TFL, and low AS) developed during the second (late) phase of the biphasic fevers and LPS-hypothermia (endotoxin shock), and it was termed the late phase syndrome . Occurring at different stages of the systemic inflammatory response and developing through different coping patterns {fight/flight (energy expenditure) vs . depression/withdrawal (energy conservation)}, the two syndromes represent two different types of adaptation to infection and have different biological significance . Viewing sickness as a dynamic entity is justified clinically . Such a dynamic approach to the problem resolves several contradictions in the current concept of sickness. Am J Physiol, 1996 Jul, 271(1 Pt 1), G97 - 103 A diet containing glycine improves survival in endotoxin shock in the rat; Ikejima K et al.; In this study, we investigated the effects of a glycine-containing diet (5%) on mortality and liver injury due to intravenous injection of endotoxin {Escherichia coli lipopolysaccharide (LPS)} in Sprague-Dawley rats in vivo . Fifty percent of the rats fed control diet died within 24 h after an intravenous injection of LPS (10 mg/kg), whereas feeding the rats glycine totally prevented mortality and markedly reduced an LPS-induced elevation of serum transaminase levels, hepatic necrosis, and lung injury . The elevation in serum tumor necrosis factor-alpha (TNF-alpha) due to LPS was also blunted and delayed significantly by glycine feeding . In a two-hit model (hepatic ischemia-reperfusion and injection of sublethal LPS), all rats fed control diet died, whereas 83% of glycine-fed animals survived with a significant reduction in transaminases and improved liver and lung histology . LPS elevated intracellular Ca2+ concentration ({Ca2+}i) in cultured Kupffer cells, an effect blocked almost completely by glycine . Glycine most likely reduces injury and mortality by preventing the LPS-induced elevation of {Ca2+}i in Kupffer cells, thereby minimizing toxic eicosanoid and cytokine production. Am J Physiol, 1996 Jul, 271(1 Pt 1), C385 - 90 Enhanced NO production during Mg deficiency and its role in mediating red blood cell glutathione loss; Mak IT et al.; The effect of dietary Mg deficiency on nitric oxide (NO) production and its role in mediating oxidative depletion of red blood cell (RBC) glutathione in rats were investigated . Male Sprague-Dawley rats were placed on Mg-deficient or Mg-sufficient diets for up to 3 wk . Plasma nitrate plus nitrite levels, determined by the Escherichia coli reductase/Griess reagent procedures, increased 1.7-fold during the 1st wk and increased 2- to 2.4-fold during the 2nd and 3rd wk on the Mg-deficient diet . In association, substantial losses (approximately 50%) of RBC glutathione occurred during the 2nd and 3rd wk . Administration of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in drinking water (0.5 mg/ml) effectively blunted the increases in plasma nitrate/nitrite during Mg deficiency . Concomitantly, losses of RBC glutathione exhibited by Mg-deficient rats were significantly attenuated . Packed RBCs, obtained from Mg-deficient but not from Mg-sufficient animals, displayed a prominent nitrosyl hemoglobin signal detected by electron spin resonance spectroscopy; the signals of the samples from the L-NAME-treated Mg-deficient rats were greatly reduced . With isolated RBCs, losses of the glutathione could be induced directly by peroxynitrite or 3-morpholinosydnonimine, which generates NO + .O2-, but not by NO (from sodium nitroprusside) alone, in a concentration-dependent manner . The results clearly indicate that NO overproduction occurs and participates in RBC glutathione loss during Mg deficiency . Because neutrophil activation also occurs, we suggest that NO might interact with superoxide anions to form peroxynitrite, which then directly oxidizes RBC glutathione. Plant J, 1996 Jul, 10(1), 83 - 90 Expression of Escherichia coli branching enzyme in tubers of amylose-free transgenic potato leads to an increased branching degree of the amylopectin; Kortstee AJ et al.; In order to increase the branching degree of potato tuber starch, the gene encoding branching enzyme (glgB) of Escherichia coli was expressed in the amylose-free potato mutant . The E . coli glgB was cloned in the binary vector pBIN19 under the transcriptional control of the potato Granule Bound Starch Synthase (GBSS) promoter and transitpeptide sequence . The E . coli glgB was cloned behind the two N-terminal amino acids of the GBSS mature protein, creating a chimeric protein . Transgenic plants were obtained which expressed the E . coli branching enzyme as was shown by the presence of mRNA and protein in the tubers . Correctly processed protein was found both in the soluble and starch granule bound protein fraction . Analysis of the starch showed an increase in the branching degree (DE) of up to 25% more branchpoints . The increase in the number of branchpoints was due to the presence of more short chains, with a degree of polymerization (DP) of 16 glucose-residues or less in the amylopectin . Changes in other characteristics of the starch, such as average chain length (CL) and lambda max, indicated a more branched structure for starch of transformed plants as well. J Gen Virol, 1996 Jul, 77 ( Pt 7), 1421 - 3 Characterization of the gene encoding core protein VP6 of two African horsesickness virus serotypes; Turnbull PJ et al.; The genes encoding the inner core protein VP6 of African horsesickness virus (AHSV) serotypes 3 and 6 have been cloned and sequenced . The genes are 1169 nucleotides in length and both encode a largely hydrophilic protein of 369 amino acids . The VP6 amino acid sequence is highly conserved between the two serotypes with an overall similarity of 95 percent . Comparison of the AHSV VP6 amino acid sequences with those of bluetongue virus serotype 10 VP6 revealed that it is 41 amino acids longer with an overall amino acid identity of 29 percent . The similarity is mainly confined to a short but highly conserved 13 amino acid region at the N terminus, a short seven amino acid region at the C terminus and a 22 amino acid region close to the C terminus . Within this last region is a smaller 11 amino acid region from 318 to 328 with a 91 percent similarity to the Rep helicase of Escherichia coli. Microbiology, 1996 Jul, 142 ( Pt 7), 1833 - 40 Transcriptional regulation of the Escherichia coli rhaT gene; Via P et al.; Transcriptional regulation of the rhaT gene, one of the operons forming the rhamnose regulon in Escherichia coli, was studied by fusing its complete or deleted promoter to the reporter gene lacZ . Analysis of beta-galactosidase activities induced in these constructions grown under different conditions predicted the presence of two putative control elements: one for the RhaS regulatory protein and activating the gene not only by L-rhamnose but also by L-lyxose or L-mannose, the other for cAMP-catabolite repression protein and activating this gene in the absence of glucose . Anaerobiosis increased the promoter function two- to threefold with respect to the aerobic condition . Experiments involving complementation of strains containing the rhaT-promoter fusion and carrying a deletion in the rhaS and/or rhaR genes with plasmids bearing the rhamnose regulatory genes showed that rhaT is controlled by a regulatory cascade, in which RhaR induces rhaSR and the accumulated RhaS directly activates rhaT. Microbiology, 1996 Jul, 142 ( Pt 7), 1819 - 24 RNA polymerase, PurR and MetR interactions at the glyA promoter of Escherichia coli; Lorenz E et al.; In Escherichia coli, the MetR and PurR proteins positively and negatively regulate glyA gene expression, respectively . A DNase I footprint analysis showed that both proteins bind independently to the glyA control region . The PurR protein blocks RNA polymerase (RNAP) from binding to the glyA promoter . The presence of hypoxanthine, the co-repressor of PurR, increases the ability of PurR to prevent RNAP binding, providing a model for repression of the glyA gene by PurR . In contrast, MetR alters the RNAP footprint pattern of the glyA control region . In addition, the MetR footprint is increased in the presence of RNAP, suggesting that the two proteins might interact. Microbiology, 1996 Jul, 142 ( Pt 7), 1659 - 65 The bglX gene located at 47.8 min on the Escherichia coli chromosome encodes a periplasmic beta-glucosidase; Yang M et al.; A new Escherichia coli gene, bgIX, encoding a beta-D-glucosidase (EC 3.2.1.21) has been characterized . The bgIX gene is located adjacent to the dld gene at 47.8 min or 2225 kb on the E . coli chromosome . The sequence of a 2.6 kb DNA fragment from this region revealed a large open reading frame encoding a protein of 765 amino acids . The BgIX protein was purified from the periplasm, and amino-terminal sequencing suggests that the mature protein is derived from cleavage of a 20 residue signal peptide . A search of the sequence databases revealed that BgIX is a member of a large family of beta-glucosidases from a variety of bacteria and fungi, and the plant Arabidopsis thaliana . It differs from the other four E . coli phospho-beta-glucosidases in sequence and in its periplasmic rather than cytoplasmic location . The BgIX enzyme has a Km of 18 +/- 1 mM and a Vmax of 3 +/- 0.7 mumol min-1 for the colorimetric substrate o-nitrophenyl beta-D-glucopyranoside. Br J Haematol, 1996 Jul, 94(1), 191 - 7 A novel mutation in the ferrochelatase gene associated with erythropoietic protoporphyria; Imoto S et al.; Erythropoietic protoporphyria (EPP) is a hereditary disorder caused by mutations of the ferrochelatase gene . We investigated a Japanese patient with a dominant form of erythropoietic protoporphyria for a ferrochelatase mutation . Sequence analysis of the proband's ferrochelatase cDNA revealed a T to C point mutation at nucleotide 557 . This mutation resulted in the replacement of Ile by Thr at amino acid position 186, a novel mutation in erythropoietic protoporphyria . An increase in ferrochelatase activity was not observed in the crude extract of E . coli over-expressing the mutant protein compared with the control, whereas a marked increase in activity was observed in that over-expressing the wild type . Prediction of the secondary structure of ferrochelatase suggested that the Ile186-->Thr mutation changed the original beta-sheet structure to an alpha helix in the region including amino acid residue of mutation . We conclude that, in the patient, the Ile186-->Thr mutation had abolished enzyme activity, possibly by disrupting the secondary structure, thereby causing erythropoietic protoporphyria. RNA, 1996 Jul, 2(7), 674 - 81 Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm; Gaur RK et al.; The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P . In general agreement with previous data, all affected sites were localized in acceptor stem and T arm . But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E . coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences . In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop . These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes . Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)) . This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA. RNA, 1996 Jul, 2(7), 652 - 63 A chloroplast transcript lacking the 3' inverted repeat is degraded by 3'-->5' exoribonuclease activity; Drager RG et al.; Chlamydomonas reinhardtii strains harboring deletions of the chloroplast atpB 3' inverted repeat (IR) are weakly phototrophic due to reduced accumulation of discrete atpB transcripts and the chloroplast ATPase beta-subunit protein . A sequence of 18 guanosine residues, which can impede a 3'-->5' exoribonuclease in vitro, is able to substitute for the atpB IR in vivo . Strains containing the poly-guanosine tract in place of the atpB 3' IR are phototrophic and accumulate near wild-type levels of discrete atpB transcripts and the ATPase beta-subunit protein . Because these atpB transcripts contain the 18 guanosine residues, and the poly-guanosine tract is not a terminator of transcription, the accumulation of discrete atpB transcripts is likely the result of impediment of 3'-->5' exoribonuclease activity . These findings support a model in which atpB transcripts lacking the 3' IR are degraded by 3'-->5' exoribonuclease activity, and demonstrate that the poly-guanosine tract can be used to study chloroplast RNA metabolism in vivo. Mol Biol Evol, 1996 Jul, 13(6), 864 - 72 Synonymous codon bias is related to gene length in Escherichia coli: selection for translational accuracy? Eyre-Walker A. The levels of synonymous codon bias is shown to be positively correlated to gene length in Escherichia coli genes which are thought to be expressed at similar levels; these are genes whose products are present in multimeric proteins in equimolar amounts . It is argued that the positive correlation could be caused by selection to avoid missense errors during translation . Since the cost of producing a protein is proportional to its length, selection in favor of codons which increase accuracy should be greater in longer genes, and long genes should therefore have higher synonymous codon bias . It is also shown that there is variation in synonymous codon use which is independent of either expression level, gene length, amino acid composition, or chromosomal location . This variation is consistent with selection for translational accuracy but may have other origins. Ann R Coll Surg Engl, 1996 Jul, 78(4), 350 - 3 Pathogenesis of pancreatic infection; Widdison AL; John Hunter studied comparative anatomy of the pancreas but was unaware of pancreatic infection which is now the leading cause of mortality in pancreatitis . This was investigated using a feline model of pancreatitis . Pathogens spread to the healthy and inflamed gland from many sources including colon, gallbladder, or a septic focus and by various routes including the circulation, reflux into the pancreatic duct or by transmural migration from the colon . Colonisation risk was proportional to necrosis and inflammation, confirming clinical observations . These studies showed that pathogens frequently colonised the pancreas, but infection developed only in animals with pancreatitis . In cats with pancreatitis, phagocytic function was reduced by 28% . This was probably owing to phagocytic capacity being overwhelmed by protease-antiprotease complexes because, in humans, granulocyte and lymphocyte function was normal . These experiments suggested that it would be difficult to prevent pancreatic colonisation, but indicated some types of therapy may have potential . These were investigated using this animal model of pancreatic infection . Treatment with either cefotaxime or levamisole (an immunostimulant) were effective . However, the anti-inflammatory drug dopamine, which reduced inflammation, did not eradicate all pathogens. Hepatology, 1996 Jul, 24(1), 219 - 25 Role of Kupffer cells and the spleen in modulation of endotoxin-induced liver injury after partial hepatectomy; Suzuki S et al.; The hypothesis that both activated Kupffer cells and the spleen may be responsible for endotoxin-induced liver injury following partial hepatectomy was investigated . Male rats were divided into a sham group receiving laparotomy alone and three groups receiving a two-thirds hepatectomy; one group was given normal saline (NS) solution as a vehicle control, one group received intravenous gadolinium chloride (GC group) (7 mg/kg body weight) for 2 days before intravenous injection of endotoxin to inhibit Kupffer cell phagocytosis, and the third group simultaneously underwent splenectomy and partial hepatectomy (SH group) . As endotoxin, lipopolysaccharide (LPS) (1 mg/kg body weight) was administered intravenously 2 days after surgery . In the GC and SH groups, phagocytic activity was reduced to approximately 40% of that in the sham group . The highest plasma tumor necrosis factor alpha (TNF-alpha) level (8,544 +/- 1,223 pg/mL) was observed in the NS group at 1 hour after LPS administration, and the level was significantly reduced by GdCl3 or splenectomy (P < 0.05) . Inhibition of Kupffer cell function and splenectomy attenuated functional and structural liver damage associated with the decreased hepatic infiltration of polymorphonuclear leukocytes (PMNs) and reduced priming of circulating PMNs in the early stage of endotoxemia following partial hepatectomy . Consequently, the 24-hour survival rate of the SH and GC groups was significantly improved to 50% and 80%, respectively (P < .05), while that of the NS group was 12.5% . These findings indicate that the modification of inflammatory mediator generation by splenectomy or inhibition of Kupffer cell function may be beneficial for the prevention of endotoxin-induced liver injury after partial hepatectomy. Eur J Biochem, 1996 Jul 1, 239(1), 229 - 34 Kinetics of phosphorolysis of 3-(beta-D-ribofuranosyl)adenine and 3-(beta-D-ribofuranosyl)hypoxanthine, non-conventional substrates of purine-nucleoside phosphorylase; Bzowska A et al.; The properties of two non-conventional substrates of the calf-spleen and Escherichia coli purine nucleoside phosphorylases (PNP), 