Escherichia coli

J Biol Chem, 1992 Feb 5, 267(4), 2388 - 92
Site-directed mutagenesis of the phosphate-binding consensus sequence in Escherichia coli adenylosuccinate synthetase; Liu F et al.; Adenylosuccinate synthetases from different sources contain an N-terminal glycine-rich sequence GDEGKGK, which is homologous to the conserved sequence GXXXXGK found in many other guanine nucleotide-binding proteins or enzymes . To determine the role of this sequence in the structure and function of Escherichia coli adenylosuccinate synthetase, site-directed mutagenesis was performed to generate five mutant enzymes: G12V (Gly12----Val), G15V (Gly15----Val), G17V (Gly17----Val), K18R (Lys18----Arg), and I19T (Ile19----Thr) . Comparison of the kinetic properties of the wild-type enzyme and those of the mutant enzymes revealed that the sequence is critical for enzyme activity . Replacement of Gly12, Gly15, or Gly17 with Val, or replacement of Lys18 with Arg, resulted in significant decreases in the kcat/Km values of the enzyme . Because the consensus sequence GXXXXGK(T/S) has been found in many GTP-binding proteins, isoleucine at position 19 in the E . coli adenylosuccinate synthetase was changed to threonine to produce the sequence GDEGKGKT . This mutation, which more closely resembles the consensus sequence, resulted in a 160-fold increase in the Km value for substrate GTP; however, there were no great changes for the other two substrates, IMP and aspartate . Based on these data, we suggest that the N-terminal glycinerich sequence in E . coli adenylosuccinate synthetase plays a more important role in enzyme catalysis than in substrate binding . In addition, a hydrophobic amino acid residue such as isoleucine, leucine, or valine, rather than threonine, may play a critical role in GTP binding in adenosuccinate synthetase . These findings suggest that the glycine-rich sequence in adenylosuccinate synthetase functions differently relative to those in other GTP binding proteins or enzymes.

J Biol Chem, 1992 Feb 5, 267(4), 2244 - 50
Insertional mutagenesis of Bordetella pertussis adenylate cyclase; Ladant D et al.; We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase . A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide . This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation . We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein . All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes . These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies . Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation . In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding . By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.

J Biol Chem, 1992 Feb 5, 267(4), 2209 - 13
IciA protein, a specific inhibitor of initiation of Escherichia coli chromosomal replication; Hwang DS et al.; Specific binding of IciA protein to the 13-mers in the origin of a minichromosome (oriC) inhibits initiation of replication in vitro by blocking the opening of this region effected by the initiator DnaA protein (Hwang, D.S., and Kornberg, A . (1990) Cell 63, 325-331) . Isolation of the iciA gene (Thony, B., Hwang, D.S., Fradkin, L., and Kornberg, A . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 4066-4070) has made possible the construction of an IciA-overproducing strain, which in turn has simplified the isolation of a large quantity of the protein, indistinguishable from that of the wild-type strain . Based on sedimentation and gel filtration, the IciA protein is an elongated dimer of a 33.4-kDa subunit . The specific binding of IciA protein to the 13-mers was stable for 2 h at 30 degrees C . The amounts of IciA protein, detected by immunoassays, increased 4-fold compared with levels (about 100 dimers) in log-phase cells whereas levels of DnaA protein decreased upon entry of cells into the stationary phase.

J Mol Biol, 1992 Feb 5, 223(3), 811 - 4
GMC oxidoreductases . A newly defined family of homologous proteins with diverse catalytic activities; Cavener DR; Sequence comparison of Drosophila melanogaster glucose dehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus niger glucose oxidase and Hansenula polymorpha methanol oxidase indicates that these four diverse flavoproteins are homologous, defining a new family of proteins named the GMC oxidoreductases . These enzymes contain a canonical ADP-binding beta alpha beta-fold close to their amino termini as found in other flavoenzymes . This domain is encoded by a single exon of the D . melanogaster glucose dehydrogenase gene.

J Mol Biol, 1992 Feb 5, 223(3), 721 - 42
Refined structure of elongation factor EF-Tu from Escherichia coli; Kjeldgaard M et al.; The crystal structure of trypsin-modified elongation factor Tu from Escherichia coli, in complex with the cofactor guanosine diphosphate has been refined to a crystallographic R-factor of 19.3%, at 2.6 A resolution . In the model described, the root-mean-square deviation from ideality is 0.019 A for bond distances and 3.9 degrees for angles . The protein consists of three domains: an alpha/beta domain (residues 1 to 200), containing the binding site of the GDP cofactor, and consisting of a six-stranded beta-pleated sheet, six alpha-helices, and two all-beta domains (residues 209 to 299 and 300 to 393), belonging to the tertiary structural class of antiparallel beta-barrels . The GDP-binding domain has a folding that is found in other GDP-binding proteins . Elongation factor Tu interacts with proteins, nucleic acids and nucleotides, making this molecule well suited as a model system for the study of these interactions.

J Mol Biol, 1992 Feb 5, 223(3), 637 - 50
Identification of a nonapeptide motif in the vimentin head domain involved in intermediate filament assembly; Herrmann H et al.; The assembly of soluble vimentin subunits into intermediate filaments (IFs) is dependent on information located in the amino-terminal domain . Using site-directed mutagenesis of a Xenopus laevis vimentin cDNA and an Escherichia coli production system to obtain pure mutated protein, we have identified, in the head domain, a nine amino acid motif (SSYRRIFGG), evolutionarily conserved from amphibia to man, which plays an important role in the orderly formation of IFs . Exchanges in the central di-arginine and in the two aromatic residues interfere with IF assembly of vimentin in vitro: on assembly under standard assembly conditions (160 mM-NaCl) most of the protein is included in dense aggregates, with a variable and minor proportion of IFs, whereas at lower ionic concentrations short and incomplete IF-like structures are formed . The deletion of the whole motif results in a protein that under standard assembly conditions (e.g . 160 mM-NaCl) predominantly and rapidly precipitates into large aggregates of non-IF material, whereas at lower ionic strength (e.g . 50 mM-NaCl) both IFs and dense aggregates are formed simultaneously . Our results show that the mutated protein can assume different forms at the same time and under the same conditions . This motif alone is insufficient for the formation of normal IFs as demonstrated by a mutant in which the motif has been brought closer to the alpha-helical rod domain by deletion of 55 internal amino acid residues . Corresponding observations have been made, by immunofluorescence microscopy, upon transfection of cultured epithelial cells lacking vimentin IFs . The importance of the head domain motif for the assembly and higher-order arrangement of IFs is discussed.

J Biol Chem, 1992 Feb 5, 267(4), 2605 - 9
Amino acids conserved in interleukin-1 receptors (IL-1Rs) and the Drosophila toll protein are essential for IL-1R signal transduction; Heguy A et al.; The cytoplasmic domain of the human T cell-type interleukin-1 receptor (hIL-1R) is not involved in the binding, internalization, or nuclear localization of interleukin-1 (IL-1), but is essential for signal transduction . We have previously localized a 50-amino acid region (residues 477-527) critical for IL-1-mediated activation of the interleukin-2 promoter in T cells . This region displays a striking degree of amino acid conservation in human, murine, and chicken IL-1Rs . Here we report the results of a site-directed mutational analysis of the cytoplasmic domain of the hIL-1R . We have introduced single-amino acid substitutions at positions conserved in all three receptors and at nonconserved positions and identified key amino acids for IL-1R function in signal transduction . Three basic (Arg431, Lys515, and Arg518) and 3 aromatic (Phe513, Trp514, and Tyr519) amino acids that are conserved in human, murine, and chicken IL-1Rs could not be replaced without abolishing IL-1R-mediated signal transduction . A substitution at another conserved position (Pro521) reduces significantly the ability of the IL-1R to transmit the IL-1 signal . Nonconserved residues could be replaced without affecting signal transduction . The cytoplasmic domain of the IL-1R is related to that of the Drosophila Toll protein, with a 26% identity and a 43% similarity in amino acid sequence . The amino acids shown to be essential for IL-1R function are conserved in the Toll protein . Our experimental data indicate that the amino acid sequence similarity between the IL-1R and the Drosophila toll protein reflects a functional homology between the two proteins.

J Biol Chem, 1992 Feb 5, 267(4), 2507 - 11
Pore-forming activity of OmpA protein of Escherichia coli; Sugawara E et al.; Escherichia coli outer membrane protein OmpA was purified to homogeneity, as a monomer, from a K12 derivative deficient in both OmpF and OmpC porins . When proteoliposomes reconstituted from the purified OmpA, phospholipids, and lithium dodecyl sulfate were tested for permeability to small molecules by osmotic swelling, it was found that OmpA produced apparently nonspecific diffusion channels that allowed the penetration of various solutes . The pore-forming activity was destroyed by the heat denaturation of the OmpA protein, and the use of an OmpA-deficient mutant showed that the activity was not caused by copurifying contaminants . The size of the OmpA channel, estimated by comparison of diffusion rates of solutes of different sizes, was rather similar to that of E . coli OmpF and OmpC porins, i.e . about 1 nm in diameter . The rate of penetration of L-arabinose caused by a given amount of OmpA protein, however, was about a hundredfold lower than the rate produced by the same amount of E . coli OmpF porin . The addition of large amounts of lithium dodecyl sulfate to the reconstitution mixture increased the permeability through the OmpA channel, apparently by facilitating the correct insertion of OmpA into the bilayer.

J Biol Chem, 1992 Feb 5, 267(4), 2337 - 44
Toxic effects of high levels of ppGpp in Escherichia coli are relieved by rpoB mutations; Tedin K et al.; A controversy has surrounded the questions of whether or not guanosine tetraphosphate (ppGpp) is a specific inhibitor of bacterial rRNA and tRNA synthesis, especially during normal exponential growth, and whether the RNA polymerase is the target of ppGpp action . To answer these questions, a pBR322-derived plasmid, pKT28, was constructed that carries the Escherichia coli relA gene encoding a ppGpp synthetase under control of the lacUV5 promoter . The plasmid was used to transform the ppGpp reporter strain VH271 in which expression of beta-galactosidase from an rrnB P1 promoter is inhibited by ppGpp . In the presence of high concentrations of lac inducer, bacteria of the transformed strain accumulate ppGpp with the result that synthesis of rRNA and beta-galactosidase is inhibited and growth ceases . At low concentrations of inducer, growth is only reduced and cells form small white colonies on X-gal indicator plates . After continued incubation, these colonies form blue sectors of faster growing mutant cells . Phage P1 transduction experiments showed that these mutants have mutations cotransducing with rpoB, the gene encoding the beta-subunit of RNA polymerase . One particular mutant strain, KT13, had acquired partial resistance to ppGpp inhibition of rRNA synthesis . The mutation in this strain was cloned by in vivo recombination into an rpoB plasmid . The presence of this plasmid conferred increased resistance to overproduction of ppGpp . These results suggest that ppGpp is a specific inhibitor of rRNA synthesis, even in the absence of amino acid starvation, and that RNA polymerase is involved as the target of ppGpp action.