3-(beta-D-ribofuranosyl)adenine (RibfAde) and 3-(beta-D-ribofuranosyl)hypoxanthine (RibfHyp), are described . In contrast to Ado, RibfAde is a substrate for the mammalian enzyme . With the calf enzyme, the pseudo-first-order rate constants (Vmax/K(m)) for phosphorolysis of RibfAde and RibfHyp are 3% and 13%, respectively, that for phosphorolysis of Ino, while for E . coli PNP the corresponding values are 22% and 30%, respectively . The Michaelis constants (K(m)) for RibfAde were 800 microM (calf PNP) and 150 microM (E . coli PNP) . For RibfHyp, the corresponding K(m) values were 220 microM and 260 microM . Two well-characterized inhibitors of calf spleen PNP {9-(2-fluoro-3,4-dihydroxybutyl)guanine} and E . coli PNP (formycin A) were found to inhibit phosphorolysis of RibfAde and RibfHyp with the same inhibition constants as for Ino . Moreover, the inhibition was competitive, which indicates that phosphorolysis of 3-beta-nucleosides occurs at the same active site as for the natural substrate Ino . In particular, the substrate properties of both 3-beta-nucleosides are consistent with their binding to the enzyme in the conformation anti to the imidazole ring about the glycosidic bond, which is superimposable on the structure of natural 9-beta-nucleosides in the conformation anti to the pyrimidine ring . The results are examined in relation to present concepts regarding the binding of substrates and inhibitors at the active site(s) of these enzymes. Eur J Biochem, 1996 Jul 1, 239(1), 214 - 9 A deletion in the first cysteine-rich repeat of the low-density-lipoprotein receptor leads to the formation of multiple misfolded isomers; Djordjevic JT et al.; The ligand-binding domain of the low-density-lipoprotein (LDL) receptor comprises seven cysteine-rich repeats, each approximately 40 amino acids long . The deletion of two amino acids (Asp26 and Gly27) from the first of these repeats (LB1), leads to a defective LDL receptor, and the clinical syndrome of familial hypercholesterolemia {Leitersdorf, E., Hobbs, H . H., Fourie, A . M., Jacobs, M., van der Westhuyzen, D.R . & Coetzee, G.A . (1988) Proc . Natl Acad . Sci . USA 85, 7912-7916} . Receptors which reach the cell surface fail to bind IgG-C7, a conformation-specific monoclonal antibody directed to LB1 . To determine the effects of the two-amino-acid deletion on the folding of the LB1 of the LDL receptor, we have expressed LB1 and the mutant repeat, des-Asp26, Gly27-LB1, as recombinant (rLB1 and des-Asp26, Gly27-rLB1) peptides, and have determined their ability to fold in vitro . Unlike rLB1, which folded into a single isomer that was recognized by IgG-C7 and had three disulfide bonds, des-Asp26, Gly27-rLB1 folded into an equilibrium mixture of four isomers . Each of these isomers contained three disulfide bonds, but none were recognized by IgG-C7 . We suggest that LDL receptors in the endoplasmic reticulum (ER) of the cell also fold into an equilibrium mixture of distinct receptor molecules, each with an abnormally folded isomer of des-Asp26, Gly27-LB1, and that the retarded transport of receptors to the cell surface arises because only a subset of the isomers reaches the cell surface. Blood, 1996 Jul 1, 88(1), 174 - 83 Effects of verocytotoxin-1 on nonadherent human monocytes: binding characteristics, protein synthesis, and induction of cytokine release; van Setten PA et al.; The epidemic form of the hemolytic uremic syndrome (HUS) has been associated with a verocytotoxin producing Escherichia coli infection . Endothelial cell damage of glomeruli and arterioles of the kidney plays a central role in the pathogenesis of HUS . A number of observations in vivo and in vitro indicate that inflammatory mediators contribute to this process . In this study we investigated the binding of 125I-verocytotoxin-1 (VT-1) to freshly isolated human nonadherent monocytes as well as the nature of the ligand to which VT-1 binds on monocytes . On the average, freshly isolated monocytes have 0.07 x 10(5) specific binding sites for 125I-VT-1 per cell . Preincubation of nonadherent monocytes with bacterial lipopolysaccharide (LPS) caused a 23- to 30-fold increase of specific binding sites for VT-1 as shown by Scatchard plot analysis . Thin-layer chromatography of extracted neutral glycolipids of the cells and subsequent binding of 125I-VT-1 showed that human monocytes bind VT-1 to a globotriaosylceramide (Gb3) species that is different from that found on endothelial cells, probably a short-chain fatty acyl Gb3 or an alpha-OH-Gb3 . In addition, we evaluated the functional consequences of VT-1 binding to human monocytes by investigating the effects of VT-1 on the total protein synthesis and, specifically, the production of the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 . We observed that VT-1 did not inhibit overall protein synthesis, nor under basal conditions, neither after stimulation with LPS, in contrast to previous observations with endothelial cells . Furthermore, we found that VT-1 induces the synthesis of the cytokines IL-1 beta, TNF-alpha, IL-6, and IL-8 in nonstimulated monocytes by a LPS-independent cell activation . The increase in the production of cytokines was parallelled by an increase in mRNA, as was demonstrated for IL-6 by reverse transcription-polymerase chain reaction . These data suggest that inflammatory mediators locally produced by VT-1-stimulated monocytes may contribute to the pathogenic mechanism of the HUS. FEMS Microbiol Rev, 1996 Jul, 18(4), 301 - 17 Adaptation to life at micromolar nutrient levels: the regulation of Escherichia coli glucose transport by endoinduction and cAMP; Ferenci T; Free-living bacteria are expert in adapting to variations in nutrient availability, often using an array of transport systems of different affinities to scavenge for particular substrates (multiport) . This review concentrates on the regulation of expression of different transporters contributing to multiport in response to varying nutrient levels . A novel mechanism of controlling bacterial transport affinity under sugar limitation is described . In particular, switching from glucose-rich to glucose-limited conditions results in Escherichia coli orchestrating outer membrane changes as well as the induction of a periplasmic binding protein-dependent (ABC-type) transport system . The changes leading to the high affinity transport pathway are directed towards uptake of rapidly utilisable concentrations and are optimal close to 10-6 M medium glucose . High affinity transport is absent under both glucose-rich 'feast' and glucose-starved 'famine' conditions hence high affinity transporters are not simply repressed by excess nutrient . Rather, the improvement in glucose scavenging involves induction of genes in 2 distinct regulons (mgl/gal and mal/lamB) through synthesis of 2 different endogenous inducer molecules (galactose, maltotriose) . Endoinducer levels are tightly controlled by extracellular glucose concentration at different glucose-limited growth rates . Aside from endoinducers, the elevated intracellular level of cAMP plays a role in induction of the high-affinity pathway but cAMP-mediated relief from catabolite repression is not itself sufficient for high affinity transport . In contrast to the repressive role of glucose when present at millimolar concentrations, micromolar glucose also leads to the induction of transport systems for other sugars, further broadening the scavenging potential of nutrient-limited bacteria for other substrates. Infect Immun, 1996 Jul, 64(7), 2881 - 4 The Hha protein as a modulator of expression of virulence factors in Escherichia coli; Mourino M et al.; We constructed hha derivatives from both a clinical uropathogenic Escherichia coli isolate (strain FVL4) and a wild E . coli strain causing bovine diarrhea (strain CCB21) and analyzed the effect of the hha allele on the expression of the different virulence factors exhibited by these strains . Expression of hemolysin and of the Vir antigen was altered in hha mutants . Whereas production of hemolysin by strain FVL4 was repressed both at a low temperature and at high osmolarity, the hha allele accounted for a significant increase of hemolysin production under these conditions . Also, the low temperature-sensitive expression of the Vir adhesin was modified in hha mutants, which were able to express this adhesin at a low temperature . Expression of other virulence factors, such as cytotoxic necrotizing factor type 1 and 2 toxins, remained unmodified in hha derivatives of strains FVL4 and CCB21. Infect Immun, 1996 Jul, 64(7), 2877 - 80 Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity; Bauerfeind P et al.; Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration in humans, synthesizes urease, a nickel metalloenzyme, as its most abundant protein . NixA, a high-affinity nickel transport protein, allows synthesis of catalytically active urease when coexpressed with H . pylori urease in an Escherichia coli host . To determine whether NixA is essential for the production of active urease in H . pylori, nixA was insertionally inactivated with a kanamycin resistance cassette (aphA) and this construct was electroporated into H . pylori ATCC 43504; allelic exchange mutants were selected on kanamycin-containing medium . The nixA mutation, confirmed by PCR, reduced urease activity by 42% (140 +/- 70 micromol of NH3/min/mg of protein in the mutant versus 240 +/- 100 micromol of NH3/min/mg of protein in the parent (P = 0.037) . Rates of nickel transport were dramatically reduced (P = 0.0002) in the nixA mutant (9.3 +/- 3.7 pmol of Ni2+/min/10(8) bacteria) of H . pylori as compared with the parent strain (30.2 +/- 8.1 pmol of Ni2+/min/10(8) bacteria) . We conclude that NixA is an important mediator of nickel transport in H . pylori . That residual nickel transport and urease activity remain in the nixA mutant, however, provides evidence for the presence of a redundant transport system in this species. Infect Immun, 1996 Jul, 64(7), 2857 - 60 The GafD protein of the G (F17) fimbrial complex confers adhesiveness of Escherichia coli to laminin; Saarela S et al.; Escherichia coli IHE11088(pRR-5) expressing the G (F17) fimbria adhered to immobilized laminin as well as to reconstituted basement membranes . No adhesion was seen with the plasmidless strain IHE11088 or with the deletion derivative IHE11088(pHUB110), which expresses the G-fimbrial filament with a defective GafD lectin and lacks N-acetyl-D-glucosamine-specific binding . Adhesion of IHE11088(pRR-5) to laminin and to reconstituted basement membranes was specifically inhibited by N-acetyl-D-glucosamine, and adhesion was abolished after N-glycosidase F treatment of laminin . The results show that the GafD lectin binds to laminin carbohydrate and suggest a novel function for the F17 fimbria in binding to mammalian basement membranes. Infect Immun, 1996 Jul, 64(7), 2808 - 11 Characterization of the acid resistance phenotype and rpoS alleles of shiga-like toxin-producing Escherichia coli; Waterman SR et al.; Shiga-like toxin-producing Escherichia coli (SLTEC) strains are an important group of enteric pathogens . In this study we have examined the abilities of 58 SLTEC isolates to survive at pH 2.5 and found 13 of these isolates to be defective in acid resistance . Introduction of rpoS on a plasmid conferred acid resistance to the majority of the acid-sensitive isolates . The rpoS genes from two of these isolates were sequenced; both isolates contained lesions in the rpoS gene resulting in a nonfunctional RpoS . These results show that mutant rpoS alleles exist in natural populations of E . coli . Such mutations may play an important role in determining the infective dose of SLTEC and suggest that isolates may vary in infectivity. Infect Immun, 1996 Jul, 64(7), 2695 - 9 Molecular cloning and characterization of Coccidioides immitis antigen 2 cDNA; Zhu Y et al.; Previous experiments have provided evidence that Coccidioides immitis antigen 2 (Ag2) is a major T-cell-reactive component of mycelia and spherule cell walls . Here we report the identification and cloning of the cDNA that encodes Ag2 from a lambda ZAP cDNA expression library constructed from spherule-derived RNA . DNA sequence analysis established that the 1,255-bp clone contains a 174-bp 5' untranslated region, a 582-bp open reading frame which encodes for a protein consisting of 194 amino acids, and a 375-bp 3' untranslated region, including a poly(A) tail . The recombinant Ag2 protein has a predicted molecular mass of 19.5 kDa and contains an 18-amino-acid N terminus which has been tentatively identified as a signal peptide . The Ag2 cDNA was ligated into the pGEX-4T-3 vector and expressed in Escherichia coli TG-1 cells as a glutathione S-transferase fusion protein . The recombinant fusion protein showed reactivity with sera from patients with coccidioidomycosis and elicited delayed-type footpad hypersensitivity responses in Coccidioides-immune mice . These results suggest that the Ag2 cDNA can be used for the large-scale production of this immunologically important protein. Infect Immun, 1996 Jul, 64(7), 2635 - 42 Identification and characterization of CS20, a new putative colonization factor of enterotoxigenic Escherichia coli; Valvatne H et al.; An enterotoxigenic Escherichia coli (ETEC) strain producing a previously undescribed putative colonization factor was isolated from a child with diarrhea in India . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial heat extracts revealed a polypeptide band of 20.8 kDa when the bacteria were grown at 37 degrees C which was absent after growth at 22 degrees C . A specific rabbit antiserum raised against the purified 20.8-kDa protein bound specifically to the fimbriae, as shown by immunoelectron microscopy, and inhibited bacterial adhesion to tissue-cultured Caco-2 cells . Transformation with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator for several ETEC fimbriae, induced hyperexpression of the 20.8-kDa fimbrial subunit and a substantial increase in the proportion of bacterial cells that were fimbriated . The N-terminal amino acid sequence of the polypeptide showed 65 and 60% identity to the PCFO20 and 987P fimbriae of human and porcine ETEC, respectively . We propose the term CS20 for this new putative colonization factor of human ETEC. Infect Immun, 1996 Jul, 64(7), 2532 - 9 Characterization of an adherence and antigenic determinant of the ArgI protease of Porphyromonas gingivalis which is present on multiple gene products; Curtis MA et al.; This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antibodies (MAbs) produced to be specific for Porphyromonas gingivalis whole cells which we had previously shown to bind to epitopes recognized by sera from periodontitis patients . Preliminary data had suggested that the arginine-specific proteases of P . gingivalis (ArgI, ArgIA, and ArgIB) contained the antigenic determinants of four of these antibodies (MAbs 1A1, 2B/H9, 7D5, and 3B1) . The location of the binding sites was examined with purified P . gingivalis enzymes and recombinant regions of the ArgI polyprotein expressed by subclones of the prpR1 gene in Escherichia coli XL-1 Blue cells . All four antibodies were reactive with protein determinants within the beta subunit, a hemagglutinin and/or adhesin component, of the ArgI dimer . MAb 1A1 strongly inhibited the agglutination of human erythrocytes by P . gingivalis W50 culture supernatant, suggesting that the binding site for this antibody contains residues which are critical for the interaction with the erythrocyte surface . The determinant for MAb 1A1 was examined further by construction of a set of truncated forms of the beta component expressed as fusion proteins with glutathione S-transferase at the N terminus . Analysis of these constructs mapped the binding site for MAb 1A1 to PrpRI residues G-907 to T-931, GVSPKVCKDV TVEGSNEFAP VQNLT . Western blot (immunoblot) analysis of P . gingivalis whole-cell proteins demonstrated that MAb 1A1 reacts with several proteins in the Mr range of 20,000 to 120,000 . Furthermore, an oligonucleotide probe corresponding to the coding sequence for the region of the ArgI beta component containing the MAb 1A1 binding site hybridized to multiple bands on genomic digests of P . gingivalis DNA . These data indicate that the MAb 1A1 epitope may be a component of a binding domain common to multiple gene products of this organism and may thus represent a functionally important target of the host's specific immune response to P . gingivalis in periodontal disease. Biochem J, 1996 Jul 1, 317 ( Pt 1), 305 - 11 Temperature-dependence of open-complex formation at two Escherichia coli promoters with extended -10 sequences; Burns HD et al.; We have studied the formation of open complexes between purified RNA polymerase from Escherichia coli and DNA fragments carrying the galP1 promoter, a promoter with an extended -10 region . Unusually, these complexes are formed readily at low temperatures . This low-temperature opening is unaffected by deletions of either upstream or downstream promoter sequences . We conclude that low-temperature open-complex formation is due to specific base sequences in and just upstream of the extended -10 region . In contrast, open complexes are not formed at low temperatures with DNA fragments carrying the E . coli cysG promoter, which also has an extended -10 region . This demonstrates that an extended -10 sequence alone is not sufficient for low-temperature opening . Additionally, we report the temperature dependence of a hybrid galP1-cysG promoter, the related galP2 and galP3 promoters and a derivative of galP1 with an improved -10 hexamer sequence. Biochem J, 1996 Jul 1, 317 ( Pt 1), 267 - 72 Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine; Mewies M et al.; In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN . The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme . Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222 . The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K) . Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin . A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis . Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP . Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase . The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass . The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed. Biochem J, 1996 Jul 1, 317 ( Pt 1), 179 - 85 Cloning and biochemical characterization of the cyclophilin homologues from the free-living nematode Caenorhabditis elegans; Page AP et al.; Cyclosporin A (CsA) is the most widely used immunosuppressive agent, whose properties are exerted via an interaction with cyclophilin, resulting in down-regulation of signal-transduction events in the T-cell . Cyclophilin is identical with peptidylprolyl cis-trans isomerase (PPI; EC 5.2.1.8), an enzyme which catalyses the isomerization between the two proline conformations in proteins, thereby acting as a catalyst in protein-folding events . Several reports indicate that CsA has potent anti-parasitic activity, effective against both protozoan and helminth species . In order to understand the various biological roles that cyclophilins play we have initiated a study of these proteins in the genetically tractable nematode Caenorhabditis elegans . Here we describe the cloning and characterization of 11 cyclophilin genes (cyp-1 to -11) derived from this nematode; this is currently the greatest number of isoforms described in a single species . Southern blotting and physical mapping indicated that these genes are dispersed throughout the nematode genome . A high degree of conservation exists between several isoforms, which also share characteristics with the ubiquitous isoforms previously described . The remaining isoforms are divergent, having altered CsA-binding domains and additional non-cyclophilin domains, which may impart compartmental specificity . Ten of these isoforms have been expressed in Escherichia coli, and the resultant fusion proteins have been examined biochemically for PPI activity, which they all possess . Isomerase activity is highest in the conserved and lowest in divergent isoforms, perhaps indicating a more specific substrate for the latter . Analysis of the C . elegans cyp genes will provide answers as to the roles played by cyclophilins in protein folding and signal transduction. Biochem J, 1996 Jul 1, 317 ( Pt 1), 135 - 40 Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein; Niemann G et al.; A southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment . Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified . The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated . The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp . In the 5' non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified . The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni(2+)-chelating affinity chromatography . The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa . On the basis of gel filtration analyses it is shown that the active enzyme is a dimer . The specific enzyme activity was determined to be 4.2 mumol CO2/min/mg protein . The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity . The Km value for L-ornithine was determined as 44 microM . The Ki values for putrescine, alpha-diffluoromethylornithine, alpha-hydrazino-ornithine and alpha-methylornithine were calculated as 51, 34, 0.34 and 42 microM respectively. Nucleic Acids Res, 1996 Jul 1, 24(13), 2592 - 6 Detection of multiple conformations of the E-domain of 5S rRNA from Escherichia coli in solution and in crystals by NMR spectroscopy; Grune M et al.; NMR spectroscopy of the E-domain fragment of Escherichia coli 5S rRNA indicates that this molecule exists in solution as either a stem-loop or as a duplex with two U-U base pairs in the bulge region . At temperatures below 27 degrees C, interconversion between the monomeric and dimeric forms in solution occurs on a time scale of weeks and allows the preparation of samples on which NMR structure determinations can be carried out on predominantly monomeric or dimeric species . The NMR results obtained provide comparison data for the distinction between A- and B-form E.coli 5S rRNA and for the possible kinetics of conversion between these forms . NMR evidence is presented that the duplex form also exists in crystals and suggestions are made for means to obtain stem-loop conformations of E-domain and other small RNA stem-loop sequences in crystals. Nucleic Acids Res, 1996 Jul 1, 24(13), 2525 - 7 Preference for guanosine at first codon position in highly expressed Escherichia coli genes . A relationship with translational efficiency; Gutierrez G et al.; The variation in base composition at the three codon sites in relation to gene expressivity, the latter estimated by the Codon Adaptation Index, has been studied in a sample of 1371 Escherichia coli genes . Correlation and regression analyses show that increasing expression levels are accompanied by higher frequencies of base G at first, of base A at second and of base C at third codon positions . However, correlation between expressivity and base compositional biases at each codon site was only significant and positive at first codon position . The preference for G-starting codons as gene expression level increases is discussed in terms of translational optimization. Nucleic Acids Res, 1996 Jul 1, 24(13), 2505 - 10 Proofreading in trans by an aminoacyl-tRNA synthetase: a model for single site editing by isoleucyl-tRNA synthetase; Jakubowski H; Editing of errors in amino acid selection by an aminoacyl-tRNA synthetase prevents attachment of incorrect amino acids to tRNA, thereby greatly enhancing accuracy of translation of the genetic code . Editing of the non-protein amino acid homocysteine, a frequent type of an error-correcting process, involves reaction of the side chain sulfhydryl group of homocysteine with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone . Here, it is shown that isoleucyl-tRNA synthetase (IleRS), which occasionally misactivates homocysteine in vitro and in vivo, catalyzes reactions of activated isoleucine with organic thiols (analogues of the side chain of homocysteine) . That these enzymatic reactions occur between Ile-tRNAIle or Ile-AMP (bound in the synthetic sub-site) and a thiol (an analogue of the side chain of homocysteine, bound in the editing sub-site), indicates that the two sub-sites are physically close on the surface of IleRS, forming a single synthetic/editing active site of the enzyme . Although IleRS.Val-AMP undergoes thiolysis as efficiently as do IleRS.Ile-AMP and IleRS.Ile-tRNAIle, IleRS.Val-tRNAIle does not react with thiols . These and other data suggest that the mischarged valine residue in IleRS.Val-tRNAIle is, most likely, positioned off the enzyme. Nucleic Acids Res, 1996 Jul 1, 24(13), 2498 - 504 Dominant negative mutator mutations in the mutL gene of Escherichia coli; Aronshtam A et al.; The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function . It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity . An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo . As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis . None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive . All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage . DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein . In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs . All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations. Nucleic Acids Res, 1996 Jul 1, 24(13), 2463 - 9 Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A; Swaminathan N et al.; R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence . Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends . In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites . The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli . The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon . Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme . Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain. J Exp Med, 1996 Jul 1, 184(1), 265 - 70 Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus; Crameri R et al.; A cDNA encoding an allergenic protein was isolated from an Aspergillus fumigatus (A . fumigatus) cDNA library displayed on the surface of filamentous phage . Serum immunoglobulin E (IgE) from A . fumigatus-sensitized individuals was used to enrich phage-expressing gene products binding to IgE . One of the cDNAs encoded a 26.7-kD protein that was identified as a manganese superoxide dismutase (MnSOD) sharing 51.5% identity and 67.2% homology to the corresponding human enzyme . Both human and A . fumigatus MnSOD coding sequences were expressed in Escherichia coli as {His}6-tagged fusion proteins and purified by Ni(2+)-chelate affinity chromatography . The two recombinant MnSODs were both recognized by IgE antibodies from subjects allergic to the A . fumigatus MnSOD and elicited specific immediate type allergic skin reactions in these individuals . Moreover, both human and A . fumigatus MnSOD induced proliferation in peripheral blood mononuclear cells of A . fumigatus-allergic subjects who showed specific IgE responses and reacted in skin tests to MnSOD . These observations provide evidence for autoreactivity to the human MnSOD in allergic persons sensitized to an environmental allergen from A . fumigatus who share a high degree of sequence homology to the corresponding human enzyme. J Clin Invest, 1996 Jul 1, 98(1), 192 - 8 Efficacy of treatment with the iron (III) complex of diethylenetriamine pentaacetic acid in mice and primates inoculated with live lethal dose 100 Escherichia coli; Molina L et al.; The iron (III) complex of diethylenetriamine pentaacetic acid (DTPA iron {III}) protected mice and baboons from the lethal effects of an infusion with live LD100 Escherichia coli . In mice, optimal results were obtained when DTPA iron (III) was administered two or more hours after infection . Prevention of death occurred in spite of the fact that the adverse effects of TNF-alpha were well underway in the mouse model . The half-life of DTPA iron (III) was 51 +/- 9 min in normal baboons; primary clearance was consistent with glomerular filtration . In septic baboons, survival was observed after administration of two doses of DTPA iron (III) at 2.125 mg/kg, the first one given before, or as late as 2 h after, severe hypotension . Administration of DTPA iron (III) did not alter mean systemic arterial pressure, but did protect baboons in the presence of high levels of TNF-alpha and free radical overproduction . Furthermore, exaggerated production of nitric oxide was attenuated . The mechanism of protection with DTPA iron (III) is not obvious . Because of its ability to interact in vitro with free radicals, its poor cell permeability, and its short half-life, we postulate that DTPA iron (III) and/or its reduced form may have protected the mice and baboons by sequestration and subsequent elimination of free radicals (including nitric oxide) from their systems. Int Arch Allergy Immunol, 1996 Jul, 110(3), 282 - 7 High-level expression of tree pollen isoallergens in Escherichia coli; Weiss C et al.; cDNAs coding for the major allergen of alder (Alnus glutinosa) pollen Aln g 1, for nine isoforms of Bet v 1, the major birch (Betula verrucosa) pollen allergen, and for four isoforms of Cor a 1, the major allergen of hazel (Corylus avellana) pollen, were inserted into the plasmid pMW175 or pMW 172 and expressed in Escherichia coli as recombinant non-fusion proteins . These constructs produced between 20 and 160 mg protein/l . The recombinant tree pollen isoallergens were tested in immunoblots for their antibody binding properties . For this purpose, we used two monoclonal antibodies (BIP 1 and BIP 4) raised against natural Bet v 1, a polyclonal rabbit anti-recombinant Bet v 1a, as well as serum IgE from allergic patients . Our results show that this expression system is suitable for the production of milligram amounts of tree pollen isoallergens which can be used for the characterization of allergenic epitopes recognized by T and B cells. J Bacteriol, 1996 Jul, 178(13), 3982 - 4 The binding site of the IclR repressor protein overlaps the promoter of aceBAK; Pan B et al.; In Escherichia coli, repression of the aceBAK operon is mediated by the IclR protein . We used an in vitro oligonucleotide selection technique to determine the consensus recognition sequence for MR . Mutational analysis confirmed the contribution of this sequence to repression in vivo and identified the -35 element of the promoter. J Bacteriol, 1996 Jul, 178(13), 3974 - 7 PstB protein of the phosphate-specific transport system of Escherichia coli is an ATPase; Chan FY et al.; The PstB protein of the phosphate-specific transport (Pst) system of Escherichia coli bound and hydrolyzed ATP, producing ADP . Urea-treated denatured PstB did not bind ATP . The N-terminal amino acid sequence of the immune serum-precipitable PstB protein was determined, and it corresponded to that deduced from the DNA sequence. J Bacteriol, 1996 Jul, 178(13), 3971 - 3 Structure modification induced in the narG promoter by binding of integration host factor and NARL-P; Zhang X et al.; Interaction of integration host factor (IHF) with linear DNA fragments containing the narG promoter region induced an apparent sharp bend in the DNA centered at the IHF-binding site . Binding of NARL-P to two sites adjacent to the IHF site did not induce bending or modify the apparent bending induced by IHF. J Bacteriol, 1996 Jul, 178(13), 3957 - 61 The activity of the high-affinity K+ uptake system Kdp sensitizes cells of Escherichia coli to methylglyoxal; Ferguson GP et al.; Expression of the Kdp system sensitizes cells to methylglyoxal (MG) whether this electrophile is added externally or is synthesized endogenously . The basis of this enhanced sensitivity is the maintenance of a higher cytoplasmic pH (pHi) in cells expressing Kdp . In such cells, MG elicits rapid cytoplasmic acidification via KefB and KefC, but the steady-state pHi attained is still too high to confer protection Lowering pHi further by incubation with acetate increases the sensitivity of cells to MG. J Bacteriol, 1996 Jul, 178(13), 3917 - 25 RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli; Kido M et al.; We have isolated suppressor mutants that suppress temperature-sensitive colony formation and anucleate cell production of a mukB mutation . A linkage group (smbB) of the suppressor mutations is located in the rne/ams/hmp gene encoding the processing endoribonuclease RNase E . All of the rne (smbB) mutants code for truncated RNase E polypeptides lacking a carboxyl-terminal half . The amount of MukB protein was higher in these rne mutants than that in the rne+ strain . These rne mutants grew nearly normally in the mukB+ genetic background . The copy number of plasmid pBR322 in these rne mutants was lower than that in the rne+ isogenic strain . The results suggest that these rne mutations increase the half-lives of mukB mRNA and RNAI of pBR322, the antisense RNA regulating ColE1-type plasmid replication . We have demonstrated that the wild-type RNase E protein bound to polynucleotide phosphorylase (PNPase) but a truncated RNase E polypeptide lacking the C-terminal half did not . We conclude that the C-terminal half of RNase E is not essential for viability but plays an important role for binding with PNPase . RNase E and PNPase of the multiprotein complex presumably cooperate for effective processing and turnover of specific substrates, such as mRNAs and other RNAs in vivo. J Bacteriol, 1996 Jul, 178(13), 3893 - 8 Nitrate reductase activity and heterocyst suppression on nitrate in Anabaena sp . strain PCC 7120 require moeA; Ramaswamy KS et al.; Mutants of Anabaena sp . strain PCC 7120 that form heterocysts when grown on nitrate-containing media were isolated following nitrosoguanidine mutagenesis . Six independent mutants were isolated, and the characterization of one mutant, strain AMC260, which forms 6 to 8% heterocysts in the presence of nitrate, is presented . A 1.8-kb chromosomal fragment that complemented the AMC260 mutant was sequenced, and a 1.2-kb open reading frame, named moeA, was identified . The deduced amino acid sequence of the predicted Anabaena sp . strain PCC 7120 MoeA polypeptide shows 37% identity to MoeA from Escherichia coli, which is required for the synthesis of molybdopterin cofactor . Molybdopterin is required by various molybdoenzymes, such as nitrate reductase . Interruption of the moeA gene in Anabaena sp . strain PCC 7120 resulted in a strain, AMC364, that showed a phenotype similar to that of AMC260 . We show that AMC260 and AMC364 lack methyl viologen-supported nitrate reductase activity . We conclude that the inability of the moeA mutants to metabolize nitrate results in heterocyst formation on nitrate-containing media . Northern (RNA) analysis detected a 1.5-kb moeA transcript in wild-type cells grown in the presence or absence of a combined nitrogen source. J Bacteriol, 1996 Jul, 178(13), 3885 - 92 Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage; Slupska MM et al.; We have cloned the human mutY gene (hMYH) from both genomic and cDNA libraries . The human gene contains 15 introns and is 7.1 kb long . The 16 exons encode a protein of 535 amino acids that displays 41% identity to the Escherichia coli protein, which provides an important function in the repair of oxidative damage to DNA and helps to prevent mutations from oxidative lesions . The human mutY gene maps on the short arm of chromosome 1, between p32.1 and p34.3. J Bacteriol, 1996 Jul, 178(13), 3877 - 84 FtsZ ring formation in fts mutants; Addinall SG et al.; The formation of FtsZ rings (Z rings) in various fts mutants was examined by immunoelectron microscopy and immunofluorescence . In two temperature-sensitive ftsZ mutants which form filaments with smooth morphology, the Z ring was unable to form . In ftsA, ftsI, and ftsQ mutants, which form filaments with an indented morphology, Z rings formed but their contraction was blocked . These results indicate that fully functional ftsA, ftsQ, and ftsI genes are not required for Z-ring formation and are unlikely to have a role in localization of the Z ring . The results also suggest that one function of the Z ring is to localize the activity of other fts gene products. J Bacteriol, 1996 Jul, 178(13), 3840 - 5 Plasmid recombination by the RecBCD pathway of Escherichia coli; Zaman MM et al.; Previously, we demonstrated that exonuclease I-deficient strains of Escherichia coli accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids carrying the chi sequence (5'- GCTGGTGG-3') (M . M . Zaman and T . C . Boles, J . Bacteriol . 