J Biol Chem, 1992 Feb 5, 267(4), 2240 - 3
The tRNA species for redundant genetic codons NNU and NNC . A thought on the absence of phenylalanine tRNA with AAA anticodon in Escherichia coli; Gavini N et al.; The redundant genetic codons NNU and NNC (where N is A, T, G, or C) specify the same amino acid and are decoded by their cognate tRNAs, which contain either a guanosine or a modified base in the wobble position of the anticodons . Since tRNAs with an adenosine in the wobble position of the anticodon, which are complementary to the NNU codons, are not found naturally, we have generated a tRNA(Phe) with AAA anticodon and examined how an adenosine in the wobble position would affect its biological function in Escherichia coli . We found that the tRNA(Phe) with GAA anticodon (wild-type) repressed the expression of the pheA gene via tRNA(Phe)-mediated attenuation of transcription, whereas the tRNA(Phe) with AAA anticodon did not influence the expression of the pheA gene . Furthermore, elevated levels of tRNA(Phe)(AAA) did not support the growth of an E . coli strain carrying a temperature-sensitive mutation in the pheS gene at 42 degrees C . Since the presence of a multicopy plasmid carrying the gene that encodes tRNA(Phe)(GAA), a substrate for phenylalanyl tRNA synthetase, enables the E . coli strain carrying the pheS(Ts) mutation to grow at 42 degrees C, the above observation suggests that unlike tRNA(Phe)(GAA), tRNA(Phe)(AAA) is not a good substrate for phenylalanyl-tRNA synthetase . Therefore, we postulate that the presence of adenosine at the wobble position of anticodons was specifically eliminated and the tRNAs with guanosine or a modified base in the wobble position were selected to decode both NNU and NNC codons in E . coli.

J Mol Biol, 1992 Feb 5, 223(3), 743 - 67
Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure; Neri D et al.; The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10% . With these protein preparations the three-dimensional structure was determined in solution . The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r . data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER . A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates . The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r . data . The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core . The n.m.r . structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63 . Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r . samples . Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution . Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.

J Biol Chem, 1992 Jan 25, 267(3), 1962 - 8
The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia; Horie Y et al.; Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys) . Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys) . However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight . Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria . We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142 . These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography . Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity . Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants . Cysteamine treatment and removal of the carboxyl-terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys) . The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both . The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7 . The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia.

J Biol Chem, 1992 Jan 25, 267(3), 1840 - 5
Insect glutathione S-transferases . Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides; Fournier D et al.; Two classes of glutathione transferases have been identified and purified from Musca domestica . The first, designated as GST1, migrates as a single band of 28 kDa in SDS-gel electrophoresis, and the second, designated as GST2, migrates as a 32-kDa band . Antisera prepared against each class have no immunological cross-reactivity, and heterodimeric associations between the two classes have not been detected . Each class is composed of several isoforms: GST1 is composed of forms with isoelectric points from 4 to 9, whereas all the forms of GST2 have acidic pI values . Screening of cDNA libraries yielded clones coding for GST1, and the gene was sequenced and expressed in Escherichia coli . The high activity found in an insecticide-resistant strain (Cornell R) is correlated with high level of GST1 transcript.

Biochemistry, 1992 Feb 4, 31(4), 1153 - 8
Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesis; Altamirano MM et al.; The nucleotide sequence of the nagB gene in Escherichia coli, encoding glucosamine-6-phosphate deaminase, located four cysteinyl residues at positions 118, 219, 228, and 239 . Chemical modification studies performed with the purified enzyme had shown that the sulfhydryl groups of two of these residues form a vicinal pair in the enzyme and are easily modified by thiol reagents . The allosteric transition to the more active conformer (R), produced by the binding of homotropic (D-glucosamine 6-phosphate or 2-deoxy-2-amino-D-glucitol 6-phosphate) or heterotropic (N-acetyl-D-glucosamine 6-phosphate) ligands, completely protected these thiols against chemical modification . Selective cyanylation of the vicinal thiols with 2-nitro-5-(thiocyanato)benzoate, followed by alkaline hydrolysis to produce chain cleavage at the modified cysteines, gave a pattern of polypeptides which allowed us to identify Cys118 and Cys239 as the residues forming the thiol pair . Subsequently, three mutated forms of the gene were constructed by oligonucleotide-directed mutagenesis, in which one or both of the cysteine codons were changed to serine . The mutant proteins were overexpressed and purified, and their kinetics were studied . The dithiol formed by Cys118 and Cys239 was necessary for maximum catalytic activity . The single replacements and the double mutation affected catalytic efficiency in a similar way, which was also identical to the effect of the chemical block of the thiol pair . However, only one of these cysteinyl residues, Cys239, had a significant role in the allosteric transition, and its substitution for serine reduced the allosteric interaction energy, due to a lower value of KT.

Biochemistry, 1992 Feb 4, 31(4), 1142 - 7
Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D; Conner GE et al.; Procathepsin D is the intracellular aspartyl protease precursor of cathepsin D, a major lysosomal enzyme . Procathepsin D is rapidly processed inside the cell, and, thus, examination of its proteolyic activation and structure has been difficult . To study this proenzyme, a nonglycosylated form of the human fibroblast procathepsin D was expressed in Escherichia coli, refold in vitro, and purified by affinity chromatography on pepstatinyl agarose . Sequence analysis of the refolded, autoactivated enzyme allowed determination of the autoproteolytic cleavage site . The sequence surrounding this cleavage site between residues LeuP26 and IleP27 (in the "pro" region) resembled the first cleavage site found during activation of other aspartyl proteases . Thus, the autoactivated procathepsin D is analogous to the pepsin activation intermediate, which has been termed pseudopepsin . The enzymatic activity, thermal and pH stability, and fluorescence spectra of pseudocathepsin D were compared to mature, predominantly two-chain, cathepsin D isolated from human placenta . The results indicated that pseudocathepsin D and mature enzyme have a similar Km toward a peptide substrate and cleave a protein substrate at identical sites . Temperature stability of the recombinant enzyme was similar to that of the tissue-derived enzyme . However, the recombinant enzyme had increased stability at low pH when compared to the glycosylated tissue-derived two-chain cathepsin D . Fluorescence spectra of the recombinant and tissue-derived enzymes were identical . Thus, the absence of asparagine-linked oligosaccharides and the presence of the remaining segment of propeptide did not significantly alter the structural and enzymatic properties of the enzyme.

Biochemistry, 1992 Feb 4, 31(4), 1119 - 24
SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study; Breukink E et al.; SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli {Lill, R., Dowhan, W., & Wickner, W . (1990) Cell 60, 271-280} . SecA insertion into the phospholipid bilayer could a role in this process . We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique . It was established that SecA is surface-active and can insert into lipid monolayers . This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin . Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes . High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer . ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers . ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced . AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers . The physiological significance of these results in protein translocation is discussed.

Biochemistry, 1992 Feb 4, 31(4), 954 - 8
Fidelity of HIV-1 reverse transcriptase copying RNA in vitro; Ji JP et al.; The genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse transcriptase . We developed an assay for reverse transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro . A lacZ alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription . The corresponding DNA template was used to examine errors by reverse transcriptase during second-strand DNA synthesis . With both templates, the mutations introduced by reverse transcriptase were identified by their mutant phenotypes in an M13 lacZ alpha-complementation assay . We found that the reverse transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates . The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template . The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28,000 and 37,000, respectively . The most frequent mutations produced by HIV-1 RT copying the RNA template were C----T transitions and G----T transversions resulting from misincorporation of dAMP.

Biochemistry, 1992 Feb 4, 31(4), 1020 - 30
Interaction of Tn501 mercuric reductase and dihydroflavin adenine dinucleotide anion with metal ions: implications for the mechanism of mercuric reductase mediated Hg(II) reduction; Cummings RT et al.; The flavoprotein Tn501 mercuric reductase (MerA) catalyzes the reduction of Hg(II) to Hg(0) through the intermediacy of the tightly bound two-electron-reduced cofactor FADH- . To gain insight into the MerA mechanism, the interaction of the holoenzyme or free FADH- with various metal ions was investigated . The free two-electron-reduced FAD cofactor, FADH-, readily reduces a variety of metal ions, provided they have suitably high redox potentials . For Hg(II) with various ligands, the rate of reduction is inversely proportional to the stability of the Hg(II)-ligand complex . These results are consistent with the free cofactor reducing metal ions by an outer-sphere electron transfer mechanism . In contrast, MerA can tightly bind several redox labile metal ions, but only Hg(II) is reduced . The inability of MerA to reduce these bound metal ions may suggest that MerA differs from free FADH- and utilizes an inner-sphere electron transfer mechanism in Hg(II) reduction.

FEBS Lett, 1992 Feb 3, 297(1-2), 77 - 80
A new gene of the f1 operon of Y . pestis involved in the capsule biogenesis; Karlyshev AV et al.; The DNA sequence determination of the f1 operon between the genes encoding the F1 subunit (caf1) and chaperone-like protein (caf1M) revealed a large open reading frame that codes for a polypeptide similar to some E . coli proteins involved in the biogenesis of fimbria . The deletion and in trans complementation analyses showed that this gene is not necessary for extracellular transport of the F1 subunit but plays a role in the capsule assembly.

Mol Biochem Parasitol, 1992 Feb, 50(2), 295 - 306
Three beta-tubulin cDNAs from the parasitic nematode Haemonchus contortus; Geary TG et al.; Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles . Furthermore, certain residues of beta-tubulin seem to be critical for this mechanism . Although the benzimidazoles selectively affect nematode vs . mammalian beta-tubulin, the molecular basis for this differential action is not known . To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of beta-tubulin cDNAs from the ruminant parasite, Haemonchus contortus . We have cloned and sequenced three beta-tubulin cDNAs from this organism, beta 12-16, beta 12-164, and beta 8-9 . The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes . beta 8-9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus . By comparing the sequences of these and other nematode beta-tubulins with mammalian beta-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined . Sequences very similar or identical to beta 8-9 and beta 12-16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H . contortus . However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to beta 8-9.

Mol Biochem Parasitol, 1992 Feb, 50(2), 285 - 94
Cloning of a cDNA encoding phosphoenolpyruvate carboxykinase from Haemonchus contortus; Klein RD et al.; Biochemical and metabolic data have led to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes . To facilitate the determination of the molecular basis for host vs parasite differences in PEPCK, we have cloned a cDNA encoding this enzyme from a parasitic nematode of ruminants, Haemonchus contortus . H . contortus PEPCK was cloned by functional complementation of a PEPCK-, malic enzyme- strain of Escherichia coli (E1786) using an egg stage H . contortus cDNA library in lambda ZAPII . Selection was for growth on malate as the sole carbon source (malate+ phenotype) . We isolated a plasmid, pPEPCK, which reproducibly confers a malate+ phenotype in E1786 . The sequence of the 2.0-kb EcoRI insert of pPEPCK predicts a 612-amino acid protein which shows about 74% similarity to Drosophila melanogaster and chicken PEPCK . Extracts of E1786{pPEPCK}, but not E1786, contain IDP- or GDP-dependent PEPCK enzyme activity . Sequence analysis revealed that the open reading frame (ORF) in pPEPCK lacked a 5' initiation codon and was probably expressed as an in-frame fusion protein with beta-galactosidase . A strategy combining library screening with PCR analysis of positive clones led to the identification of a clone encoding 6 additional NH2-terminal amino acids, including a Met, which, by comparison with known PEPCK amino acid sequences, is likely to be the translation initiation site.

Mol Biochem Parasitol, 1992 Feb, 50(2), 245 - 54
Ferritins of Schistosoma mansoni: sequence comparison and expression in female and male worms; Dietzel J et al.; Recombinant clones of Schistosoma mansoni cDNA libraries containing the complete coding regions of 2 different ferritin subunits have been isolated and sequenced . This allows for the first time a comparison of ferritin sequences from an invertebrate with those of vertebrates . The deduced amino acid sequences of both Schistosoma ferritin subunit clones show significant homology to vertebrate ferritin H chains . Similarity exceeds 50% identity and includes the recently identified ferroxidase center which is present only in H chains . However, non-conservative substitutions of amino acid residues lining the 3-fold symmetry channel were found, and a gap of 3 successive amino acids unique to the 2 Schistosoma ferritin sequences was identified . Remarkably, for each of the 2 genes, we found a conspicuous difference in the amount of ferritin transcripts between females and males: one of the genes is preferentially expressed in females, the other in males.