176:5093-5100, 1994) . Since high-molecular weight linear DNA is believed to be the natural substrate for RecBCD-mediated recombination during conjugation (A . J . Clark and K . B . Low, p . 155-215, in K . B . Low, ed., The Recombination of Genetic Material, 1988), we analyzed the recombination frequencies of chi+ and chi0 plasmids in sbcB strains . Here, we report that chi sites stimulate plasmid recombination frequency by 16-fold in sbcB strains . Chi-stimulated plasmid recombination is dependent on RecBCD but is independent of RecF pathway genes . The distribution of recombination products suggests that high-molecular-weight linear plasmid DNA is a substrate for RecBCD-mediated recombination . Surprisingly, our data also suggest that chi+ plasmids also recombine by the RecBCD pathway in rec+ sbcB+ cells. J Bacteriol, 1996 Jul, 178(13), 3755 - 62 Loss of overproduction of polypeptide release factor 3 influences expression of the tryptophanase operon of Escherichia coli; Yanofsky C et al.; Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced inhibition of Rho-mediated transcription termination . Previous studies indicated that tryptophan induction might involve leader peptide inhibition of ribosome release at the stop codon of tnaC, the coding region for the operon-specified leader peptide . In this study we examined tna operon expression in strains in which the structural gene for protein release factor 3, prfC, is either disrupted or overexpressed . We find that prfC inactivation leads to a two- to threefold increase in basal expression of the tna operon and a slight increase in induced expression . Overexpression of prfC has the opposite effect and reduces both basal and induced expression . These effects occur in the presence of glucose and cyclic AMP, and thus Rho-dependent termination rather than catabolite repression appears to be the event influenced by the prfC alterations . prfC inactivation also leads to an increase in basal tna operon expression in various rho and rpoB mutants but not in a particular rho mutant in which the basal level of expression is very high . The effect of prfC inactivation was examined in a variety of mutants with alterations in the tna leader region . Our results suggest that translation of tnaC is essential for the prfC effect . The tryptophan residue specified by tnaC codon 12, which is essential for induction, when replaced by another amino) acid, allows the prfC effect . Introducing UAG or UAA stop codons rather than the normal tnaC UGA stop codon, in a strain with an inactive prfC gene, also leads to an increase in the basal level of expression . Addition of the drug bicyclomycin increases basal operon expression of all mutant strains except a strain with a tnaC'-'lacZ fusion . Expression in the latter strain is unaffected by prfC alterations . Our findings are consistent with the interpretation that ribosome release at the tnaC stop codon can influence tna operon expression. J Bacteriol, 1996 Jul, 178(13), 3742 - 7 Membrane insertion of the F-pilin subunit is Sec independent but requires leader peptidase B and the proton motive force; Majdalani N et al.; F pilin is the subunit required for the assembly of conjugative pili on the cell surface of Escherichia coli carrying the F plasmid . Maturation of the F-pilin precursor, propilin, involves three F plasmid transfer products: TraA, the propilin precursor; TraQ, which promotes efficient propilin processing; and TraX, which is required for acetylation of the amino terminus of the 7-kDa pilin polypeptide . The mature pilin begins at amino acid 52 of the TraA propilin sequence . We performed experiments to determine the involvement of host cell factors in propilin maturation . At the nonpermissive temperature in a LepBts (leader peptidase B) host, propilin processing was inhibited . Furthermore, under these conditions, only full-length precursor was observed, suggesting that LepB is responsible for the removal of the entire propilin leader peptide . Using propilin processing as a measure of propilin insertion into the plasma membrane, we found that inhibition or depletion of SecA and SecY does not affect propilin maturation . Addition of a general membrane perturbant such as ethanol also had no effect . However, dissipation of the proton motive force did cause a marked inhibition of propilin processing, indicating that membrane insertion requires this energy source . We propose that propilin insertion in the plasma membrane proceeds independently of the SecA-SecY secretion machinery but requires the proton motive force . These results present a model whereby propilin insertion leads to processing by leader peptidase B to generate the 7-kDa peptide, which is then acetylated in the presence of TraX. Hum Genet, 1996 Jul, 98(1), 12 - 5 Assignment of the NTRK4 (trkE) gene to chromosome 6p21; Valent A et al.; Reverse transcriptase-polymerase chain reactions using foetal brain RNA with reverse and forward primers of the first, second and third NTRK4 region allowed us to obtain three amplified NTRK4 fragments . The specificity of amplified fragments was checked by digestion with restriction endonucleases AvrII, HindIII and PspII for the first, second and third regions, respectively . Each restriction site was specific for each amplified fragment . The fragment of the NTRK4 first region was also sequenced and the sequence determined was identical to the human NTRK4 sequence . The three amplified fragments were cloned in pBS . For the Southern technique, plasmid pBS-NTRK4a (with an insert of 1052 bp) detected a human 9-kb HindIII sequence which was localised unambiguously on chromosome 6 . For fluorescence in situ hybridisation, the three plasmids, pBS-NTRK4a, pBS-NTRK4b (insert 924 bp) and pBS-NTRK4c (insert 1114 bp) were pooled and used as a probe . This NTRK4 probe was localised on 6p21 . Of 50 metaphases analysed, 49 contained twin spot signals on both sister chromatids. Exp Neurol, 1996 Jul, 140(1), 14 - 20 Toxicity of replication-defective adenoviral recombinants in dissociated cultures of nervous tissue; Durham HD et al.; Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neurons in vitro and in vivo; however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time . In vitro, this may be due to direct toxicity of the protein product of the transgene or adenoviral molecules . In vivo, in addition, an immune attack by the host could eliminate the transduced cells . To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures . Cultures of dissociated murine spinal cord-dorsal root ganglia were exposed to AVR containing the Escherichia coli lacZ (E . coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells . Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1 + E3)-deleted replication-incompetent AVR at high titers {> or = 5 x 10(8) viral particles/ml or multiplicity of infection (m.o.i.) 1000} and (ii) high levels of expression of the reporter gene product, beta-galactosidase, at titers that result in 55-75% transduction efficiency (5 x 10(7)-5 x 10(8) viral particles/ml or m.o.i . 100-1000) . Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic. Chest, 1996 Jul, 110(1), 198 - 204 Transvisceral lactate fluxes during early endotoxemia; Bellomo R et al.; The pathogenesis of hyperlacticemia during sepsis is poorly understood . We investigated the role of lung, kidney, gut, liver, and muscle in endogenous lactate uptake and release during early endotoxemia in an intact, pentobarbital-anesthetized dog model (n = 14) . Ultrasonic flow probes were placed around the portal vein and hepatic, renal, and femoral arteries . After splenectomy, catheters were inserted into the pulmonary artery, aorta, and hepatic, left renal, and femoral veins . Whole blood lactate and blood gases from all catheters, organ flows, and cardiac output were measured before and 30 to 45 min after a bolus infusion of Eacherichia coli endotoxin (1 mg/kg) . After endotoxin infusion, mean arterial blood lactate level increased from 0.92 +/- 0.11 to 1.60 +/- 0.15 mmol/L (p < 0.0001) . Lung lactate flux changed from uptake to release of lactate adding a mean of 9.97 +/- 16.23 mmol/h (p < 0.05) to the systemic circulation . Liver and muscle lactate fluxes remained neutral at all times, while kidney and gut took up lactate from the circulation both before and after endotoxin infusion (mean renal uptake, 2.73 +/- 3.85 mmol/L; p < 0.001; mean gut uptake, 2.46 +/- 2.31 mmol/h; p < 0.002) . Except for the kidney, where a decrease in blood flow correlated with diminished uptake, there was no correlation between changes in transvisceral lactate fluxes and organ or systemic oxygen delivery during endotoxemia . A positive correlation between lactate uptake and oxygen consumption during endotoxemia was seen for both gut (p < 0.0001) and kidney (p < 0.002) . We conclude that, in the dog, the pathogenesis of endotoxin-induced hyperlacticemia is complex . The lung may be responsible for significant lactate release, and other viscera that normally take up lactate are unable to adequately clear this increased lactate. Am J Respir Crit Care Med, 1996 Jul, 154(1), 68 - 75 Serial measures of total body oxygen consumption in an awake canine model of septic shock; Eichacker PQ et al.; We examined serial changes in total body oxygen consumption (Vo2) in a permanently tracheotomized canine sepsis model . On Day 0, beagles had an Escherichia coli-infected (septic) or sterile (control) clot surgically placed in the peritoneum . During the 21-d study, 10 of the 16 septic animals and none of the six control animals died (p = 0.02) . After clot placement septic versus control animals had decreased mean arterial blood pressure (mm Hg; Day 1: 106 versus 128, p = 0.055; Day 2: 95 versus 125, p = 0.004, respectively) and left ventricular ejection fraction (Day 1: 0.44 versus 0.69, p = 0.0006; Day 2: 0.33 versus 0.57, p = 0.0001, respectively) . Despite significant lethality and cardiovascular dysfunction, in the septic group on Days 1 and 2, septic versus control animals had no significant differences in mean metabolic cart measured (Vo2DIR, ml/kg/min; Day 1: 11.9 versus 12.4, p = 0.81; Day 2: 14.2 versus 13.5, p = 0.72, respectively) and intravascular catheter calculated (Vo2INDIR, ml/kg/min; Day 1: 11.2 versus 11.2, p = 0.99; Day 2: 12.8 versus 15.4, p = 0.49, respectively) . On Day 1 in septic and control animals, volume infusion produced increases (p < 0.001) in oxygen delivery (Do2) . In septic and control animals these changes in Do2 were similar and were associated with similar increases in Vo2DIR (p = 0.001), and Vo2INDIR (p = 0.001) . In fact, at all time points studied (baseline, Day 1, 2, and 21), both before and after volume infusion, levels of Do2, Vo2DIR, and Vo2INDIR did not differ between septic and control animals, nor did they differ between septic survivors and nonsurvivors . Because levels of Vo2DIR and Vo2INDIR were similar in both groups, we pooled data from septic and control animals . Throughout the study, Vo2 showed a moderate association with Vo2INDIR (r = 0.55, p = 0.003), but mean Vo2DIR was lower at baseline (p = 0.001) and on Day 21 (p = 0.07) and greater on Day 2 (p < 0.01) . In summary, our techniques, which detected small changes in both Vo2DIR and Vo2INDIR occurring with volume infusion, did not demonstrate differences in these parameters comparing control and septic animals . These results in euvolemic septic animals suggest that total body Vo2 may not reflect pathogenetic mechanisms during sepsis and septic shock . Furthermore, these results suggest that although the level of total body Vo2 may reflect the effects of therapeutic interventions such as volume loading, it should not itself serve as a therapeutic target. Arch Surg, 1996 Jul, 131(7), 767 - 74 Effects of nitric oxide donor SIN-1 on oxygen availability and regional blood flow during endotoxic shock; Zhang H et al.; BACKGROUND: An excessive release of nitric oxide (NO) has been incriminated in the circulatory disturbances of septic shock . OBJECTIVE: To study the effects of an NO donor, 3-morpholinosydnonimine (SIN-1), an oxygen availability and regional blood flow during endotoxic shock to see if a beneficial effect of NO synthase inhibitors in septic shock could be conclusively demonstrated . MATERIALS AND METHODS: In 14 anesthetized and mechanically ventilated dogs, global invasive hemodynamic monitoring was completed and ultrasonic flow probes were placed around the superior mesenteric, left renal, and left femoral arteries for simultaneous measurements of regional blood flow . All dogs received Escherichia coli endotoxin, 2 mg/kg . A control group (n = 7) was administered saline at 20 mL/kg per hour, and a SIN-1 group (n = 7) was given a combination of saline with SIN-1 at successive doses of 1, 2, and 4 micrograms/kg per minute . RESULTS: Neither systemic nor pulmonary arterial pressures were influenced by SIN-1 . Cardiac index, stroke index, and left ventricular stroke work index did increase at low to moderate doses of SIN-1 but tended to decrease at the highest dose . Systemic and pulmonary vascular resistances decreased . Fractional blood flow increased in the mesenteric bed at all doses used, was not influenced in the renal bed, but decreased in the femoral bed at the highest dose . Oxygen-derived variables were similar in the 2 groups . Blood lactate and plasma concentrations of tumor necrosis factor were not significantly influenced . At the end of the SIN-1 infusion, the administration of 5 mg/kg of methylene blue increased arterial pressure, pulmonary arterial pressure, and systemic and pulmonary vascular resistances but decreased cardiac index and regional blood flow . CONCLUSIONS: The administration of low to moderate doses of the NO donor SIN-1 can significantly increase cardiac index and superior mesenteric blood flow without deleterious effects on arterial pressure in this model of endotoxic shock . These findings support the hypothesis that NO is essential to maintain organ blood flow even during endotoxic shock. J Virol, 1996 Jul, 70(7), 4819 - 24 Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics; LaFemina RL et al.; The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies . Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro . We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing . When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein . The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter . The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate) . This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides . Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease. J Virol, 1996 Jul, 70(7), 4805 - 10 Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli; Chartier C et al.; Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli and eukaryotic cells . This report describes a novel system based on the cloning and manipulation of the full-length adenovirus genome as a stable plasmid in E . coli, by using the bacterial homologous recombination machinery . The efficiency and flexibility of the method are illustrated by the cloning of the wild-type adenovirus type 5 genome, the insertion of a constitutive promoter upstream from the E3 region, the replacement of the E1 region by an exogenous expression cassette, and the deletion of the E1 region . All recombinant viral DNAS were shown to be fully infectious in permissive cells, and the modified E3 region or the inserted foreign gene was correctly expressed in the infected cells. J Trauma, 1996 Jul, 41(1), 61 - 71; discussion 71-2 Acute ethanol intoxication and endotoxemia after trauma; Woodman GE et al.; To determine actions of acute intoxication on pathophysiologic responses to trauma, anesthetized and ventilated mongrel pigs received a 20% solution of ethanol (EtOH) by an intravenous (IV group; 2 g/kg, n = 8) or an oral (PO group; 3 g/kg, n = 12 x 60 minutes) route of administration, or the lactated Ringer's vehicle (LR group; n = 12) . After 60 minutes, all were subjected to soft tissue injury and 30 to 35% hemorrhage, 60-minute shock, and then resuscitation, with shed blood plus supplemental LR . After 3 days, host defense was challenged with Escherichia coli lipopolysaccharide (LPS); (1 microgram/kg x 30-minutes IV) . The supplemental resuscitation was identical (50-53 mL/kg/hours), but posttraumatic acidosis was observed in the IV group and the PO group (base deficit = 4.4 +/- 1.3 and 5.5 +/- 0.9 mEq/L) and not in the LR group . After 3 days, the acid-base equilibrium was restored, but a difference in host defense was unmasked by LPS . In the LR group, LPS-evoked pulmonary vasoconstriction was followed by decreased compliance and ventilation-perfusion mismatch, which was associated at 3 to 5 hours with a base deficit, reduced SVO2, and reduced PO2 (-0.5 +/- 0.2 mEq/L, 46 +/- 1%, 127 +/- 1 mm Hg) . These changes were blunted in the PO group (2.0 +/- 0.1 mEq/L, 56 +/- 1%, 183 +/- 4 mm Hg) and potentiated in the IV group (-4.3 +/- 0.5 mEq/L, 40 +/- 2%, 60 +/- 2 mm Hg), even though more fluid was required to maintain systemic arterial and cardiac filling pressures following LPS administration in the IV (40 +/- 6 mL/kg/ hours) versus the LR or PO groups (31 +/- 5 or 23 +/- 3) . The PO versus LR differences could not be attributed to enteral nutrition because an isocaloric solution of 50% dextrose had no effect versus LR solution . EtOH caused neutropenia following trauma, relative to LR solution, but the IV versus PO differences could not be discriminated on the basis of neutrophil or lymphocytes counts, nor CD18 receptor expression, nor renal or hepatic dysfunction . However, T4 lymphocytes and cortisol, a nonspecific index of inflammation, were higher for at least 24 hours after trauma with IV, relative to PO or LR . Blood EtOH was similar with IV or PO during resuscitation (100-120 mg/dL), but the kinetics were different prior to trauma . With PO, blood EtOH slowly accumulated to a steady state plateau, the level of which was higher with no anesthesia or no trauma . With IV, blood EtOH peaked at 275 mg/dL and then exponentially declined with a rate that was not influenced to a major extent by trauma or by anesthesia . Therefore: 1) EtOH absorption is impaired during trauma (in part because of reduced gut blood flow); 2) acute EtOH intoxication at the time of trauma altered neutrophils, plasma cortisol, and T4 lymphocytes during recovery and host defense to a superimposed LPS challenge . The apparently favorable effect of PO versus IV EtOH on the response to endotoxemia after trauma probably reflects differences in the kinetics of blood EtOH in the interval before reperfusion but a "first pass" effect (metabolism in the gut or liver) might also explain the data. Crit Care Med, 1996 Jul, 24(7), 1233 - 7 Dopexamine maintains intestinal villus blood flow during endotoxemia in rats; Schmidt H et al.; OBJECTIVE: To determine the influence of dopexamine, a synthetic catecholamine ligand for dopaminergic and beta 2-adrenergic receptors, on alterations of the intestinal villus microcirculation in a model of normotensive endotoxemia . DESIGN: Randomized, controlled trial . SETTING: Experimental laboratory . SUBJECTS: Twenty-one male Wistar rats . INTERVENTIONS: Rats were treated with a continuous infusion of dopexamine (2.5 micrograms/kg/min; N = 7; group A) or 0.9% saline (n = 7; group B) during a study period of 120 mins . Both groups were given endotoxin (Escherichia coli lipopolysaccharide; 1.5 mg/kg Iv) over 60 mins . Animals in the control group (n = 7; group C) received a volume-equivalent infusion of 0.9% saline . Total volume substitution in all groups was 15 mL/kg/hr . MEASUREMENTS AND MAIN RESULTS: Blood flow in the intestinal villi of the distal ileum was determined using in vivo videomicroscopy at baseline, and 60 and 120 mins after the endotoxin challenge . These blood flow determinations were done by an observer who was unaware of the previous treatment of the animals . In addition, mean arterial pressure was monitored at baseline, and 15, 30, 45, 60, 75, 90, 105, and 120 mins later . The administration of 1.5 mg/kg endotoxin alone (group B) resulted in a reduction of the intestinal villus blood flow to 74.8 +/- 9.5% of baseline after 60 mins, and to 61.1 +/- 8.5% of baseline after 120 mins (baseline: 7.4 +/- 0.6 nL/min; 60 mins: 5.3 +/- 0.8 nL/min; 120 mins: 4.4 +/- 0.5 nL/min; p < .05) . This reduction of blood flow was associated with a decrease in the arteriolar diameters by 13.8 +/- 2.5% after 60 mins, and by 17.1 +/- 4.3% after 120 mins (p < .05 vs . baseline) . In contrast, villus blood flow in the dopexamine group (group A) did not show statistically significant changes during the entire study period, despite the administration of endotoxin (baseline: 8.2 +/- 0.6 nL/min; 60 mins: 7.3 +/- 0.8 nL/min; 120 mins: 7.8 +/- 0.5 nL/min) . No vasoconstriction of the villus arterioles was noted in this group . In control animals (group C), the blood flow (baseline: 8.1 +/- 1.6 nL/min; 60 mins: 7.6 +/- 1.4 nL/min: 120 mins: 7.8 +/- 1.4 nL/min) and the arteriolar diameters remained unchanged throughout the observation period . Mean arterial pressure did not differ between groups: it remained unaltered in all groups during the entire study period . CONCLUSIONS: Dopexamine maintains intestinal villus arterial perfusion and prevents the vasoconstriction in villus arterioles during early normotensive endotoxemia . Therefore, further studies in critically ill patients will have to determine whether the early prophylactic use of dopexamine can limit ischemia and prevent the development of multiple organ failure. Crit Care Med, 1996 Jul, 24(7), 1117 - 24 Experimental human endotoxemia increases cardiac regularity: results from a prospective, randomized, crossover trial; Godin PJ et al.; OBJECTIVE: To determine whether human endotoxemia is associated with a loss of the physiologic beat-to-beat variability of heart rate . DESIGN: Prospective, randomized, crossover, single-blind study . SETTING: Clinical research center in a federal, nonuniversity hospital . SUBJECTS: Healthy volunteers . INTERVENTIONS: Intravenous administration of reference (Escherichia coli) endotoxin or saline placebo, with or without previous administration of oral ibuprofen . MEASUREMENTS AND MAIN RESULTS: Electrocardiograms were continuously recorded and digitized using series of 1000 beat epochs of R-R intervals over 8 hrs . Analyses included measures in the time domain (standard deviation), frequency domain (power spectra), and a measure of regularity (approximate entropy) . Endotoxin administration was associated with loss of variability by all measures . This loss of variability remained significant even with administration of ibuprofen, which blocked the development of fever and endotoxin-related symptoms . CONCLUSIONS: Infusion of endotoxin into human volunteers causes loss of heart rate variability, as measured by standard deviation and power spectra, as well as an increase in heart rate regularity, as measured by approximate entropy . Changes in approximate entropy occurred earlier than changes in other heart rate variability measures and may be a useful means of detecting early sepsis . This reduction in regularity is consistent with a model in which the pathogenesis of multiple organ system dysfunction syndrome involves the physiologic uncoupling of vital organ systems. Cancer Res, 1996 Jul 1, 56(13), 2891 - 5 Overexpression of tissue inhibitor of metalloproteinases-2 retroviral-mediated gene transfer in vivo inhibits tumor growth and invasion; Imren S et al.; We have demonstrated previously that overexpression of tissue inhibitor of metalloproteinases-2 (TIMP-2), an inhibitor of matrix-degrading metalloproteinases, not only inhibits the invasive and metastatic behavior of tumor cells but also significantly decreases tumor growth in vivo (Y . A . DeClerck et at, Cancer Res., 52: 701-708, 1992) . This latter effect was found to be dependent on the ability of TIMP-2 to prevent the degradation of the collagen matrix (A . M . Montgomery et al., Cancer Res., 54: 5467-5473, 1994) . In this report, we have overexpressed TIMP-2 in tumor tissue by retroviral-mediated gene transfer into tumor cells by co-injecting s.c . in nude mice tumorigenic c-Ha-ras-transfected rat embryo fibroblasts with irradiated packaging cells producing high titer retroviral vectors containing the human TIMP-2 cDNA . The growth rate of tumors derived from cells co-injected with the TIMP-2 vector producer cells was significantly slower than the growth rate of tumors derived from cells co-injected with packaging cells producing a retrovirus containing the Escherichia coli beta-galactosidase gene . The transduction efficiency was estimated at 13%, and the production of a functional human TIMP-2 in tumor cells transduced with the TIMP-2-containing vector was documented . Furthermore, histological analysis of tumors derived from tumor cells co-injected with the TIMP-2 vector producer cells revealed the presence of a thick connective tissue capsule and a lack of local invasion . The data indicate that retroviral-mediated transduction of TIMP-2 cDNA into a limited population of tumor cells in vivo is sufficient to increase the accumulation of connective tissue proteins in tumor tissue, to inhibit growth, and to prevent local invasion. Nephrol Dial Transplant, 1996 Jul, 11(7), 1332 - 7 Haemolytic uraemic syndrome following bone marrow transplantation . Case report and review of the literature; Verburgh CA et al.; Thrombotic microangiopathy (TMA) can be a late complication of bone marrow transplantation (BMT) . A patient is described in whom the haemolytic uraemic syndrome developed 10 months after BMT and who died of E . coli sepsis while on maintenance haemodialysis . The literature is reviewed, regarding clinical presentation, incidence, pathogenesis and therapy . TMA can be observed, after an interval of 3-12 months, in about 6-26% of patients following BMT . Reported cases vary considerably in clinical severity, from mild presentations to severe TMA with high mortality rates despite intensive therapy . Important pathogenetic roles are ascribed to the conditioning total body irradiation and the use of cyclosporin A, but other factors may be involved as well . Next to supportive therapy, plasma exchange and the use of ACE inhibitors may be of value in treating BMT-associated TMA. Mutagenesis, 1996 Jul, 11(4), 327 - 33 MX100, a new Escherichia coli tester strain for use in genotoxicity studies; Kranendonk M et al.; The development of a new Escherichia coli tester strain for use in metabolic and mechanistic studies of genotoxins, strain MR2101/pKR11, has recently been reported . This strain, a derivative of the E . coli K12 laboratory strain AB1157, has sensitivity towards the detection of base-substitution mutagenesis, monitored by the reversion of arginine auxotrophy {argE3, (ochre)} . Besides arginine, MK2101/pKR11 is auxotrophic for histidine (hisG4), leucine (leuB6), proline (DeltaproA) and threonine (thr-1) . MX100 was developed to overcome the auxotrophy for four amino acids of MR2101/pKR11 which are non-essential for the mutagenic responsiveness of the strain . We restored the biosynthesis for these four amino acids in MR2101/pKR11, resulting in strain MX100 . This strain showed an almost 2-fold increase in mutagenic activity relative to MR2101/pKR11 with a set of diagnostic mutagens (aflatoxin B1, benzo{a}pyrene, 4-nitroquinoline-1-oxide, 2,7-dimethyl-benz{a}anthracene and others) and was further characterized with other types of mutagens in which it showed sensitivity towards the detection of oxidative (H2O2t-butyl-hydroperoxide, cumene-hydroperoxide, KO2) and carbonyl mutagens (methylglyoxal, malondialdehyde) . As MX100 seems to have the right characteristics of a versatile genotoxicity tester strain and due to the extensive genetic and physiological knowledge of E . coli K12 in general and AB1157 in particular, we propose that MX100 could serve as mother strain for the development of specialized tester strains, of interest in studies of metabolism and/or mechanism of action of genotoxic carcinogens. EMBO J, 1996 Jul 1, 15(13), 3442 - 7 Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase; Kavli B et al.; Uracil-DNA glycosylase (UDG) protects the genome by removing mutagenic uracil residues resulting from deamination of cytosine . Uracil binds in a rigid pocket at the base of the DNA-binding groove of human UDG and the specificity for uracil over the structurally related DNA bases thymine and cytosine is conferred by shape complementarity, as well as by main chain and Asn204 side chain hydrogen bonds . Here we show that replacement of Asn204 by Asp or Tyr147 by Ala, Cys or Ser results in enzymes that have cytosine-DNA glycosylase (CDG) activity or thymine-DNA glycosylase (TDG) activity, respectively . CDG and the TDG all retain some UDG activity . CDG and TDG have kcat values in the same range as typical multisubstrate-DNA glycosylases, that is at least three orders of magnitude lower than that of the highly selective and efficient wild-type UDG . Expression of CDG or TDG in Escherichia coli causes 4- to 100-fold increases in the yield of rifampicin-resistant mutants . Thus, single amino acid substitutions in UDG result in less selective DNA glycosylases that release normal pyrimidines and confer a mutator phenotype upon the cell . Three of the four new pyrimidine-DNA glycosylases resulted from single nucleotide substitutions, events that may also happen in vivo. EMBO J, 1996 Jul 1, 15(13), 3430 - 41 A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases; Truniger V et al.; The functional significance of the conserved motif 'YxGG/A', located between the 3'-5' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, has been studied by site-directed mutagenesis in phi29 DNA polymerase . Single substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity . Nine mutants showed an altered polymerase/3'-5' exonuclease balance on a template/primer DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization . Interestingly, these three different phenotypes could be obtained by mutating a single amino acid at the 'YxGG/A' motif . All different phenotypes could be directly related to defects in DNA binding at a particular active site . Thus, a high pol/exo ratio was related to a poor stability at the 3'-5' exonuclease active site . On the contrary, a low pol/exo ratio or null polymerization capacity was related to a poor stability at the polymerization active site and either a normal or an increased accessibility to the exonuclease active site . These results allow us to propose that this motif, located in the connecting region between the N-terminal and C-terminal domains, has a primary role in DNA binding, playing a critical role in the coordination or cross-talk between synthesis and degradation. EMBO J, 1996 Jul 1, 15(13), 3256 - 66 Stable binding of the herpes simplex virus ICP47 protein to the peptide binding site of TAP; Tomazin R et al.; The herpes simplex virus (HSV) ICP47 protein inhibits the MHC class I antigen presentation pathway by inhibiting the transporter associated with antigen presentation (TAP) which translocates peptides across the endoplasmic reticulum membrane . At present, ICP47 is the only inhibitor of TAP . Here, we show that ICP47 produced in bacteria can block human, but not mouse, TAP, and that heat denaturation of ICP47 has no effect on its ability to block TAP . ICP47 inhibited peptide binding to TAP without affecting ATP binding, consistent with previous observations that the peptide binding and ATP binding sites of TAP are distinct . ICP47 bound to TAP with a higher affinity (KD approximately 5 x 10(-8) M) than did peptides, and ICP47 did not dissociate from TAP . ICP47 was not transported by TAP and remained sensitive to proteases added from the cytosolic surface of the membrane . Peptides acted as competitive inhibitors of ICP47 binding to TAP, and this inhibition required a 100- to 1000-fold molar excess of peptide . These results demonstrate that ICP47 binds to a site which includes the peptide binding domain of TAP and remains bound to this site in a stable fashion. Mol Cell Biol, 1996 Jul, 16(7), 3884 - 92 The p53-binding protein 53BP2 also interacts with Bc12 and impedes cell cycle progression at G2/M; Naumovski L et al.; Using the yeast two-hybrid system, we have isolated a cDNA (designated BBP, for Bcl2-binding protein) for a protein (Bbp) that interacts with Bcl2 . Bbp is identical to 53BP2, a partial clone of which was previously isolated in a two-hybrid screen for proteins that interact with p53 . In this study, we show that specific interactions of Bbp/53BP2 with either Bcl2 or p53 require its ankyrin repeats and SH3 domain . These interactions can be reproduced in vitro with bacterially expressed fusion proteins, and competition experiments indicate that Bcl2 prevents p53 from binding to Bbp/53BP2 . BBP/53BP2 mRNA is abundant in most cell lines examined, but the protein cannot be stably expressed in a variety of cell types by transfection . In transiently transfected cells, Bbp partially colocalizes with Bcl2 in the cytoplasm and results in an increased number of cells at G2/M, possibly accounting for the inability to obtain stable transfectants expressing the protein . These results demonstrate that a single protein can interact with either Bcl2 or p53 both in yeast cells and in vitro . The in vivo significance of these interactions and their potential consequences for cell cycle progression and cell death remain to be determined. Mol Cell Biol, 1996 Jul, 16(7), 3714 - 9 Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair; Wei D et al.; Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair . However, recent studies of repair of cyclobutane pyrimidine dimers or benzo{a}pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation . To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR . To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE . The principal DNA adduct formed by either agent involves guanine . We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA . The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts . These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved . However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions . Instead, the rate of repair at a particular nucleotide position depends on the interaction between the specific adduct conformation and the local DNA conformation at that nucleotide. Mol Cell Biol, 1996 Jul, 16(7), 3668 - 78 A novel methyltransferase (Hmt1p) modifies poly(A)+-RNA-binding proteins; Henry MF et al.; RNA-binding proteins play many essential roles in the metabolism of nuclear pre-mRNA . As such, they demonstrate a myriad of dynamic behaviors and modifications . In particular, heterogeneous nuclear ribonucleoproteins (hnRNPs) contain the bulk of methylated arginine residues in eukaryotic cells . We have identified the first eukaryotic hnRNP-specific methyltransferase via a genetic screen for proteins that interact with an abundant poly(A)+-RNA-binding protein termed Npl3p . We have previously shown that npl3-1 mutants are temperature sensitive for growth and defective for export of mRNA from the nucleus . New mutants in interacting genes were isolated by their failure to survive in the presence of the npl3-1 allele . Four alleles of the same gene were identified in this manner . Cloning of the cognate gene revealed an encoded protein with similarity to methyltransferases that was termed HMT1 for hnRNP methyltransferase . HMT1 is not required for normal cell viability except when NPL3 is also defective . The Hmt1 protein is located in the nucleus . We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro . These findings now allow further exploration of the function of this previously uncharacterized class of enzymes. Mol Cell Biol, 1996 Jul, 16(7), 3637 - 50 Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7; Bardwell L et al.; Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae . To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo . When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in the absence of any additional yeast proteins . These complexes were specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7 . Neither the kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste7 and Kss1 were necessary for complex formation . Ste7-Kss1 (and Ste7-Fus3) complexes were present in yeast cell extracts and were undiminished in extracts prepared from a ste5delta-ste11delta double mutant strain . In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or pheromone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissociation of the high-affinity complex was shown to be required for either phosphorylation event . Deletions of Ste7 in the region required for its stable association with Kss1 and Fus3 in vitro significantly decreased (but did not eliminate) signaling in vivo . These findings suggest that the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ensure fidelity in signal transmission and to insulate one signaling pathway from another. Mol Cell Biol, 1996 Jul, 16(7), 3338 - 49 Determinants of DNA-binding specificity of ETS-domain transcription factors; Shore P et al.; Several mechanisms are employed by members of transcription factor families to achieve sequence-specific DNA recognition . In this study, we have investigated how members of the ETS-domain transcription factor family achieve such specificity . We have used the ternary complex factor (TCF) subfamily as an example . ERK2 mitogen-activated protein kinase stimulates serum response factor-dependent and autonomous DNA binding by the TCFs Elk-1 and SAP-la . Phosphorylated Elk-1 and SAP-la exhibit specificities of DNA binding similar to those of their isolated ETS domains . The ETS domains of Elk-1 and SAP-la and SAP-2 exhibit related but distinct DNA-binding specificities . A single residue, D-69 (Elk-1) or V-68 (SAP-1), has been identified as the critical determinant for the differential binding specificities of Elk-1 and SAP-1a, and an additional residue, D-38 (Elk-1) or Q-37 (SAP-1), further modulates their DNA binding . Creation of mutations D38Q and D69V is sufficient to confer SAP-la DNA-binding specificity upon Elk-1 and thereby allow it to bind to a greater spectrum of sites . Molecular modelling indicates that these two residues (D-38 and D-69) are located away from the DNA-binding interface of Elk-1 . Our data suggest a mechanism in which these residues modulate DNA binding by influencing the interaction of other residues with DNA. Curr Microbiol, 1996 Jul, 33(1), 60 - 6 Cloning and Characterization of Two Closely Linked Cellulase Genes from Cellvibrio mixtus Kahler CM, Pemberton JM. A 16.5-kb BamHI fragment of the Cellvibrio mixtus chromosome was found to direct carboxymethylcellulase, xylanase, and avicel hydrolysis . Two closely linked genes were subcloned from this insert . The gene, cmcI, was cloned as a 2.7-kb fragment and expressed in Escherichia coli . It encoded an enzyme of approximately 74 kDa which degraded carboxymethylcellulose and xylan but did not attack the microcrystalline cellulose substrate avicel . A second cellulase capable of degrading avicel, encoded by exoI, was found 5.5 kb downstream of cmcI . Two translation products of 53.7 kDa and 51.5 kDa were produced in E . coli strains expressing exoI . Northern analysis of total mRNA of C . mixtus grown on avicel, with a probe generated from cmcI, showed that cmcI and exoI were not cotranscribed in an operon. Microb Ecol, 1996 Jul, 32(1), 11 - 21 The Effect of Temperature on Viability of Carbon- and Nitrogen-Starved Escherichia coli Nelson SM, Attwell RW, Dawson MM, Smith CA. Escherichia coli was grown in a defined medium at optimum temperature and then transferred to each of five different starvation regimes at 5°C, 20°C, or 37°C, for 1000 hours . Cells were maintained with growth-limiting amounts of carbon or nitrogen, or without either or both nutrients . Bacterial cell viability was assessed by dilution plating, the reduction of 2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT), direct viable counts (DVC), and microcolony development . The recoverability of cells on solid medium declined most rapidly, and to the greatest extent in most cases, in cultures maintained at 37°C . Only nitrogen-starved cells maintained at 5°C became completely nonculturable . The reduction of INT consistently indicated higher numbers of viable cells compared to the other methods in all cultures . The viabilities of carbon- and nitrogen-limited cells, assessed by all methods, were similar to one another at each of the temperatures . Viability was lowest at 37°C . Nutrient-downshifted cells also followed a temperature-dependent pattern of survival with viability lowest at 37°C . Morphological differences were noted at different temperatures but were most obvious for nitrogen-starved cells at 37°C, which increased in length. Virology, 1996 Jul 1, 221(1), 120 - 9 Ligation of double-stranded and single-stranded {oligo(dT)} DNA by vaccinia virus DNA ligase; Odell M et al.; Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized . The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG . Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT) . This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases . The enzyme requires high ATP concentrations with a Km for the overall ligation of a ssDNA substrate of 0.8 mM . The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described. Virology, 1996 Jul 1, 221(1), 98 - 112 Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus; Mardassi H et al.; Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a small enveloped virus containing a positive-strand RNA genome, possesses at least three major structural proteins designated N, M, and E . The N protein is considered as the major component of the nucleocapsid, whereas M and E are membrane-associated . Previous studies using peptide-specific antibodies assigned these proteins to ORFs 7, 6, and 5, respectively . In the present report, monospecific antisera raised against Escherichia coli-expressed ORFs 5, 6, and 7 products were used to study the synthesis and processing of PRRSV structural proteins in the highly permissive MARC-145 cell line . Treatment of viral proteins with various glycosidases showed that only E was modified by N-linked glycans . Pulse-chase experiments revealed that intracellular transport of the major envelope glycoprotein was delayed in the premedial Golgi compartment . During the first 30 min of chase, E undergoes a gradual downward shift of its apparent molecular weight, thought to result from trimming of the mannose-rich glycan structures . Once E is transported to the medial Golgi or proximal elements, some molecules undergo complete processing of all their high-mannose N-linked oligosaccharides to complex type, while in other molecules only a fraction of N-linked glycans are terminally glycosylated . These two differentially glycosylated forms of E were found to be incorporated into extracellular virions . In cells and virions, both M and E were shown to occur in heterodimeric complexes linked by disulfide bonds . The oligomerization process, as analyzed from pulse-chase experiments, showed that M and E are incorporated into M-E complexes with different kinetics and efficiencies, in a fashion similar to their counterparts in equine arteritis virus . Apparently, all steps of E protein N-glycans processing proceed after its association with M which occurs in the endoplasmic reticulum (ER) . In the infected cells, E and M appear highly membrane-associated, while N is predominantly cytosolic. Arch Biochem Biophys, 1996 Jul 1, 331(1), 55 - 62 Characterization of an acyl-CoA-binding protein from Arabidopsis thaliana; Engeseth NJ et al.; A cDNA clone was obtained from Arabidopsis thaliana that encodes a protein containing 92 amino acid residues with high sequence identity (57%) to bovine acyl-CoA-binding protein (ACBP) . The coding sequence of this clone was expressed in Escherichia coli and the gene product (10.4 kDa) was purified . The recombinant A . thaliana ACBP (rAthACBP) was shown to bind acyl-CoA esters and protect acyl-CoAs from degradation by microsomal acyl-hydrolases . Antibodies that were raised to rAthACBP recognized the native Arabidopsis ACBP and also cross-reacted with a number of other plant ACBPs, including rapeseed (Brassica napus) ACBP . The pattern of expression and level of the gene product were examined in various tissues of Arabidopsis and Brassica using Western blotting . A . thaliana tissues contained between 3 and 143 micrograms AthACBP g(-1) FW depending on the tissue (0.4 to 14 nmol g(-1) FW) . Developing B . napus seeds underwent a 12-fold increase in ACBP levels during seed maturation (20 to 250 micrograms ACBP g(-1) FW); the highest concentration occurring near the peak of triacylglycerol accumulation (26 nmol g(-1) FW. Arch Biochem Biophys, 1996 Jul 1, 331(1), 9 - 14 The effects of nitric oxide on electron transport complexes; Welter R et al.; The effect of nitric oxide on mitochondrial electron transfer complexes was studied by comparing the activities of nitric oxide-treated and untreated, deoxygenated samples of purified beef heart succinate-cytochrome c reductase, succinate-ubiquinone reductase, and ubiquinol-cytochrome c reductase . More than 90% of succinate-cytochrome c reductase activity is lost during nitric oxide treatment . The activity of the succinate-ubiquinone reductase component of succinate-cytochrome c reductase decreases 95%, while the ubiquinol-cytochrome c reductase component is unaffected by nitric oxide . This inactivation is due primarily to the destruction of iron-sulfur clusters from succinate-ubiquinone reductase . When purified beef heart succinate-ubiquinone reductase was treated with nitric oxide, virtually all activity was irreversibly lost . The electron paramagnetic resonance (EPR) spectra of the treated complex showed typical iron-nitric oxide complex signals, confirming that inactivation is due to destruction of the iron-sulfur clusters . Similar results were obtained with purified Escherichia coli succinate-ubiquinone reductase . Pure beef heart ubiquinol-cytochrome c reductase treated with nitric oxide loses 40% of its initial activity, but regains most of it (90-100 % after 24 h of incubation at 0 degrees C in the absence of nitric oxide . This suggests that ubiquinol-cytochrome c reductase is protected from nitric oxide when complexed with succinate-ubiquinone reductase or that when split from succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase undergoes a conformational change which allows access of nitric oxide to the Rieske iron-sulfur center . Such access is not possible when ubiquinol-cytochrome c reductase is complexed with succinate-ubiquinone reductase . The loss of ubiquinol-cytochrome c reductase activity correlates with a decrease in the Rieske protein EPR signal intensity without formation of any new EPR signal . The Rieske iron-sulfur cluster signal is recovered after 24 h incubation in the absence of nitric oxide. Cancer, 1996 Jul 1, 78(1), 30 - 4 Phase II clinical trial with 5-fluorouracil, recombinant interferon-alpha-2b, and cisplatin for patients with metastatic or regionally advanced carcinoma of the esophagus; Wadler S et al.; BACKGROUND: Recombinant interferon-alpha (IFN) augments the cytotoxicity of both 5-fluorouracil (5-FU) and cisplatin in vitro . A phase II study of 5-FU and IFN resulted in response rates of 25-27% in patients with metastatic esophageal carcinoma . METHODS: A Phase II trial was initiated to determine the clinical utility of a three-drug combination (FIP) in patients with regionally advanced or metastatic esophageal carcinoma . Eligibility included biopsy-proven Stage III or IV squamous cell carcinoma or adenocarcinoma of the esophagus with no prior chemotherapy, adequate performance status, nutritional status, bone marrow, hepatic and renal function, and signed informed consent . Patients were treated in the exact sequence of IFN==>cisplatin==>5-FU . Patients received 5-FU, 750 mg/m2/day for 5 days followed by weekly bolus therapy at the same dose; cisplatin, 100 mg/m2 on Day 1, followed by weekly therapy, 25 mg/m2 over the course of 1 hour; and IFN, 10 MU subcutaneously 3 times/week beginning on Day 1 . All patients received sargramostim (granulocyte-macrophage colony-stimulating factor, Escherichia coli-derived), 5 micrograms/kg subcutaneously 5 times/week . No patients received radiotherapy . RESULTS: Twenty-four patients were enrolled; 23 were eligible, and 1 was excluded on pathology review (patient was found to have a leiomyoblastoma) . The demographics of the population were: median age, 63 years (range, 43-73 years); 18 male patients; squamous cell carcinoma: adenocarcinoma ratio, 22:1, and Stage III:IV ratio, 10:13 . Grade 3-4 National Cancer Institute Common Toxicity Criteria toxicities included: leukopenia (13), thrombocytopenia (14), and infection (9) . Grade 3 diarrhea, mucositis, and