Hybridoma, 1992 Feb, 11(1), 71 - 86
Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin; van Duijnhoven HL et al.; Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin . As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein . Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin . Four of the monoclonal antibodies did not recognize mouse furin . All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis . Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter . This approach was necessary, since physiological levels of fur gene encoded proteins appeared to be very low . In cells transfected with human or mouse fur cDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies . Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.

Am Rev Respir Dis, 1992 Feb, 145(2 Pt 1), 388 - 93
Polyethylene glycol-conjugated superoxide dismutase attenuates septic lung injury in guinea pigs; Suzuki Y et al.; Reactive oxygen species (ROS), including superoxide anions, play an important role in mediating acute lung injury . We examined whether polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) attenuates lung injury in Escherichia coli-treated guinea pigs . Twenty-four guinea pigs were divided into four groups: (1) control group; (2) septic group, in which live E . coli (2 x 10(9)/kg) were injected intravenously; (3) pretreatment group, in which PEG-SOD (2,000 IU/kg) was injected intravenously 15 min before E . coli; and (4) posttreatment group, in which PEG-SOD (2,000 IU/kg) was injected intravenously 30 min after E . coli . Lung injury was assessed by the concentration ratio of 125I-labeled albumin in lung tissue and bronchoalveolar lavage (BAL) fluid relative to plasma (L/P and BAL/P), lung wet-to-dry weight ratio, and the number of neutrophils in BAL fluid . Plasma half-life of PEG-SOD in normal guinea pigs was 13.5 h . L/P, lung wet-to-dry weight ratio, and the number of neutrophils in BAL fluid decreased in both pretreatment and posttreatment groups compared with the septic group . BAL/P decreased in the pretreatment group but not in the posttreatment group compared with the septic group . After the animal model studies, we investigated the effect of PEG-SOD on the human neutrophil extracellular generation of ROS stimulated by phorbol myristate acetate (PMA) in lucigenin-dependent chemiluminescence (CL) . PEG-SOD at concentrations greater than or equal to 0.1 U/ml inhibited PMA-induced CL in a dose-dependent manner . We also examined the effect of PEG-SOD on the neutrophil intracellular generation of ROS using flow cytometry to assess intracellular hydroethidine oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Am Rev Respir Dis, 1992 Feb, 145(2 Pt 1), 348 - 54
Regional and systemic oxygen delivery/uptake relations and lactate flux in hyperdynamic, endotoxin-treated dogs; Curtis SE et al.; Pathologic oxygen supply dependency (PO2SD) may be etiologic in multisystem organ failure (MSOF) and has been related to mortality in sepsis . Although elevated lactate levels are generally assumed to be a marker of anaerobiosis in these patients, endotoxin may increase serum lactate by inactivation of pyruvate dehydrogenase (PDH), unrelated to tissue PO2 . We hypothesized that regional lactate flux may correlate poorly with local oxygen delivery in sepsis . This study examined both the whole-body (WB) and regional (isolated hind limb L and gut G) responses to endotoxin infusion in terms of oxygen delivery, oxygen uptake, and lactate flux in 12 pentobarbital-anesthetized dogs . To separate hypoxia-induced lactate production from that related to inactivation of PDH by endotoxin, half the dogs received dichloroacetate (DCA), a PDH activator . After endotoxin and volume resuscitation, each animal had low systemic vascular resistance with normal to high cardiac output . Despite adequate oxygen delivery to WB, L, and G, arterial lactate levels rose significantly . A 30-min hypoxic challenge (12% FIO2) did not increase lactate levels but did increase WB O2 uptake . DCA normalized lactate levels without influencing oxygen delivery and uptake relations . These data show that lactate levels in endotoxic states may be a poor marker of tissue hypoxia and may be more related to PDH activity.

Tijdschr Diergeneeskd, 1992 Feb 1, 117(3), 82 - 6
{Necropsy findings in fattening pigs during the period 1979-1989}; Bethlehem M et al.; Necropsy findings of 1041 fattening pigs, during the period of 1979-1989 are presented . The pigs originated from one veterinary practice association . Enterotoxicosis associated with E . coli serotype O149:K91:K88 was the most prevalent diagnosis, followed by fibrinous pleuropneumonia due to Actinobacillus pleuropneumoniae . A decrease was noticed for Aujeszky disease during the late years . After 1985 an increase was found for torsio mesenterialis . In 1.7% of the cases no post mortem diagnosis could be made.

Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 900 - 4
The third chromophore of DNA photolyase: Trp-277 of Escherichia coli DNA photolyase repairs thymine dimers by direct electron transfer; Kim ST et al.; Photolyases repair pyrimidine dimers in DNA by converting the light energy of 300- to 500-nm photons into chemical energy . Enzymes from various organisms contain two chromophore cofactors (FADH2 and either methenyltetrahydrofolate or 8-hydroxy-5-deazaflavin) that absorb the low-energy photons and initiate splitting of the cyclobutane ring by a radical mechanism . Here, we show that, in addition to these two chromophores, in the far UV range, direct excitation of one specific tryptophan residue (out of 15 total) in the polypeptide chain of Escherichia coli photolyase leads to splitting of the cyclobutane ring with high quantum yield (phi = 0.56), independent of the other chromophores . The specific tryptophan residue responsible for photosensitized repair was identified as Trp-277 by site-specific mutagenesis.

Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 1138 - 42
Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy; Lynch CM et al.; Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli beta-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene . Direct gene transfer by infusion of virus into rat carotid arteries was not observed . However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful . Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 1123 - 7
Isolation of a src homology 2-containing tyrosine phosphatase; Plutzky J et al.; Tyrosine phosphorylation is controlled by the opposing actions of tyrosine kinases and phosphotyrosine phosphatases (PTPs) . src homology 2 domains (SH2) are found in several types of signaling proteins, including some tyrosine kinases . These domains bind phosphotyrosyl proteins and thus help promote signal transduction . Using mixed oligonucleotide-directed polymerase chain reactions, two previously undescribed rat PTP cDNA fragments were generated . Through subsequent screening of rat megakaryocyte and human erythroleukemia libraries, we obtained a full-length coding sequence for one of these fragments . This cDNA, SH-PTP1, encodes a tyrosine phosphatase containing two highly conserved SH2 domains . SH-PTP1, with a 2.4-kilobase mRNA, a predicted open reading frame of 595 amino acids, and a structure suggesting a nontransmembrane protein, is expressed primarily in hematopoietic and epithelial cells . When expressed in Escherichia coli, SH-PTP1 possesses PTP activity . The structure of SH-PTP1 establishes an additional branch of the tyrosine phosphatase family and suggests mechanisms through which tyrosine phosphatases might participate in signal transduction pathways.

Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 1016 - 20
In vivo duplication of genetic elements by the formation of stem-loop DNA without an RNA intermediate; Ohshima A et al.; Gene duplication through cDNA synthesis by reverse transcriptase is believed to have played an important role in the diversification of genomes during evolution . Here, we demonstrate that a genomic DNA sequence can be duplicated in vivo as a result of template switching . When an inverted repeat (IR) structure was inserted in a site downstream from a ColE1 plasmid origin of DNA replication, transformation of Escherichia coli cells with this plasmid resulted in the production of a new DNA fragment encompassing the region from the origin to the center of the IR structure . The structure of this DNA molecule is composed of a long stem-loop formed by a single-stranded DNA, in which the loop is formed by the IR structure . The DNA fragment is designated slDNA, for stem-loop DNA . The experiments in this study suggest that during DNA replication, template switching at the stem-loop structure formed by the IR structure gives rise to slDNA utilizing the nascent DNA strand or the parental strand as a template . The mechanistic implications of slDNA synthesis, and its possible roles in genome evolution, are discussed.

J Bacteriol, 1992 Feb, 174(4), 1197 - 204
Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478; Whelan KF et al.; A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, SalI, and XhoI . The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids . All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map . A region involved in incompatibility was cloned and mapped . The location of a previously unreported arsenite resistance gene was also determined . The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb SalI fragment of R478.

J Bacteriol, 1992 Feb, 174(4), 1119 - 23
Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli; Walker MS et al.; The effects of mutations in the -10, -35, and Fnr box regions of the narGHJI promoter of Escherichia coli were determined by assaying the expression of beta-galactosidase from narG::lacZ fusion plasmids under aerobic and anaerobic conditions . A 1-base change in the -10 hexamer completely abolished expression, whereas a 3-base change to create the consensus TATAAT resulted in significant aerobic as well as anaerobic expression . A mutation in the putative -35 hexamer did not affect anaerobic expression but reduced aerobic expression from the construction with the -10 consensus sequence . A mutation in the Fnr box severely reduced anaerobic expression but did not affect aerobic expression . When the complete 5' region of the nar operon including the NarL box was present, nitrate stimulated both aerobic and anaerobic expression . Stimulation of expression by nitrate occurred in an fnr mutant but not in a narL mutant . We conclude that the rate of transcription of the nar operon is dependent on two distinct modes of transcription . One mode, which occurs at low levels, depends on the -10 and -35 hexamer sequences and is dramatically enhanced by changing the -10 sequence to the consensus TATAAT . The second depends on the -10 and Fnr box sequences but is independent of the -35 sequence . This second mode occurs at a very high level under anaerobic conditions when Fnr is activated and is also enhanced by changing the -10 sequence to the consensus TATAAT . NarL, activated by nitrate, stimulated both modes of transcription, indicating that it does not act through Fnr but that it directly affects the interaction of RNA polymerase with the promoter.

Eur J Biochem, 1992 Feb 1, 203(3), 641 - 53
Biogenesis of the yeast vacuole (lysosome) . Mutation in the active site of the vacuolar serine proteinase yscB abolishes proteolytic maturation of its 73-kDa precursor to the 41.5-kDa pro-enzyme and a newly detected 41-kDa peptide; Hirsch HH et al.; The codon of the catalytic serine in the active site of the vacuolar serine proteinase yscB (PrB) was changed to alanine, yielding the mutant gene prb1-Ala519 . Following replacement of the wild-type PRB1 allele with prb1-Ala519, only a 73-kDa molecule was detected by immunoprecipitation with PrB-specific antiserum . The size of the mutant molecule corresponds to the unprocessed cytoplasmic precursor (pre-super-pro-PrB), as detected in sec61 mutants, when translocation into the endoplasmic reticulum is blocked . However, the mutant molecule is completely translocated into the secretory pathway, as indicated by protection from proteinase K digestion in spheroplast lysates in the absence of detergent . When N-glycosylation was inhibited in prb1-Ala519 mutant cells by tunicamycin, a smaller molecule of about 71 kDa appeared consistent with single N-glycosylation and signal-sequence cleavage of the translocated mutant PrB molecule in the endoplasmic reticulum . Thus, the active-site mutation prevents the wild-type processing of the N-glycosylated 73-kDa precursor of PrB to the 41.5 kDa pro-PrB in the endoplasmic reticulum . In order to characterize the processing of wild-type super-pro-PrB in more detail, we generated antibodies against the non-enzymatic superpeptide domain of the 73-kDa precursor expressed in Escherichia coli . We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDS/PAGE is generated in the endoplasmic reticulum . Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wild-type cells . Correspondingly, the superpeptide was virtually undetectable by immunoblotting wild-type cell extracts . In contrast, no degradation of radioactively labeled 41-kDa superpeptide was observed within 60 min in mutant strains deficient in the vacuolar proteinase yscA (PrA), in which maturation of vacuolar pro-PrB to active PrB is blocked . Accordingly, superpeptide antigenic material was readily detected by immunoblotting cell extracts and enriched in vacuolar preparations of PrA deficient mutant cells . These results indicate that the superpeptide and pro-PrB travel to the vacuole, where the superpeptide is rapidly degraded upon pro-PrB activation to PrB . Using purified vacuoles, rapid degradation of the superpeptide was reconstituted in vitro by addition of either mature PrA or mature PrB . However, the PrA-triggered in vitro degradation of the superpeptide required PrB activity, as this process was inhibited in the presence of the PrB inhibitor chymostatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Biochem, 1992 Feb 1, 203(3), 483 - 91
Three-dimensional 15N-1H-1H and 15N-13C-1H nuclear-magnetic resonance studies of HPr a central component of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli . Assignment of backbone resonances; van Nuland NA et al.; We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr . The protein was uniformly enriched with 15N and 13C to overcome spectral overlap . Complete assignments were obtained for the backbone 1H, 15N and 13C resonances, using three-dimensional heteronuclear 1H NOE 1H-15N multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation 1H-15N multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on 15N-enriched HPr and an additional three-dimensional triple-resonance 1HN-15N-13C alpha correlation spectroscopy (HNCA) experiment on 13C, 15N-enriched HPr . Many of the sequential backbone 1H assignments, as derived from two-dimensional NMR studies {Klevit, R.E., Drobny, G.P . & Waygood, E.B . (1986) Biochemistry 25, 7760-7769}, were corrected . Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact.