vomiting occurred in 6 patients, 4 patients, and 1 patient, respectively . There were two instances of sudden death, likely related to tumor progression . Major responses occurred in 15 of 23 patients (65%; 95% confidence interval, 43%, 85%) (1 complete response, 14 partial responses) . The median survival was 8.6 months; with a median follow-up of 26 months, estimated 30-month survival was 31% . CONCLUSIONS: This regimen, although moderately toxic, has substantial activity in metastatic and regionally advanced squamous cell carcinoma of the esophagus . Further investigations should be conducted to determine the role of IFN in the treatment of esophageal carcinoma. DNA Res, 1996 Jun 30, 3(3), 137 - 55 A 718-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 12.7-28.0 min region on the linkage map; Oshima T et al.; The 718,122 base pair sequence of the Escherichia coli K-12 genome corresponding to the region from 12.7 to 28.0 minutes on the genetic map is described . This region contains at least 681 potential open reading frames, of which 277 (41%) have been previously identified, 147 (22%) are homologous to other known genes, 139 (20%) are identical or similar to the hypothetical genes registered in databases, and the remaining 118 (17%) do not show a significant similarity to any other gene . In this region, we assigned a cluster of cit genes encoding multienzyme citrate lyase, two clusters of fimbrial genes and a set of lysogenic phage genes encoding integrase, excisionase and repressor in the e14 genetic element . In addition, a new valine tRNA gene, designated valZ, and a family of long directly repeated sequences, LDR-A, -B and -C, were found. J Biol Chem, 1996 Jun 28, 271(26), 15443 - 50 The hepatitis B virus transactivator protein, HBx, interacts with single-stranded DNA (ssDNA) . Biochemical characterizations of the HBx-ssDNA interactions; Qadri I et al.; Human hepatitis B virus X protein, HBx, is widely acknowledged as a transcriptional transactivator . While HBx has been shown to increase gene expression in trans, it is generally believed that it does not bind double-stranded DNA . Using several experimental approaches, we show that HBx interacts with single-stranded DNA in a manner that is not sequence-specific . Various heterologous single-stranded DNA (ssDNA) oligonucleotides were able to compete in HBx-ssDNA interactions in gel shift assays . Escherichia coli non-sequence-specific, single-stranded DNA binding protein, E . coli SSB, displaced the HBx-ssDNA interactions, confirming the ability of HBx to interact with single-stranded DNA in a non-sequence-specific manner . We have further characterized the HBx-ssDNA interactions under various biochemical conditions . These include the effects of mono- and divalent cations, the effect of cardiolipin and heparin, pH and temperature dependence, and variations in the incubation time . HBx bound more tightly to d(pyrimidines)25 than to d(purines)25, a property that is characteristic of other single-stranded DNA-binding proteins (SSBs) . Collectively the results presented here provide the first evidence of HBx's interaction with ssDNA . The biochemical parameters of these interactions were similar to those of known viral and cellular SSBs. J Biol Chem, 1996 Jun 28, 271(26), 15336 - 40 Probing a role of subunit IV of the Escherichia coli bo-type ubiquinol oxidase by deletion and cross-linking analyses; Saiki K et al.; Subunit IV of the Escherichia coli bo-type ubiquinol oxidase is a 12-kDa membrane protein encoded by the cyoD gene and is conserved in the bacterial heme-copper terminal oxidases . To probe the functional role of subunit IV, we carried out deletion analysis and chemical cross-linking experiments with a homobifunctional and cleavable reagent . Spectroscopic properties of the mutant oxidases suggest that the C-terminal two-third (Val45 to His109) containing helices II and III is essential for the functional expression of the oxidase complex and for the CuB binding to the heme-copper binuclear center in subunit I . Cross-linking studies indicate that subunit IV is in close vicinity to subunit III . Based on these observations, we propose that subunit IV is present in a cleft formed by subunits I and III and assists the CuB binding to subunit I during biosynthesis or assembly of the oxidase complex. J Biol Chem, 1996 Jun 28, 271(26), 15776 - 81 Differential sensitivities of portions of the mRNA for ribosomal protein S20 to 3'-exonucleases dependent on oligoadenylation and RNA secondary structure; Coburn GA et al.; The 3'-exonucleolytic decay of the mRNA for ribosomal protein S20 has been reconstituted in vitro using purified RNase II and crude extracts enriched for polynucleotide phosphorylase (PNPase) activity . We show that RNase II can catalyze the degradation of the 5' two-thirds of the S20 mRNA and that prior oligoadenylation of the 3' termini of truncated S20 mRNA substrates can significantly stimulate the initiation of degradation by RNase II . The intact S20 mRNA is, however, insensitive to attack by RNase II and polyadenylation of its 3'-end cannot overcome the natural resistance of the S20 mRNA to RNase II . Complete degradation of either the entire S20 mRNA without prior endonucleolytic cleavage or the 3'-terminal 147-residue fragment is dependent on both oligoadenylation and PNPase activity . Moreover, this process can take place in the absence of RNase E activity . Our data point to the importance of oligoadenylation in facilitating 3'-exonucleolytic activity and indicate that there are alternative degradative pathways . The implications for mRNA decay are discussed. J Biol Chem, 1996 Jun 28, 271(26), 15407 - 13 Refined crystal structures of guanine nucleotide complexes of adenylosuccinate synthetase from Escherichia coli; Poland BW et al.; Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A . Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position . The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base . The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group . Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site . The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites . The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate . No electron density, however, for the corresponding diphosphate nucleoside is observed . In addition, electron density for bound Mg2+ is absent in these nucleotide complexes . The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase . The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase. J Biol Chem, 1996 Jun 28, 271(26), 15656 - 61 The ordered assembly of the phiX174-type primosome . III . PriB facilitates complex formation between PriA and DnaT; Liu J et al.; The properties of two mutant PriA proteins, PriA C439Y and PriA C445Y have been used to reveal the role of PriB during assembly of the phiX174-type primosome . The replication defects of both mutant PriA proteins could be rescued by high concentrations of DnaT . Analysis of the formation of intermediate complexes in primosome assembly and the effect of PriB on PriA binding to DNA demonstrated that the mutant PriA proteins could not form a PriA-PriB complex on DNA carrying a primosome assembly site . Consequently, the mutant proteins also could not form PriA-PriB-DnaT complexes at concentrations of DnaT sufficient to form such a complex with wild-type PriA . In addition, PriB was found to stabilize wild-type but not mutant PriA proteins on DNA . At high concentrations of DnaT, both mutant and wild-type PriA proteins could form a PriA-DnaT complex and support PriB-independent phiX174 complementary strand DNA replication . Thus, during primosome assembly, PriB facilitates complex formation between PriA and DnaT. J Biol Chem, 1996 Jun 28, 271(26), 15649 - 55 The ordered assembly of the phiX174-type primosome . II . Preservation of primosome composition from assembly through replication; Ng JY et al.; Gel filtration chromatography was used to isolate both preprimosomal and primosomal complexes formed on single-stranded DNA-binding protein-coated phiX174 DNA by the combination of PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG . The presence and relative amounts of primosomal proteins in these complexes were determined by Western blotting . Protein-DNA complexes isolated (i) after assembly in the presence of 10 microM ATP, (ii) after preprimosome movement in the presence of 1 mM ATP, (iii) after priming in the presence of the four ribonucleoside triphosphates, or (iv) after complementary strand DNA replication in the presence of the DNA polymerase III holoenzyme all had the same protein composition; preprimosomes contained PriA, PriB, PriC, DnaT, and DnaB, whereas primosomes included DnaG . The stable association of DnaG with the protein-DNA complex could be attributed partially to its ability to remain bound to the primers synthesized . In the absence of PriC, the efficiencies of priming and replication were reduced by one-third and one-half, respectively, even though PriC was not required for the formation of stable protein-DNA complexes on a 304-nucleotide-long single strand of DNA containing a primosome assembly site (Ng, J . Y., and Marians, K . J . (1996) J . Biol . Chem . 271, 15642-15648) . We hypothesize that maintenance of the primosome on the replicated DNA may provide a mechanism to allow primosomes to participate in the resolution of recombination intermediates and intermediates formed during double strand break repair by permitting the re-establishment of a replication fork. J Biol Chem, 1996 Jun 28, 271(26), 15642 - 8 The ordered assembly of the phiX174-type primosome . I . Isolation and identification of intermediate protein-DNA complexes; Ng JY et al.; The phiX-type primosome was discovered during the resolution and reconstitution in vitro of the complementary strand DNA replication step of the phiX174 viral life cycle . This multienzyme bidirectional helicase-primase complex can provide the DNA unwinding and Okazaki fragment-priming functions at the replication fork and has been implicated in cellular DNA replication, repair, and recombination . We have used gel mobility shift assays and enhanced chemiluminescence Western analysis to isolate and identify the pathway of primosome assembly at a primosome assembly site (PAS) on a 300-nucleotide-long single-stranded DNA fragment . The first three steps do not require ATP and are as follows: (i) PriA recognition and binding to the PAS, (ii) stabilization of the PriA-PAS complex by the addition of PriB, and (iii) formation of a PriA-PriB-DnaT-PAS complex . Subsequent formation of the preprimosome involves the ATP-dependent transfer of DnaB from a DnaB-DnaC complex to the PriA-PriB-DnaT-PAS complex . The final preprimosomal complex contains PriA, PriB, DnaT, and DnaB but not DnaC . A transient interaction between the preprimosome and DnaG generates the five-protein primosome . As described in an accompanying article (Ng, J . Y., and Marians, K . J . (1996) J . Biol . Chem . 271, 15649-15655), when assembled on intact phiX174 phage DNA, the primosome also contains PriC. J Biol Chem, 1996 Jun 28, 271(26), 15486 - 90 Specificity of DnaK for arginine/lysine and effect of DnaJ on the amino acid specificity of DnaK; de Crouy-Chanel A et al.; Molecular chaperones form a class of proteins that bind selectively to nascent, unfolded, misfolded, or aggregated polypeptides and are involved in protein folding, protein targeting to membranes, and protein renaturation after stress . Chaperones70, including the DnaK chaperone of Escherichia coli, interact specifically with peptides enriched in internal hydrophobic residues, with a preference for positively charged peptides . We previously reported that DnaK interacts with the hydrophobic amino acids Ile, Leu, Val, Ala, Phe, Trp, and Tyr . In the present study, we show that DnaK also possesses a specific binding site for the positively charged amino acids arginine and lysine . Furthermore, the binding of arginine and lysine to DnaK is strengthened when its hydrophobic binding sites are occupied . The specificity of DnaK for Arg/Lys is supported by DnaK-peptide binding studies; the homopolypeptides poly-Arg and poly-Lys interact with DnaK, contrasting with other hydrophilic homopolypeptides, and hydrophobic peptides interact more strongly with DnaK if they contain Arg/Lys at their N terminus . Interestingly, the cochaperone DnaJ attenuates the interaction of DnaK with hydrophobic amino acids while strengthening its interaction with arginine or lysine . The interaction of DnaK with both hydrophobic sequences and with arginine and lysine, and its modulation by DnaJ, may have important implications in both protein folding and protein insertion into membranes. J Biol Chem, 1996 Jun 28, 271(26), 15393 - 400 A single point mutation in CTP synthetase of Chlamydia trachomatis confers resistance to cyclopentenyl cytosine; Wylie JL et al.; A Chlamydia trachomatis strain (L2/CPEC) resistant to the cytotoxic effects of cyclopentenyl cytosine (CPEC) was isolated by a stepwise selection procedure . This strain showed an approximate 350-fold increase in resistance to CPEC . Sequencing of the gene encoding CTP synthetase from this resistant strain revealed a single point mutation, resulting in a change of amino acid 149 from Asp to Glu . This appeared to be the only mutation in L2/CPEC, because no changes in CTP transport, CTP synthetase expression, or incorporation of CPEC into DNA or RNA could be detected . The mutation in the chlamydial CTP synthetase resulted in a loss of CTP feedback inhibition . This was demonstrated both in vivo using Escherichia coli cells carrying the cloned gene, and an in vitro assay using partially purified preparations of CTP synthetase . As a result of the loss of feedback inhibition, E . coli cells carrying the CPECR CTP synthetase showed a 22-fold increase in their CTP pools . However, examination of the CTP pools of L2/CPEC revealed no change in CTP levels when compared with wild type C . trachomatis. J Biol Chem, 1996 Jun 28, 271(26), 15590 - 6 Self-activation of recombinant human lysosomal procathepsin D at a newly engineered cleavage junction, "short" pseudocathepsin D; Beyer BM et al.; To obtain a recombinant model of human cathepsin D with kinetic properties that are identical with native human liver enzyme, we have addressed the significant differences in structure and catalytic function between naturally occurring enzyme and bacterially derived pseudocathepsin D . Human procathepsin D was expressed in a baculovirus system to obtain correctly folded, glycosylated enzyme that upon acidification completely converts to the active intermediate, pseudocathepsin D . The oligosaccharide moieties of this recombinant enzyme contributed to about 5% of the apparent molecular mass of the enzyme, and the carbohydrate composition was quite similar to the native material . However, specificity constants (kcat/Km) of this glycosylated pseudoform for several synthetic chromogenic substrates were considerably less (33%-50%) than those for the native enzyme and were virtually identical with those observed with nonglycosylated pseudocathepsin D . A cleavable junction suitable for self-processing at the normal maturation point of human cathepsin D was engineered into procathepsin D according to known specificity requirements of this enzyme, and the construct was expressed using baculovirus . Following experiments that demonstrated that the new proenzyme failed to process to the expected point, the new cleavage junction was moved 6 residues toward the amino terminus of procathepsin D and expressed in Escherichia coli . After refolding, the protein containing the newly engineered junction self-processed, generating a shortened mutant form of pseudocathepsin D that is 6 residues longer at the amino terminus than the native material . The kinetic properties of this newly engineered pseudoform proved to be identical with those of the native enzyme, thus establishing an improved recombinant model for this important aspartic proteinase. J Biol Chem, 1996 Jun 28, 271(26), 15549 - 57 Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site; Kim JH et al.; Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli exhibits a basal PRPP-independent glutaminase activity having a kcat/Km that is 0.3% of fully active enzyme . Binding of PRPP activates the enzyme by a structural change that lowers the Km for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5-phosphoribosylamine . By analysis of the x-ray structure of the glutamine site containing bound 6-diazo-5-oxonorleucine, a glutamine affinity analog, and by site-directed mutagenesis we have identified residues important for glutamine binding, catalysis, and coupling with PRPP . Tyr74 is a key residue in the coupling between the sites for glutamine in the NH2-terminal domain and PRPP in the COOH-terminal domain . Arg73 and Asp127 have roles in glutamine binding . The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys1 to participate as a proton acceptor in formation of the thiolate needed for nucleophilic attack on the carboxamide of glutamine, nor as a general acid for amide nitrogen