Clin Orthop, 1992 Feb, (275), 243 - 7
Arthroscopic management of pyarthrosis; Parisien JS et al.; Sixteen patients (average age, 38 years; range, 20-63 years) with pyarthrosis were treated during a ten-year period by arthroscopic techniques consisting of joint debridement and application of suction drains, combined with appropriate antibiotics . There were 13 knees, two shoulders, and one ankle in the series . At the first visit to the authors' institution, patients typically had fever, leukocytosis, elevated sedimentation rate, and localized joint findings, such as generalized tenderness, swelling, effusion, and painful, limited range of motion in almost every joint involved . Most patients were seen two to five days after the onset of symptoms . After the initial culture and sensitivity were obtained and broad-spectrum antibiotics were administered, all patients were taken to the operating room on an emergency basis . At an average follow-up evaluation of 36 months (range, 14-48 months), the results have been excellent to good, without evidence of recurrence.

J Cell Biol, 1992 Feb, 116(4), 957 - 65
Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil; de Cuevas M et al.; Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain . The stalk domain has sequence features characteristic of alpha-helical coiled coils . To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli . When observed by EM, this protein formed a rodlike structure 40-55 nm long that was occasionally bent at a hingelike region near the middle of the molecule . An additional EM study and a chemical cross-linking study showed that this protein forms a parallel dimer and that the two chains are in register . Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha-helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C . From these results, we conclude that the stalk of kinesin heavy chain forms an alpha-helical coiled coil structure . The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha-helical structure in the stalk is less stable than the rest . By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E . coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C . We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.

Diabetes, 1992 Feb, 41(2), 183 - 6
Cloning and expression of IDDM-specific human autoantigens; Rabin DU et al.; A DNA cloning approach was taken to identify islet cell protein antigens that are recognized specifically by insulin-dependent diabetes mellitus (IDDM) sera . A human islet cDNA library was generated and screened with diabetic sera . In this article, identification of two clones is described . Proteins expressed by these lambda phages appeared to react specifically with newly diagnosed diabetic sera . Islet cell antibody 12 (ICA12) was tested by Western blotting . ICA512 was not reactive with sera in the Western format but was specifically immunoprecipitated by diabetic sera from an Escherichia coli extract.

Cell Immunol, 1992 Feb, 139(2), 531 - 40
Marker profile, enzyme activity, and function of a human myelomonocytic leukemia cell line; Kavai M et al.; Morphological and functional characteristics of a permanent human leukemia cell line (DD) that possesses myelomonocytic features were investigated . The cells bear a second type Fc gamma receptor and form rosettes with sheep erythrocytes sensitized with rabbit IgG (EA) . However, the surface-bound EA is not internalized . The cell line lacks the surface markers CD2, CD19, CD14, HLA-DR, Fc gamma receptor I, Fc gamma receptor III, and CR3 . alpha 1-Antitrypsin, lysozyme, Factor XIII a subunit of blood coagulation, and acid phosphatase reactions were negative . A terminal differentiation of the DD cell line was observed when the expression of CD14, CR3, Fc gamma receptor I, and Fc gamma receptor III was induced . The DD cells induced with 12-O-tetradecanoylphorbol-13-acetate or Escherichia coli lipopolysaccharide can internalize EA via Fc gamma receptor II and complement-coated yeast in the function of the inducers . The phagocytic ability appears to be parallel with the appearance of enzymes which participate in phagocytosis.

Mol Cell Biol, 1992 Feb, 12(2), 455 - 67
Segments of the POU domain influence one another's DNA-binding specificity; Aurora R et al.; The ubiquitously expressed mammalian POU-domain protein Oct-1 specifically recognizes two classes of cis-acting regulatory elements that bear little sequence similarity, the octamer motif ATGCAAAT and the TAATGARAT motif . The related pituitary-specific POU protein Pit-1 also recognizes these two motifs but, unlike Oct-1, binds preferentially to the TAATGARAT motif . Yet in our assay, Pit-1 still binds octamer elements better than does the octamer motif-binding protein Oct-3 . The POU domain is responsible for recognizing these diverse regulatory sequences through multiple DNA contacts that include the two POU subdomains, the POU-specific region, and the POU homeodomain . The DNA-binding properties of 10 chimeric POU domains, in which different POU-domain segments are derived from either Oct-1 or Pit-1, reveal a high degree of structural plasticity; these hybrid proteins all bind DNA well and frequently bind particular sites better than does either of the parental POU domains . In these chimeric POU domains, the POU-specific A and B boxes and the hypervariable POU linker can influence DNA-binding specificity . The surprising result is that the influence a particular segment has on DNA-binding specificity can be greatly affected by the origin of other segments of the POU domain and the sequence of the binding site . Thus, the broad but selective DNA-binding specificity of Oct-1 is conferred both by multiple DNA contacts and by dynamic interactions within the DNA-bound POU domain.

J Exp Med, 1992 Feb 1, 175(2), 461 - 9
Search for hepatitis B virus cell receptors reveals binding sites for interleukin 6 on the virus envelope protein; Neurath AR et al.; The major target organ for hepatitis B virus (HBV) is the liver . However, cells other than hepatocytes, including peripheral blood lymphocytes and monocytes, may become infected with HBV . The cell receptor binding site was assigned to the preS(21-47) segment of the HBV envelope protein . HBV receptors were detected on human liver and hepatoma cells, on B lymphocytes, and, as shown here, on monocytes, and T cell lines, activated by Escherichia coli lipopolysaccharide and concanavalin A, respectively . The cell receptors for HBV have not been characterized until now . The detection of HBV receptors and their "activation antigen" characteristic on distinct cells suggested paths for identification of the receptors with already defined cell surface proteins . This search revealed that interleukin 6 contains recognition sites for the preS(21-47) sequence and mediates HBV-cell interactions . Thus, HBV belongs to a group of viruses utilizing cytokines or cytokine receptors for replication and interference with the host immune system.

J Am Coll Cardiol, 1992 Feb, 19(2), 433 - 40
Evaluation of thrombolytic and systemic effects of the novel recombinant plasminogen activator BM 06.022 compared with alteplase, anistreplase, streptokinase and urokinase in a canine model of coronary artery thrombosis; Martin U et al.; The thrombolytic and systemic effects of BM 06.022 were evaluated and compared with those of alteplase, anistreplase, streptokinase and urokinase in a canine model of coronary artery thrombosis . BM 06.022 consists of the kringle-2 and protease domains of human tissue plasminogen activator (t-PA) and is unglycosylated because of its expression in Escherichia coli cells . Thrombus formation in anesthetized open chest dogs was induced by electrical injury to the intimal surface of the left circumflex coronary artery at a high level site of obstruction . In heparinized dogs, none of six vehicle-treated animals exhibited reperfusion . Reperfusion was achieved in four of six dogs at 18.3 +/- 6 min after intravenous bolus injection of 140 kU/kg (0.24 mg/kg) of BM 06.022, whereas four of six dogs exhibited reperfusion later (p less than 0.05) at 76.5 +/- 16.1 min during infusion of 1.33 mg/kg of alteplase (0.13 mg/kg as initial bolus injection, followed by 0.66 mg/kg over 1 h and 0.53 mg/kg over 2 h) . Significantly later (p less than 0.05) reperfusion than that achieved with BM 06.022 was achieved in five of six dogs at 57.8 +/- 12.1 min after intravenous injection of 0.4 U/kg of anistreplase . Streptokinase (21,000 IU/kg over 60 min) and urokinase (20,000 IU/kg as an intravenous bolus injection, followed by 20,000 IU/kg over 89 min) each induced reperfusion in three of six dogs but at 67 +/- 12 and 84.3 +/- 17.1 min (p less than 0.05 vs . BM 06.022), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1992 Feb, 174(3), 998 - 1006
Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase; Barr GC et al.; The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184 . The cloned DNA complemented a mutation in the osmZ locus of E . coli, which codes for HNS . However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E . coli cya gene, which codes for adenylate cyclase . Acquisition of the cya mutations was independent of RecA . These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene . A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus . These sequences appear to contain at least two genetically active regions.

J Bacteriol, 1992 Feb, 174(3), 953 - 61
Suppression of oxidative envelope damage by pseudoreversion of a superoxide dismutase-deficient mutant of Escherichia coli; Imlay JA et al.; Mutants of Escherichia coli that are devoid of superoxide dismutase (SOD) fail to grow in aerobic minimal medium . This is largely because of the O2- sensitivities of several amino acid biosynthetic pathways, since amino acid supplements can restore growth, albeit at a slow rate . We now report that growth in amino acid-supplemented medium can be further stimulated by the presence of extracellular osmolytes . Osmolytes also partially suppress the amino acid requirements of the SOD mutant . These data suggest that the combination of oxidative injury and turgor pressure permeabilizes the cell envelope and that critical metabolites, including the limiting products of damaged biosynthetic pathways, escape from the cell . External osmolytes may offer protection by countervailing the usual turgor pressure and thus stabilizing the damaged envelope . This model is consistent with the previous observation that deficiency of cell wall components is lethal to SOD mutants . A pseudorevertant that can grow at a moderate rate in normosmotic medium without amino acid supplementation has been obtained (J . A . Imlay and I . Fridovich, Mol . Gen . Genet . 228:410-416, 1991) . Analysis suggests that the suppressor mutation allows the envelope either to resist or to tolerate oxidative lesions . Study of the pseudorevertant may illuminate the molecular basis of this oxidative envelope injury.

J Bacteriol, 1992 Feb, 174(3), 921 - 9
FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions; Nilsson L et al.; In Escherichia coli transcription of the tRNA operon thrU (tufB) and the rRNA operon rrnB is trans-activated by the protein FIS . This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tufB) and the P1 promoter of the rrnB operon . There are indications that this type of regulation is representative for the regulation of more stable RNA operons . In the present investigation we have studied UAS-dependent transcription activation of the thrU (tufB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up . In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines . Concomitantly, a peak of the cellular FIS concentration is observed . Cells in the stationary phase are depleted of FIS . The rather abrupt increase of transcription activation depends on the nutritional quality of the medium . It is not seen in minimal medium . After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured . This peak gets higher as the medium gets more strongly enriched . We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tufB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists . This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon . Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that wild-type cells . Presumably it is controlled by the ribosome feedback regulatory system . cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS . This activation is constant throughout the entire growth cycle and is independent of nutritional factors . The well-known growth rate-dependent control, displayed by exponentially growing cells studied under various nutritional conditions, is governed by two regulatory mechanisms: repression, presumably by ribosome feedback inhibition, and stimulation by trans activation . FIS allows very fast bacterial growth.

J Bacteriol, 1992 Feb, 174(3), 914 - 20
Cloning and nucleotide sequence of a gene (ompS) encoding the major outer membrane protein of Legionella pneumophila; Hoffman PS et al.; The major outer membrane protein of Legionella pneumophila is composed of 28- and 31-kDa subunits cross-linked by interchain disulfide bonds . The oligomer is covalently anchored to the underlying peptidoglycan via the 31-kDa subunit . We have cloned the structural gene ompS encoding both proteins . Oligonucleotide probes synthesized from the codons of the N-terminal amino acid sequence of purified 28- and 31-kDa subunits were used to identify cloned sequences . A 2.9-kb HindIII fragment cloned into pBluescript (clone H151) contained the ompS gene . Nucleotide sequence analysis revealed an open reading frame of 891 bp encoding a polypeptide of 297 amino acids . A leader sequence of 21 amino acids was identified, and the mature protein contained 276 amino acids . The deduced amino acid sequence of OmpS matched the experimentally determined amino acid sequence (32 amino acids), with the exception of two cysteine residues . The deduced amino acid sequence was rich in glycine and aromatic amino acids and contained four cysteine residues, two in the amino terminus and two in the carboxy region . Primer extension analysis (total RNA from L . pneumophila) identified the transcription start at 96 to 98 bp upstream of the translation start, but no Escherichia coli-like promoter sequences were evident . While an mRNA transcript from clone H151 was detected, no cross-reactive protein was detected by immunoblotting with either monoclonal or polyclonal antibody . Attempts to subclone the gene in the absence of the putative promoter region (i.e., under the control of the lac promoter) proved unsuccessful, possibly because of overproduction lethality in E . coli . The ompS DNA sequence was highly conserved among the serogroups of L . pneumophila, and related species also exhibited homology in Southern blot analysis at a moderately high stringency . Evidence is presented to suggest that this gene may be environmentally regulated in L . pneumophila.

J Bacteriol, 1992 Feb, 174(3), 867 - 72
The narJ gene product is required for biogenesis of respiratory nitrate reductase in Escherichia coli; Dubourdieu M et al.; Respiratory nitrate reductase purified from the cell membrane of Escherichia coli is composed of three subunits, alpha, beta, and gamma, which are encoded, respectively, by the narG, narH, and narI genes of the narGHJI operon . The product of the narJ gene was deduced previously to be a highly charged, acidic protein which was not found to be associated with any of the purified preparations of the enzyme and which, in studies with putative narJ mutants, did not appear to be absolutely required for formation of the membrane-bound enzyme . To test this latter hypothesis, the narJ gene was disrupted in a plasmid which contained the complete narGHJI operon, and the operon was expressed in a narG::Tn10 insertion mutant . The chromosomal copy of the narJ gene of a wild-type strain was also replaced by the disrupted narJ gene . In both cases, when nar operon expression was induced, the alpha and beta subunits accumulated in a form which expressed only very low activity with either reduced methyl viologen (MVH) or formate as electron donors, although an alpha-beta complex separated from the gamma subunit is known to catalyze full MVH-linked activity but not the formate-linked activity associated with the membrane-bound complex . The low-activity forms of the alpha and beta subunits also accumulated in the absence of the NarJ protein when the gamma subunit (NarI) was provided from a multicopy plasmid, indicating that NarJ is essential for the formation of the active, membrane-bound complex . When both NarJ and NarI were provided from a plasmid in the narJ mutant, fully active, membrane-bound activity was formed . When NarJ only was provided from a plasmid in the narJ mutant, a cytosolic form of the alpha and beta subunits, which expressed significantly increased levels of the MVH-dependent activity, accumulated, and the alpha subunit appeared to be protected from the proteolytic clipping which occurred in the absence of NarJ . We conclude that NarJ is indispensible for the biogenesis of membrane-bound nitrate reductase and is involved either in the maturation of a soluble, active alpha-beta complex or in facilitating the interaction of the complex with the membrane-bound gamma subunit.

J Bacteriol, 1992 Feb, 174(3), 743 - 8
Role of the heat shock response in stability of mRNA in Escherichia coli K-12; Henry MD et al.; The heat shock response in Escherichia coli involves extensive induction of the heat shock proteins, with the concomitant suppression of the synthesis of the non-heat shock proteins . While the induction of the heat shock proteins has been shown to occur primarily at the transcriptional level, the suppression of non-heat shock proteins is poorly understood . We have investigated the possibility that an increased decay of non-heat shock mRNAs is a means of decreasing the synthesis of non-heat shock proteins during the heat shock response . Heat shock response-defective strains were compared with wild-type controls by several criteria to evaluate both mRNA stability and the induction of enzymes known to be involved in mRNA turnover . Our results indicate that increased mRNA decay is not a mechanism used to regulate the synthesis of non-heat shock proteins.

J Bacteriol, 1992 Feb, 174(3), 702 - 10
Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB; Karow M et al.; Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5 degrees C but not at lower temperatures . In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB . The msbB gene has been mapped to 40.5 min on the E . coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al . (Y . Kohara, K . Akiyama, and K . Isono, Cell 50:495-508, 1987) . Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB . The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature . The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles . Mutational analysis of msbB shows that the gene is not essential for E . coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30 degrees C not seen with either of the single mutants . Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances . The addition of quaternary ammonium compounds rescues the temperature-sensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2+ or Ca2+ . These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function.

J Bacteriol, 1992 Feb, 174(3), 1072 - 5
Enzymatic basis of thiol-stimulated secretion of porphyrins by Escherichia coli; Javor GT et al.; 1-Thioglycerol (TG) stimulates the synthesis of porphyrin in aerobically growing Escherichia coli . Here the levels of delta-aminolevulinate biosynthetic enzymes in untreated and TG-treated E . coli THU and PUC2 (a mutant of THU which overproduces porphyrins in the presence of thiols) cells were determined . TG treatment elevated the activity of glutamyl-tRNA reductase in both strains . The increased activity was not caused by activation of preexisting enzymes by thiols or by oxidizing agents but was dependent on new protein synthesis.

J Bacteriol, 1992 Feb, 174(3), 1060 - 2
Replication of the R6K plasmid during the Escherichia coli cell cycle; Keasling JD et al.; The cell-cycle replication pattern of the R6K plasmid has been investigated by using the membrane-elution technique to produce cells labelled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA . The high-copy plasmid R6K replicates exponentially in a cell-cycle-independent manner . A mini-R6K plasmid deleted for the ori alpha origin of replication also replicates, exponentially in a cell-cycle-independent manner.

Arch Biochem Biophys, 1992 Feb 1, 292(2), 475 - 8
Chemical modification of glucosamine-6-phosphate synthase by diethyl pyrocarbonate: evidence of histidine requirement for enzymatic activity; Badet-Denisot MA et al.; Glucosamine-6-phosphate synthase from Escherichia coli was inactivated by diethylpyrocarbonate at pH 7.3 and 4 degrees C with a second-order rate constant of 1220 M-1 min-1 . The difference spectrum of inactivated vs native enzyme had a maximum absorption at 242 nm, which is characteristic of N-carbethoxyhistidine . No trough at around 280 nm due to O-carbethoxytyrosine was observed and the sulfhydryl content of the enzyme was unchanged . Studies with {14C}diethylpyrocarbonate provided evidence that derivatization of a single histidine residue of the amino-terminal glutamine-binding domain inactivated glucosamine-6P synthase . These results are consistent with the participation of an histidine residue in a catalytic triad, Cys/His/Asp, necessary to generate ammonia from glutamine.

J Virol, 1992 Feb, 66(2), 971 - 82
Identification and expression of rpo19, a vaccinia virus gene encoding a 19-kilodalton DNA-dependent RNA polymerase subunit; Ahn BY et al.; The vaccinia virus DNA-dependent RNA polymerase subunit gene rpo19 was identified, and its expression was examined at RNA and protein levels . Antibody to the multisubunit RNA polymerase purified from virions reacted with a polypeptide with an apparent Mr of 21,000 that was synthesized in reticulocyte lysates programmed with (i) mRNA from infected cells that was isolated by hybridization to DNA subclones of the viral genomic HindIII A fragment and (ii) mRNA made in vitro by transcription of the viral open reading frame A6R . Polyclonal antiserum, raised to a recombinant protein product of the A6R open reading frame which could encode an 18,996-Da protein with an acidic N terminus, reacted with Mr-21,000 and -22,000 polypeptides that cosedimented with purified RNA polymerase . Internal sequencing of the two polypeptides confirmed that both were encoded by A6R, and the gene was named rpo19 to indicate the predicted molecular mass of the polypeptide in kilodaltons . Immunoblotting and metabolic labeling of infected cell proteins indicated that synthesis of the Mr-21,000 polypeptide started early and continued throughout virus infection, whereas the Mr-22,000 form appeared late following DNA replication . RNA analyses suggested that the rpo19 mRNA was expressed from a dual early/late promoter and that the protein-coding region of the mRNA was directly preceded by a short 5' poly(A) leader, apparently initiated within the TAAATG motif at the beginning of the open reading frame.

J Virol, 1992 Feb, 66(2), 1228 - 31
Human immunodeficiency virus type 1 and type 2 protease monomers are functionally interchangeable in the dimeric enzymes; Patterson CE et al.; Human immunodeficiency virus type 1 (HIV-1) and HIV-2 proteases are dimers of identical subunits . We made a construct for the expression of recombinant one-chain HIV-2 protease dimer, which, like the previously described one-chain HIV-1 protease dimer, is fully active . The constructs for the one-chain dimers of HIV-1 and HIV-2 proteases were modified to produce hybrid one-chain dimers consisting of both HIV-1 and HIV-2 protease monomers . Although the monomers share only 47.5% sequence identity, the hybrid one-chain dimers are fully active, suggesting that the folding of both HIV-1 and HIV-2 protease monomers is functionally similar.

J Virol, 1992 Feb, 66(2), 1066 - 73
Hemagglutinin mutations related to antigenic variation in H1 swine influenza viruses; Luoh SM et al.; The hemagglutinin (HA) of a recent swine influenza virus, A/Sw/IN/1726/88 (H1N1), was shown previously to have four antigenic sites, as determined from analysis of monoclonal antibody (MAb)-selected escape mutants . To define the HA mutations related to these antigenic sites, we cloned and sequenced the HA genes amplified by polymerase chain reaction of parent virus and MAb-selected escape mutants . The genetic data indicated the presence of four amino acid changes . After alignment with the three-dimensional structure of H3 HA, three changes were located on the distal tip of the HA, and the fourth was located within the loop on the HA . We then compared our antigenic sites, as defined by the changed amino acids, with the well-defined sites on the H1 HA of A/PR/8/34 . The four amino acid residues corresponded with three antigenic sites on the HA of A/PR/8/34 . This finding, in conjunction with our previous antigenic data, indicated that two of the four antigenic sites were overlapping . In addition, our previous studies indicated that one MAb-selected mutant and a recent, naturally occurring swine isolate reacted similarly with the MAb panel . However, their amino acid changes were different and also distant on the primary sequence but close topographically . This finding indicates that changes outside the antigenic site may also affect the site . A comparison of the HA amino acid sequences of early and recent swine isolates showed striking conservation of genetic sequences as well as of the antigenic sites . Thus, swine influenza viruses evolve more slowly than human viruses, possibly because they are not subjected to the same degree of immune selection.