transfer . Based on the x-ray model of the glutamine site and analysis of a mutant enzyme we propose that the free NH2 terminus of Cys1 functions as the proton acceptor and donor . The results indicate that the side chain of Asn101 and the backbone nitrogen of Gly102 function to stabilize a tetrahedral oxyanion resulting from attack of Cys1 on the glutamine carboxamide . Cys1, Arg73, Asn101, Gly102, and Asp127 are conserved in the NH2-terminal domain of a subfamily of amidotransferases that includes asparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes . Tyr74, on the other hand, is conserved only in glutamine PRPP amidotransferase sequences consistent with a specific role in interdomain coupling . The catalytic framework of key glutamine site residues supports the assignment of glutamine PRPP amidotransferase to a recently described Ntn (NH2-terminal nucleophile) hydrolase family of enzymes. J Biol Chem, 1996 Jun 28, 271(26), 15401 - 6 Membrane topology of the colicin A pore-forming domain analyzed by disulfide bond engineering; Duche D et al.; Four colicin A double-cysteine mutants possessing a disulfide bond in their pore-forming domain were constructed to study the translocation and the pore formation of colicin A . The disulfide bonds connected alpha-helices 1 and 2, 2 and 10, 3 and 9, or 3 and 10 of the pore-forming domain . The disulfide bonds did not prevent the colicin A translocation through the Escherichia coli envelope . However, the mutated colicins were able to exert their in vivo channel activity only after reduction of their disulfide bonds . In vitro studies with brominated phospholipid vesicles and planar lipid bilayers revealed that the disulfide bond that connects the alpha-helices 2 and 10 prevented the colicin A membrane insertion, whereas the other double-cysteine mutants inserted into lipid vesicles . The disulfide bonds that connect either the alpha-helices 1 and 2 or 3 and 10 were unable to prevent the formation of a conducting channel in presence of membrane potential . These results indicate that alpha-helices 1, 2, 3, and 10 remain at the membrane surface after application of a membrane potential. J Biol Chem, 1996 Jun 28, 271(26), 15527 - 32 Characterization of the ligand-binding domains of glutamate receptor (GluR)-B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins; Arvola M et al.; We recently reported that a functional ligand-binding site of an alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)-selective glutamate receptor (GluR)-D subunit can be expressed in insect cells as a soluble, N-glycosylated fusion protein consisting of two segments (S1 and S2) that are related by amino acid sequence to bacterial periplasmic binding proteins (Kuusinen, A., Arvola, M., and Keinanen, K., EMBO J . 14, 6327-6332) . In an attempt to further characterize the structural determinants for ligand binding, we have now expressed the ligand-binding sites of GluR-B and GluR-D subunits in Escherichia coli as soluble periplasmic proteins . The bacterially expressed S1-S2 fusion proteins bound {3H}AMPA with a high affinity (Kd of 12 nM for GluR-B, Kd of 60 nM for GluR-D) and with a ligand pharmacology typical of native AMPA receptors, indicating that N-linked glycosylation is not required for the formation or the maintenance of the ligand-binding site . The flip and flop splice variants of the GluR-D S1-S2 fusion protein bound {3H}AMPA with equal affinities, whereas deletion of the C-terminal one-third of the S2 segment including the flip/flop sequence resulted in a loss of binding activity . Our results highlight the potential of bacterial expression for the analysis of the binding site and support a close structural similarity between glutamate receptors and bacterial proteins. J Biol Chem, 1996 Jun 28, 271(26), 15736 - 42 The major catalytic subunit isoforms of cAMP-dependent protein kinase have distinct biochemical properties in vitro and in vivo; Gamm DM et al.; Two isoforms of the catalytic subunit of cAMP-dependent protein kinase, Calpha and Cbeta1, are known to be widely expressed in mammals . Although much is known about the structure and function of Calpha, few studies have addressed the possibility of a distinct role for the Cbeta proteins . The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity . Cbeta1 demonstrated higher Km values for some peptide substrates than did Calpha, but Cbeta1 was insensitive to substrate inhibition, a phenomenon that was observed with Calpha at substrate concentrations above 100 microM . Calpha and Cbeta1 displayed distinct IC50 values for the alpha and beta isoforms of the protein kinase inhibitor, protein kinase inhibitorpeptide, and the type IIalpha regulatory subunit (RIIalpha) . Of particular interest, purified type II holoenzyme containing Cbeta1 exhibited a 5-fold lower Ka value for cAMP (13 nM) than did type II holoenzyme containing Calpha (63 nM) . This latter result was extended to in vivo conditions by employing a transcriptional activation assay . In these experiments, luciferase reporter activity in COS-1 cells expressing RIIalpha2Cbeta12 holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RIIalpha2Calpha2 holoenzyme . These results provide evidence that type II holoenzyme formed with Cbeta1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously. Gene, 1996 Jun 26, 172(2), 303 - 8 Cloning, sequencing and functional expression of a DNA encoding pig cytosolic malate dehydrogenase: purification and characterization of the recombinant enzyme; Trejo F et al.; Using the polymerase chain reaction, DNA encoding cytosolic malate dehydrogenase (cMDH) has been cloned from a pig heart cDNA library . Large amounts of the enzyme (30 mg per litre of original culture) have been produced in Escherichia coli using an inducible expression vector (pKK223-3) in which the 5'-non-coding region of the gene was replaced with the tac promoter . The complete nucleotide sequence of the DNA is reported for the first time . The recombinant cMDH purified was shown to be identical to the native enzyme according to: chromatographic behaviour, isoelectric point, N-terminal amino acid sequence, and physiochemical and catalytic properties. Gene, 1996 Jun 26, 172(2), 211 - 5 A 14-kDa Arabidopsis thaliana RNA polymerase III subunit contains two alpha-motifs flanked by a highly charged C terminus; Larkin RM et al.; We have sequenced a cDNA and a gene, AtRPC14, from Arabidopsis thaliana (At) (ecotype Columbia) that encode a protein related to the yeast RNA polymerases (Pol) I and III subunits, yAC19 . Polyclonal antibodies raised against the recombinant At polypeptide (AtC14) bind to the Pol I and/or III subunits of about 13-15 kDa, but do not bind to any Pol II subunit in Pol purified from cauliflower, wheat or At . The amino acid (aa) sequence derived from the AtRPC14 cDNA and genomic clones consists of 122 aa, as compared to the 142 aa in the yeast yAC19 subunit and 143 aa in a putative Caenorhabditis elegans CeAC16 subunit . AtC14, yAC19 and CeAC16 contain a conserved sequence of about 85 aa which is related to two motifs in the alpha subunit of Escherichia coli (Ec) Pol . AtC14 lacks a highly charged N terminus of about 50 aa found in both yAC19 and CeAC16, but has a highly charged C terminus of about 30 aa not found in yAC19 and CeAC16. Gene, 1996 Jun 26, 172(2), 207 - 9 Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua; Matsushita Y et al.; A cDNA encoding farnesyl diphosphate (FPP) synthase (FPPS) has been cloned from a cDNA library of Artemisia annua . The sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with a calculated molecular weight of 39420 kDa . The deduced aa sequence of the cDNA was highly similar to FPPS from other plants, yeast and mammals, and contained the two conserved domains found in polyprenyl synthases including FPPS, geranylgeranyl diphosphate synthases and hexaprenyl diphosphate synthases . The expression of the cDNA in Escherichia coli showed enzyme activity for FPPS in vitro. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6786 - 91 Negative electrostatic surface potential of protein sites specific for anionic ligands; Ledvina PS et al.; Determination of the crystal structure of an "open" unliganded active mutant (T141D) form of the Escherichia coli phosphate receptor for active transport has allowed calculation of the electrostatic surface potential for it and two other comparably modeled receptor structures (wild type and D137N) . A discovery of considerable implication is the intensely negative potential of the phosphate-binding cleft . We report similar findings for a sulfate transport receptor, a DNA-binding protein, and, even more dramatically, redox proteins . Evidently, for proteins such as these, which rely almost exclusively on hydrogen bonding for anion interactions and electrostatic balance, a noncomplementary surface potential is not a barrier to binding . Moreover, experimental results show that the exquisite specificity and high affinity of the phosphate and sulfate receptors for unions are insensitive to modulations of charge potential, but extremely sensitive to conditions that leave a hydrogen bond donor or acceptor unpaired. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6647 - 52 Copy-choice recombination mediated by DNA polymerase III holoenzyme from Escherichia coli; Canceill D et al.; Formation of deletions by recombination between short direct repeats is thought to involve either a break-join or a copy-choice process . The key step of the latter is slippage of the replication machinery between the repeats . We report that the main replicase of Escherichia coli, DNA polymerase III holoenzyme, slips between two direct repeats of 27 bp that flank an inverted repeat of approximately equal 300bp . Slippage was detected in vitro, on a single-stranded DNA template, in a primer extension assay . It requires the presence of a short (8 bp) G+C-rich sequence at the base of a hairpin that can form by annealing of the inverted repeats . It is stimulated by (i) high salt concentration, which might stabilize the hairpin, and (ii) two proteins that ensure the processivity of the DNA polymerase III holoenzyme: the single-stranded DNA binding protein and the beta subunit of the polymerase . Slippage is rather efficient under optimal reaction conditions because it can take place on >50% of template molecules . This observation supports the copy-choice model for recombination between short direct repeats. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6621 - 5 Interaction of the two cytosolic domains of mammalian adenylyl cyclase; Whisnant RE et al.; Adenylyl cyclase activity can be reconstituted by simple mixture of the two cytosolic domains of the enzyme after their independent synthesis in Escherichia coli . We have synthesized and purified the C1a domain of type I adenylyl cyclase and the C2 domain of the type II enzyme to assess their interactions with each other and with the activators Gsalpha and forskolin . In the absence of an activator, the fragments associate with low affinity and display low catalytic activity . This basal activity can be stimulated more than 100-fold by either forskolin or activated Gsalpha . Further, the addition of these activators increases the apparent affinity of the fragments for each other . Stimulation of catalysis by Gsalpha and forskolin is synergistic . These data suggest a model wherein either Gsalpha or forskolin enhances association of the other activator with adenylyl cyclase, as well as facilitating the interaction between the C1 and C2 domains of the enzyme. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6562 - 6 Nucleator-dependent intercellular assembly of adhesive curli organelles in Escherichia coli; Hammar M et al.; Bacterial adhesion to other bacteria, to eukaryotic cells, and to extracellular matrix proteins is frequently mediated by cell surface-associated polymers (fimbriae) consisting of one or more subunit proteins . We have found that polymerization of curlin to fimbriae-like structures (curli) on the surface of Escherichia coli markedly differs from the prevailing model for fimbrial assembly in that it occurs extracellularly through a self-assembly process depending on a specific nucleator protein . The cell surface-bound nucleator primes the polymerization of curlin secreted by the nucleator-presenting cell or by adjacent cells . The addition of monomers to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomer and the surface of monomer condensation. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6504 - 9 An abundant cell-surface polypeptide is required for swimming by the nonflagellated marine cyanobacterium Synechococcus; Brahamsha B; Certain marine unicellular cyanobacteria of the genus Synechococcus exhibit a unique and mysterious form of motility characterized by the ability to swim in liquid in the absence of flagella . An abundant cell-surface-associated polypeptide that is required for swimming motility by Synechococcus sp . strain WH8102 has been identified, and the gene encoding it, swmA, has been cloned and sequenced . The predicted SwmA protein contains a number of Ca2+-binding motifs as well as several potential N-glycosylation sites . Insertional inactivation of swmA in Synechococcus sp . strain WH8102 results in a loss of the ability to translocate, although the mutant strain, Swm-1, generates torque . This suggests that SwmA functions in the generation of thrust. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6443 - 7 Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct; Duckett DR et al.; Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents . Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage . We show that the human mismatch recognition activity MutSalpha recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between O4-methylthymine and adenine . However, the protein fails to recognize 1,3-intrastrand adduct produced by trans-diamminedichloroplatinum(II) at a d(GpTpG) sequence . These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(II) adducts by MutSalpha may be involved in the cytotoxic action of this chemotherapeutic agent. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6320 - 5 Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli; Bugos RC et al.; Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage . Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity . To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli . VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins . The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da . Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa) . The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region . The E . coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts . This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction . The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6314 - 9 Cell cycle regulation and cell type-specific localization of the FtsZ division initiation protein in Caulobacter; Quardokus E et al.; Many genes involved in cell division and DNA replication and their protein products have been identified in bacteria; however, little is known about the cell cycle regulation of the intracellular concentration of these proteins . It has been shown that the level of the tubulin-like GTPase FtsZ is critical for the initiation of cell division in bacteria . We show that the concentration of FtsZ varies dramatically during the cell cycle of Caulobacter crescentus . Caulobacter produce two different cell types at each cell division: (i) a sessile stalked cell that can initiate DNA replication immediately after cell division and (ii) a motile swarmer cell in which DNA replication is blocked . After cell division, only the stalked cell contains FtsZ . FtsZ is synthesized slightly before the swarmer cells differentiate into stalked cells and the intracellular concentration of FtsZ is maximal at the beginning of cell division . Late in the cell cycle, after the completion of chromosome replication, the level of FtsZ decreases dramatically . This decrease is probably mostly due to the degradation of FtsZ in the swarmer compartment of the predivisional cell . Thus, the variation of FtsZ concentration parallels the pattern of DNA synthesis . Constitutive expression of FtsZ leads to defects in stalk biosynthesis suggesting a role for FtsZ in this developmental process in addition to its role in cell division. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6236 - 40 Targeted disruption of the Rad51 gene leads to lethality in embryonic mice; Tsuzuki T et al.; The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair . To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells . Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped . There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development . Doubly knocked-out ES cells were not detected under conditions of selective growth . These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell . The homozygous Rad51 null mutation can be categorized in cell-autonomous defects . Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.
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