Infect Immun, 1992 Feb, 60(2), 571 - 7
A vir-repressed gene of Bordetella pertussis is required for virulence; Beattie DT et al.; Coordinate regulation of gene expression in Bordetella pertussis is controlled by the products of the vir locus, BvgA and BvgS . In the presence of modulating signals such as MgSO4 and nicotinic acid, expression of vir-activated genes (vag) is reduced, while expression of vir-repressed genes (vrg) is maximal . We have cloned one of these vir-repressed genes, vrg-6, in Escherichia coli . DNA sequencing has shown that vrg-6 is contained on a single EcoRI restriction endonuclease fragment and is predicted to code for a protein of 105 amino acids with a molecular weight of 11,441 . The predicted protein product appears to have two domains, one consisting of seven hydrophobic proline-rich pentameric repeats and the other consisting of five alkaline trimeric repeats . Southern blot analysis has revealed vrg-6-homologous sequences in the chromosomes of Bordetella bronchiseptica and Bordetella parapertussis, but, unlike Bordetella pertussis, these species do not express vrg-6-homologous RNA when grown under modulating conditions . In order to assess the role of vrg gene products in B . pertussis pathogenesis, two 18323 derivatives which harbor TnphoA insertions in vrg genes were analyzed in a mouse model of respiratory infection . Strain SK6, which carries a vrg-6::TnphoA mutation, failed to induce lymphocytosis and was significantly less able to colonize lungs and trachea than its parent strain 18323 or than SK18, which harbors a TnphoA fusion in the vrg-18 locus . This is the first evidence that a vir-repressed gene may play an important role in the virulence of B . pertussis and the pathogenesis of whooping cough.

Infect Immun, 1992 Feb, 60(2), 485 - 90
An enzymatic mutant of Shiga-like toxin II variant is a vaccine candidate for edema disease of swine; Gordon VM et al.; Edema disease (ED) of weanling pigs is caused by an infection with Escherichia coli that produces Shiga-like toxin II variant (SLT-IIv) . Pathology identical to that caused by ED can be duplicated in pigs that are injected with less than 10 ng of purified SLT-IIv per kg of body weight . Therefore, SLT-IIv was mutated to create an immunoreactive form of the toxin that was significantly reduced in enzymatic activity . Initially, purified SLT-IIv was treated with formaldehyde which abrogated cytotoxic activity . Pigs were vaccinated with the toxoid (100 micrograms) to determine whether a toxoid was a viable vaccine candidate and whether young pigs were capable of mounting an immune response . Although the pigs developed a neutralizing antibody titer (1:128 to 1:512) 28 days postinjection, they also lost weight and developed ED lesions . The deleterious effect of the toxoid appeared to result from residual enzymatic activity or a reversion to a toxic form . An alternative method, site-directed mutagenesis, was employed to consistently reduce the enzymatic activity of SLT-IIv . Glutamate at position 167 of the mature A subunit was replaced by aspartate (E167D), and arginine at position 170 was replaced by lysine (R170K) . These mutations reduced cytotoxic activity 10(4)-fold and 10-fold, respectively, while the enzymatic activities were decreased 400-fold and 5-fold, respectively . The activity of a toxin that contained both mutations (SLT-IIvE167D/R170K) closely resembled that of SLT-IIvE167D . When position 167 was replaced by glutamine (E167Q), the cytotoxic activity decreased 10(6)-fold and the enzymatic activity decreased approximately 1,500-fold . Pigs that were vaccinated with purified, mutant toxin designated SLT-IIvE167Q developed a neutralizing antibody titer of 1:512 21 days postinjection, and their tissues were free of ED lesions . These data suggest that SLT-IIvE167Q may represent an effective vaccine against ED.

Infect Immun, 1992 Feb, 60(2), 455 - 64
Demonstration of a B-lymphocyte mitogen produced by the Lyme disease pathogen, Borrelia burgdorferi; Schoenfeld R et al.; Lyme disease refers to the multisymptomatic illness in humans which results from infection with the tick-borne spirochete Borrelia burgdorferi . The white-footed mouse is the major reservoir for B . burgdorferi and, upon infection, certain inbred mice develop symptoms similar to those reported in human disease . Sonicated preparations of washed spirochetes were found to have potent mitogenic activity when cultured with lymphocytes from naive C57BL/6, C3H/HeJ, or BALB/c mice . The activity of the B . burgdorferi sonicate was approximately fourfold greater than that of a similarly prepared Escherichia coli sonicate . Polymyxin B efficiently inhibited the mitogenic activity of the E . coli sonicate but only slightly inhibited that of the B . burgdorferi sonicate, suggesting that a lipid A-containing lipopolysaccharide was not responsible for the B . burgdorferi activity . Kinetic analysis indicated peak proliferation at 2 to 3 days of culturing, suggesting polyclonal activation . B- and T-lymphocyte depletion experiments indicated that the major cell type responding to the B . burgdorferi mitogen was the B lymphocyte . This mitogen stimulated murine B cells not only to proliferate but also to differentiate into antibody-secreting cells, as demonstrated by the production of immunoglobulin by stimulated splenocytes . Furthermore, the sonicated preparation stimulated the B-cell tumor line CH12.LX to secrete immunoglobulin in the absence of accessory cells . B . burgdorferi also stimulated interleukin-6 production in splenocyte cultures . The observation that B . burgdorferi can stimulate activation of and immunoglobulin production by normal B lymphocytes may directly reflect on the development of arthritis associated with persistent infection by this organism.

Infect Immun, 1992 Feb, 60(2), 416 - 27
Isolation and expression of a gene which encodes a wall-associated proteinase of Coccidioides immitis; Cole GT et al.; A chymotrypsinlike serine proteinase of Coccidioides immitis with an estimated molecular size of 34 kDa has been shown by immunoelectron microscopy to be associated with the walls of the parasitic cells of this human respiratory pathogen . The proteinase has been suggested to play a role in spherule development . We report the isolation of a 1.2-kb cDNA from an expression library of C . immitis constructed in the lambda ZAP II phage vector . The cDNA is suggested to encode the 34-kDa protein . We demonstrate identity between segments of the deduced amino acid sequence of the open reading frame of the 1.2-kb cDNA and three distinct sequences obtained from cyanogen bromide cleavage peptides of the purified proteinase . The occurrence of N-glycosyl linkage sites in the deduced sequence of 309 amino acids of the open reading frame (ORF) correlates with our identification of such linkage sites in the native glycosylated proteinase . A protein encoded by an 800-bp fragment of the 1.2-kb cDNA, which was produced by transformed Escherichia coli XL1-Blue, was recognized by the anti-34-kDa protein antibody in a Western blot (immunoblot) . Northern (RNA) hybridization of total poly(A)-containing RNA of C . immitis with the labeled 1.2-kb cDNA clone revealed a single band of approximately 1.75 kb . Partial homology was demonstrated between the deduced amino acid sequence of the ORF (927 bp) and reported sequences of alpha-chymotrypsin and chymotrypsinogens . Expression of the proteinase gene was examined by Northern dot blot analysis of total RNA from different stages of parasitic cell development in C . immitis . Maximum levels of specific mRNA were detected during early endospore wall differentiation . The 34-kDa proteinase appears to be concentrated in walls of the parasitic cells at stages of active growth . We suggest that the enzyme may participate in wall plasticization and/or intussusception or in cell wall turnover.

Exp Parasitol, 1992 Feb, 74(1), 11 - 9
Localisation of hypoxanthine phosphoribosyl transferase in the malaria parasite Plasmodium falciparum; Shahabuddin M et al.; The enzyme hypoxanthine phosphoribosyl transferase of the human malaria parasite Plasmodium falciparum has been located in parasites and parasite-infected erythrocytes by antibody probing . The probe was a polyclonal rabbit antiserum made against the parasite enzyme made in Escherichia coli . The enzyme is associated with membrane-bound compartments in merozoites and asexual blood parasites . In particular, indirect immunofluorescence studies reveal the enzyme localized in vesicle-like structures within the cytoplasm of the infected erythrocyte . This is the first time a P . falciparum protein of defined metabolic function has been tracked to a site outside the parasite cytosol . Studies on the targeting of the enzyme using a cell-free system suggests that the protein reaches its destination via a route different from the normal secretory pathway.

Immunol Cell Biol, 1992 Feb, 70 ( Pt 1), 1 - 7
Beneficial effects of endotoxin treatment on metabolism in tumour-bearing rats; Rofe AM et al.; The effects of endotoxin treatment on host metabolism in tumour-bearing rats were investigated . Metabolism in control rats (non-tumour-bearing) was slightly altered by endotoxin treatment, whereas in tumour-bearing rats a number of biochemical parameters that were initially perturbed by the presence of the tumour had returned to normal at 48 h post-treatment . The beneficial effects included increased blood glucose and insulin concentrations, and decreased ketone body, triglyceride and lactate concentrations . Potentially non-beneficial effects of endotoxin observed in both tumour-bearing and control rats included decreased plasma cholesterol, and increased plasma phosphate, potassium and alkaline phosphatase levels . Endotoxin caused haemorrhaging in the encapsulated tumour, and this was associated with histological evidence of endothelial damage, red cell infiltration into surrounding tumour tissue and a marked decrease in cell viability . The in vivo uptake of glucose by the tumour, measured by 2-deoxy {U-14C}glucose uptake, was decreased by 96% following endotoxin treatment, and this was associated with a two-fold increase in glucose uptake by muscle . It is concluded that endotoxin treatment has major effects on cell viability and the integrity of vasculature in the tumour, which limits glucose uptake by the tumour and thereby decreases the energy and substrate requirements of the tumour, thus benefiting the host . It is suggested that tumour cytotoxicity and intra-tumour haemorrhage are the result of endotoxin stimulating cytokine release from macrophages that are already activated by the presence of the tumour.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Calcium, 1992 Feb, 13(2), 123 - 30
The effect of endotoxemia on concanavalin A induced alterations in cytoplasmic free calcium in rat spleen cells as determined with Fluo-3; Hagar AF et al.; The effects of acute (3 h) and chronic (30 h) in vivo infusions of Escherichia coli endotoxin on the Ca2+ homeostasis of rat spleen cells was investigated . Conditions were established for obtaining reliable estimates of {Ca2+}i in these cells using the newly-developed Ca2+ indicator Fluo-3 . The resting {Ca2+}i of splenocytes and T lymphocyte-enriched preparations were 119 +/- 35 and 102 +/- 31 nM, respectively . Treatment of the cells with concanavalin A (Con A) resulted in a rapid increase in {Ca2+}i . The magnitude of the increase was positively correlated with the concentration of Con A, whereas the time required to reach the maximum {Ca2+}i was inversely related to the amount of Con A . The peak {Ca2+}i was attained more rapidly in splenocytes (i.e . less than or equal to 30 s) than in the T cell-enriched fraction (i.e . 1.5-2.0 min) . Both the resting {Ca2+}i and the Con A-induced increase in {Ca2+}i were similar to values previously reported for other lymphocyte cell types using different Ca2+ indicators, thereby supporting the values obtained with Fluo-3 . Infusions of saline or endotoxin prior to the isolation of the cells did not result in significant alterations of either resting {Ca2+}i or the cells' response to Con A . Since chronic infusions of endotoxin have previously been shown to cause a reduction in blastogenic responsiveness of splenocytes to Con A, these data suggest that the endotoxin-induced lesion occurs distal to the mobilization of intracellular Ca2+.

Jpn J Genet, 1992 Feb, 67(1), 17 - 27
Genetic mapping and biochemical characterization of suppressor mutations sukA and sukB for a dnaK7(Ts) mutation of Escherichia coli K-12; Itikawa H et al.; Temperature-resistant pseudorevertants were isolated from a dnaK7(Ts) mutant of Escherichia coli K-12 . Two of these pseudorevertants were shown to carry suppressor mutations, sukA and sukB, respectively . Genetic mapping by conjugation and P1-transduction revealed that these suppressor mutations were located at two distinct sites between 76 and 77 min close to the suhA and rpoH genes . Labeled cellular proteins were extracted from suppressor mutants grown at various temperatures and subjected to SDS-gel electrophoresis . Autoradiograms of the gels indicated that these suppressor mutations each resulted in increased synthesis of the heat shock protein Lon (an ATP-dependent protease, La) at both permissive and nonpermissive temperatures.

J Cell Sci, 1992 Feb, 101 ( Pt 2), 463 - 74
Cloning and expression of Drosophila HP1 homologs from a mealybug, Planococcus citri; Epstein H et al.; The mealybug chromosome cycle is one of the most dramatic examples of genomic imprinting known . In embryos that are to become male the entire paternal chromosome set becomes heterochromatic and inactive at the blastoderm stage, while the maternal set remains active and euchromatic . HP1 is a protein from Drosophila melanogaster, which binds preferentially to heterochromatin on polytene chromosomes and is likely to be a modifier of position effect variegation . This paper describes the isolation and sequencing of two cDNA clones encoding HP1 homologs from the mealybug, Planococcus citri . The protein product of the cDNA clone that was closer to HP1 in sequence was expressed as a fusion protein in Escherichia coli, and polyclonal rat antibodies were raised against it . Immunohistochemistry to mealybug squash preparations showed that this protein was a male-specific nuclear protein, but that it was not specifically associated with the heterochromatic set of chromosomes.

Biotechniques, 1992 Feb, 12(2), 264 - 9
DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase; Cano RJ et al.; A fluorometric procedure for the detection of DNA-DNA hybrids is described . The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS . This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm . DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells . Streptavidin-alkaline phosphatase conjugates were added to react with bound probe . Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed . The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated . A slope four times greater than that of background was considered positive . One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay . Similar results were obtained with whole cells . Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically . Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.

Biochem Int, 1992 Feb, 26(1), 1 - 5
A two-base-pair substitution in T7 promoter by SP6 promoter-specific base pairs alone abolishes T7 promoter activity but reveals SP6 promoter activity; Lee SS et al.; The phage T7 and SP6 RNA polymerase-promoter systems are very similar in many characteristics, but maintains stringent specificity for each . In order to identify the base pair element that distinguishes between T7 and SP6 promoters, the base pairs at -12, -10, -9, and -8 of the T7 promoter consensus sequence were changed singly and multiply to the SP6 promoter-specific base pairs, and assayed for T7 and SP6 promoter activities . The results indicate that the primary discrimination element is the base pairs at -8 and -9 . The two-base-pair substitution alone in T7 promoter by SP6-specific base pairs is sufficient to make the T7 variant be a SP6 promoter, abolishing T7 promoter activity.

Proteins, 1992 Feb, 12(2), 101 - 4
Redesigning the DNA-binding specificity of a zinc finger protein: a data base-guided approach; Desjarlais JR et al.; A peptide corresponding to the three zinc finger domains of the human transcription factor Sp1 has been expressed and found to bind a consensus Sp1 binding site with the sequence 5'-GGGGCGGGG-3' . Examination of the amino acid distributions within a large zinc finger sequence data base and chemical arguments suggested that a particular Arg to Gln sequence change might convert binding specificity to 5'-GGGGCAGGG-3' . Experimental tests of this hypothesis revealed that such a change could be induced only when two other sequence changes, deduced from examination of sequence correlations, were made as well . These results provide the most direct information to date about how zinc finger proteins might recognize adenine-containing binding sites and bear on the existence and nature of any code between zinc finger protein and binding site sequences.

Mil Med, 1992 Feb, 157(2), 55 - 8
Norfloxacin compared to trimethoprim/sulfamethoxazole for the treatment of travelers' diarrhea among U.S . military personnel deployed to South America and West Africa; Thornton SA et al.; A randomized treatment trial of travelers' diarrhea was carried out among U.S . military personnel participating in routine exercises in several port cities in South America and West Africa . A 5-day, twice daily course of either norfloxacin (400 mg) or trimethoprim/sulfamethoxazole (TMP/SMX, 160/800 mg) was given to 142 volunteers . At the end of 5 days of treatment, diarrhea had resolved in 100% of 73 patients receiving norfloxacin and 97.1% (67/69) receiving TMP/SMX . A probable bacterial pathogen was determined in 44% of 142 subjects: 49% of the norfloxacin group and 39% of the TMP/SMX group . The most common pathogens detected were enterotoxigenic Escherichia coli in 20% of cases and rotavirus in 15% . Resistance to TMP/SMX was present in 20 (27%) bacterial isolates, while no resistance to norfloxacin was found . Eight of 10 patients in the TMP/SMX treatment group who had TMP/SMX-resistant bacterial enteropathogens improved clinically . Both norfloxacin and TMP/SMX were clinically effective in the treatment of travelers' diarrhea in this military population.

Actas Urol Esp, 1992 Feb, 16(2), 144 - 7
{Prostatic abscess--new diagnostic and therapeutic guidelines}; Napal Lecumberri S et al.; Five prostatic abscesses treated in our Unit over the last three years (1988, 89, 90) are presented . Both the mode of presentation and symptoms as well as the course of treatment followed (three percutaneous drainage through the perineum, lead by ultrasound scanning, one endoscopic abscess incision with Collins' knife and one resolved with antibiotics subsequently needing TUR of the prostatic adenoma) are reviewed . The change of attitude in the treatment of prostatic abscesses (Ultrasound-lead percutaneous drainage) and the major usefulness of ultrasound scanning for diagnosis, treatment and subsequent follow-up are emphasized.

Cell Struct Funct, 1992 Feb, 17(1), 77 - 86
Intracellular localization and partial amino acid sequence of a stress-inducible 40-kDa protein in HeLa cells; Hattori H et al.; We earlier discovered a novel 40-kDa protein (hsp40) induced by heat shock and other stresses in mammalian and avian cells . In this report, we purified the hsp40 in HeLa cells, using modified two-dimensional gel electrophoresis, and determined the amino terminal amino acid sequence of this protein . The hsp40 is homologous to DnaJ, an Escherichia coli heat-shock protein, as well as to DnaJ-homologous proteins in yeast such as SCJ1, Sec63/Np11, YDJ1 and SIS1 . Indirect immunofluorescence staining using an anti-hsp40 polyclonal antibody demonstrated that hsp40 was localized faintly throughout the cell in non-heat-shocked cells and was accumulated in nuclei and nucleoli in heat-shocked cells . The intracellular localization of hsp40 was very similar to that of hsp70, suggesting that these two hsps colocalize in heat-shocked HeLa cells.

Microb Pathog, 1992 Feb, 12(2), 159 - 64
Quantitative assessment of the ability of Escherichia coli to invade cultured animal cells; Robins-Browne RM et al.; Assays to quantify bacterial invasion of epithelial cells generally fail to take account of the ability of the bacteria to adhere to the cells prior to invasion . We have developed a modified invasion assay to allow for this factor . We then used the assay to investigate diarrhoeagenic strains of Escherichia coli with differing ability to adhere to and invade HEp-2 epithelial cells . The results showed that enteroinvasive strains of E . coli were the most invasive variety, followed in order by enteropathogenic E . coli and enterotoxigenic E . coli . These findings correspond to what is known of the ability of the bacteria to invade the intestinal tract in vivo . The results also indicated that adhesins of diarrhoeagenic E . coli play no direct role in invasion, although they may facilitate invasion indirectly by promoting initial contact between bacteria and animal cells.

Microb Pathog, 1992 Feb, 12(2), 153 - 7
In vitro adherence property of cytolethal distending toxin (CLDT) producing EPEC strains and effect of the toxin on rabbit intestine; Bouzari S et al.; Sixteen cytolethal distending toxin-producing enteropathogenic Escherichia coli (CLDT+ EPEC) strains of six different serogroups were included in this study . The strains showed varying adherence patterns on HEp-2 cells, i.e . six strains showed localized adherence (LA), five strains exhibited diffuse adherence (DA) and five strains were non-adherent . Histological study of rabbit ileal segments showed that live cultures of CLDT+ EPEC did not cause lesions characteristic of attachment and effacement (AE) and the toxin effect resembled that of classical LT or CT.

Circ Shock, 1992 Feb, 36(2), 113 - 9
Xanthine derivative HWA 138 attenuates hemodynamic and oxygen uptake dysfunction secondary to severe endotoxin shock in sheep; Masouye P et al.; Continuous E . coli endotoxin infusion in five awake sheep produced hypodynamic shock characterized by decreased cardiac output, increased pulmonary vascular resistance (PVR), leukopenia, high plasma levels of lactate, thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha), and a pathological oxygen delivery-consumption relationship, i.e., a dependence of oxygen consumption on oxygen delivery, leading to death within 8-9 hr . Pretreatment with the xanthine derivative HWA 138 in six sheep prevented the endotoxin-induced fall in cardiac output and oxygen delivery, attenuated the increase in PVR and plasma 6-k-PGF1 alpha, and prolonged survival to 13-19 hr in five of the six sheep . The xanthine derivative had no effect on endotoxin-induced leukopenia, nor on the increase of plasma lactate and TXB2 levels . Although in the HWA 138 treated animals oxygen consumption increased significantly (by 59 +/- 17% at 8 hr), this increase was only transient, and at a level that was most probably insufficient to meet the increased metabolic requirements of this model . We conclude that pretreatment with HWA 138 attenuates endotoxin-induced hemodynamic and oxygen uptake disturbances and prolongs survival in this animal model of severe endotoxin shock.

Biochimie, 1992 Feb, 74(2), 195 - 205
Mammalian prolyl-tRNA synthetase corresponds to the approximately 150 kDa subunit of the high-M(r) aminoacyl-tRNA synthetase complex; Kerjan P et al.; The high-M(r) aminoacyl-tRNA synthetase complex previously purified from sheep liver differed from those isolated from several other mammalian sources by the absence of prolyl-tRNA synthetase activity and the presence of glutamyl tRNA synthetase as a polypeptide of 85 kDa instead of 150 kDa . Using a milder extraction procedure that minimizes proteolysis, we now report the isolation of a sheep liver complex that contains both prolyl-tRNA synthetase activity and the 150-kDa polypeptide . The correspondence between prolyl-tRNA synthetase and the 150-kDa polypeptide, inferred from the results of several approaches reported in this study, was further demonstrated by showing that antibodies to a free form of sheep liver prolyl-tRNA synthetase generated by endogenous proteolysis, specifically reacted with the 150-kDa components of the complexes from sheep and rabbit, but failed to react with the previously purified complex from sheep that contained neither prolyl-tRNA synthetases activity nor the 150-kDa component . Moreover, we show that the 150-kDa polypeptide is also recognized by antibodies to the 85-kDa polypeptide previously assigned to glutamyl-tRNA synthetase . The possibility that the largest subunit of the mammalian high-M(r) complexes may be a bifunctional protein encoding both glutamyl- and prolyl-tRNA synthetase activities is considered and discussed in light of the recently published sequence of the corresponding polypeptide from HeLa cells . In accordance with this prediction, we show that the amino acid sequence of the carboxyl-terminal moiety of this bifunctional polypeptide shows significant similarity to the sequence of prolyl-tRNA synthetase from Escherichia coli.

Biochimie, 1992 Feb, 74(2), 131 - 6
Aphidicolin inhibits DNA polymerizing activity but not nucleolytic activity of Escherichia coli DNA polymerase II; Ishino Y et al.; We have purified the DNA polymerase II of Escherichia coli from the recombinant strain carrying the plasmid which encodes the polB gene . We confirmed that the purified protein, of molecular weight 90,000, possesses a 3'----5' exonuclease activity in addition to DNA polymerizing activity in a single polypeptide . Its DNA polymerizing activity was sensitive to the drug aphidicoline, which is a specific and direct inhibitor of the alpha-like DNA polymerases including eukaryotic replicative DNA polymerases . Aphidicolin had no detectable effect on the 3'----5' exonuclease activity . The inhibition by aphidicolin on the polymerizing activity of polymerase II was competitive with respect to dNTP and uncompetitive with respect to template DNA . This mode of action is the same as that on eukaryotic DNA polymerase alpha . The apparent Ki value calculated from Lineweaver-Burk plots was 55.6 microM.

Sci China B, 1992 Feb, 35(2), 169 - 75
Formation study of toroidal condensation of DNA; Li AZ et al.; A detailed study of the formation of toroidal condensation produced from the 2700 base pair fragments of plasmid PUC13 DNA, induced by metal ions, is introduced . We have extracted several typical intermediate structures based on investigation by electron microscopy, and compared their size distributions . The observations suggest that the formation process of DNA condensation is the process of folding and arranging DNA chains and that of disorder-order transition . The result also indicates that the condensed particles are polymeric, but not randomly aggregative, further proving the toroidal structures are formed in certain regularities.

FEMS Microbiol Lett, 1992 Feb 1, 70(1), 9 - 13
Cloning of a DNA fragment involved in pigment production in Streptomyces avermitilis; Tibbetts MW et al.; Streptomyces avermitilis has the ability to synthesize a diffusible, brown, melanin-like pigment, a common property among many Streptomyces species . A region of the S . avermitilis chromosome involved in the production of this pigment was cloned in Escherichia coli . Production of the brown pigment was attained in E . coli, and is optimal when medium is supplemented with copper ions, tyrosine and IPTG . The cloned S . avermitilis pigment-producing DNA fragment is under the control of the lac promoter carried in the E . coli vector . The gene involved in pigment production could be used as a tool to analyse gene expression in S . avermitilis, and as an alternative cloning marker in Streptomyces-Escherichia coli vectors.

Hepatogastroenterology, 1992 Feb, 39(1), 43 - 6
Occult gallbladder perforation: an unusual complication of gallstone lithotripsy; Janowitz P et al.; Three days following extracorporeal shock-wave lithotripsy of a solitary, calcified gallstone, a 69-year-old white male patient was re-admitted with E . coli sepsis and fever of up to 39.4 degrees C . Ultrasound and CT both revealed a smooth-rimmed hypodense paravasate in the middle portion of the left liver lobe adjacent to the gallbladder, with a density identical to gallbladder fluid . The evidence for perforation was based on CT scanning, and a diagnosis of occult gallbladder perforation was made . Conservative treatment was performed successfully . Following elective cholecystectomy two months thereafter, gallbladder histology showed signs of chronic cholecystitis and E . coli was isolated in bile cultures . The paravasate had granulated and finally cicatrized . By combining ESWL and chemical dissolution, treatment of multiple, calcified and pigment gallstones is possible and this approach has become an attractive alternative therapy modality for a selected group of gallstone patients . Further assessments of the efficacy and safety of this technique are necessary . Conservative treatment of occult gallbladder perforation is possible and should be performed in high-risk patients.

Curr Genet, 1992 Feb, 21(2), 121 - 4
Transformation of the mycoparasite Parasitella simplex to neomycin resistance; Burmester A; The facultatively parasitic zygomycete Parasitella simplex was transformed to neomycin resistance by a vector, which had been developed primarily for transformation of its host Absidia glauca . This plasmid, pAmNEF21, contained the bacterial resistance gene for neomycin (NPTII) under the control of the promoter region from the gene for elongation factor 1 (tef) isolated from A . glauca . Both flanking regions of the marker gene contain parts of the structural tef gene . DNA isolated from two Parasitella transformants was re-transformed in E . coli and the resulting plasmids, pAt21 and pAt35, were analyzed . The restriction map and Southern blot analysis show that both plasmids are rearranged . They had lost the structural tef information and were found to contain new DNA fragments, which were identical in both cases . Southern blot analysis of the transformants indicates that the rearranged plasmids are present in the fungal transformants and that the changes are not the result of re-transformation in E . coli . Plasmids were only recovered after growth under selective conditions . Southern blot analysis and re-transformation with undigested transformant DNA shows that the plasmids are replicated autonomously.

No To Shinkei, 1992 Feb, 44(2), 137 - 42
{Brain injury induced by continuous infusion of endotoxin (ET) in rat--relationship between ET infusion hours and development of cerebral and visceral lesions}; Tamada F et al.; Experimental generalized Shwartzman reaction (GSR) in animals can be induced by systemic injection of bacterial endotoxin lipopolysaccharide (ET) and presents with thrombotic occlusions of small blood vessels in different organs analogous to disseminated intravascular coagulation (DIC) in man . It is known that DIC involvement of the central nervous system (CNS) in man presents with grave prognosis, but there is a paucity of information concerning CNS involvement in animals with ET induced GSR . In order to better understand the pathogenesis of CNS involvement of DIC in man, and to search for better prophylactic and therapeutic measures against DIC, animal model of DIC was induced by continuous intravenous infusion of E coli ET . A total of 56 male Donryu rats were divided into 6 groups and infused with physiologic saline (controls) and ET lipopolysaccharide at 88.5 micrograms/hour . The rats were killed at 6 different intervals from 24 to 160 hours and examined postmortem . Intracerebral hemorrhagic lesions were seen in 2 of 7 rats (29%) as early as 24 hours of ET-infusion and increased to 77% to 87% of rats receiving continuous ET infusion up to 160 hours . Formation of fibrin thrombi was uncommon in intracerebral blood vessels, but it was frequently observed in choroid plexus capillaries . Fibrin thrombi in other viscera (heart, lungs, liver, kidneys, spleen) were common but decreased toward the end of 160-hour infusion period . Results of this study showed that a continuous intravenous infusion of large dose ET lipopolysaccharide in rats produces intracerebral hemorrhagic lesions analogous to DIC changes of brain seen in man.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver, 1992 Feb, 12(1), 17 - 21
Measurements of hepatic malondialdehyde and conjugated diene concentrations in 24-hour endotoxemic rats; Enochsson LB et al.; The aim of our study was to analyze if intraperitoneally administered E-coli endotoxin (1-7 mg.kg-1) causes an increase in liver malondialdehyde (MDA) and conjugated dienes, 24 h postinjection . Blood glucose, white blood cell counts, liver edema and mortality were also measured . The results of our study show that there were no signs of increased MDA or diene production in the liver . To our surprise, blood leukocyte counts of the experimental groups were not statistically different from controls . In the endotoxemic animals there was a slight liver edema which, however, did not influence the measurements of the lipid peroxidation metabolites . Blood glucose levels were decreased in the endotoxemic animals . There was no mortality in our study, perhaps due to the fact that the rats were fed and not starved . We conclude that, in our study, a bolus dose of endotoxin i.p . does not cause any signs of increased liver lipid peroxidation 24 h postinjection . The liver seems, however, to be affected with slight edema and low blood glucose levels.

Yeast, 1992 Feb, 8(2), 79 - 82
A rapid method for localized mutagenesis of yeast genes; Muhlrad D et al.; We have developed a simple procedure for the localized mutagenesis of yeast genes . In this technique the region of interest is first amplified under mutagenic polymerase chain reaction (PCR) conditions . Cotransformation of the PCR product with a gapped plasmid containing homology to both ends of the PCR product allows in vivo recombination to repair the gap with the mutagenized DNA . This procedure is efficient, allows targeting of specific regions for mutagenesis, and requires no subcloning steps in Escherichia coli.

Mol Microbiol, 1992 Feb, 6(4), 503 - 9
Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22 kDa protein (pC) in Escherichia coli; Fuchs R et al.; We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis . The pC protein was purified from lysates of B . burgdorferi strain PKo . After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas-phase sequenator . Thus partial amino acid sequences were obtained . The deduced oligonucleotides were used as hybridization probes . After Southern blotting a reactive band in the 3 kb range of PstI-digested genomic DNA was detected . The insertion of these fragments into pUC vectors finally resulted in pc-positive Escherichia coli clones . The gene (encoding a protein with 212 amino acids) was expressed in E . coli with varying deletions at the 5' end . A sequence comparison with other outer membrane proteins of B . burgdorferi indicates a processing of pC that is similar to that of lipoproteins.

J Dairy Sci, 1992 Feb, 75(2), 415 - 22
Efficacy of an Escherichia coli J5 mastitis vaccine in an experimental challenge trial; Hogan JS et al.; An Escherichia coli (O111:B4) J5 bacterin was tested for efficacy in reducing IMI and severity of clinical coliform mastitis in an experimental challenge trial . Ten cows were immunized at drying off, 30 d after drying off, and at calving . Ten control cows were not immunized . Right front quarters of all cows were infused with a heterologous strain of E . coli approximately 30 d after calving . Vaccinated cows had lower bacterial counts in milk and lower rectal temperatures than unvaccinated controls following intramammary challenge . Milk production and DMI were more depressed in controls than in vaccinated cows . Milk SCC did not differ between experimental groups . Mean serum IgG titer to whole cell E . coli J5 was significantly greater in vaccinated than in unvaccinated cows at 30 d after drying off, day of challenge, and 7 d postchallenge . Milk IgG titer to E . coli J5 was higher at challenge in vaccinated than in control cows . Vaccination with the E . coli J5 bacterin did not prevent IMI but did reduce severity of clinical signs following intramammary experimental challenge with a heterologous E . coli strain.

Cell Mol Biol, 1992 Feb, 38(1), 1 - 10
Autophosphorylation of 70 kDa heat shock proteins; Leustek T et al.; Several members of the 70 kDa heat shock protein group are known to be phosphorylated in vivo and have recently been found to undergo a Ca(2+)-stimulated autophosphorylation . The characteristics of the autophosphorylation reaction with Escherichia coli DnaK the mitochondrial and chloroplast homologs, and the endoplasmic reticulum Bip/Grp78 are discussed . Some common features are a requirement for Ca2+, inhibition by Mg2+ and phosphorylation solely on a threonine residue . Although the role of autophosphorylation of these proteins is not clear, it is known that the level of phosphorylation of some Hsp70 proteins in vivo is responsive to stress and other cellular conditions.

Plant Mol Biol, 1992 Feb, 18(4), 653 - 62
A complete cDNA for adenine phosphoribosyltransferase from Arabidopsis thaliana; Moffatt BA et al.; An intact cDNA from Arabidopsis thaliana for adenine phosphoribosyltransferase (APRT) was isolated and sequenced . The cDNA is 729 nucleotides in length and predicts a protein of Mr 27,140 . The deduced amino acid sequence has been compared with those of other APRTs and shown to be most similar to the Escherichia coli protein . Construction of a molecular tree of the known APRT amino acid sequences indicates the A . thaliana and E . coli APRT sequences form one cluster and the currently available vertebrate and invertebrate sequences form a separate grouping . Since it is possible to select either for or against the expression of APRT, the isolation of this APRT cDNA clone will allow these selection schemes to be used in plant genetic experiments.

Alcohol Clin Exp Res, 1992 Feb, 16(1), 64 - 7
Acute ethanol intoxication prevents lipopolysaccharide-induced down regulation of protein kinase C in rat Kupffer cells; D'Souza NB et al.; Protein kinase (PK) C has been implicated in a number of cellular events, many of which are also known to be affected by ethanol (ETOH) . ETOH intoxication is also known to impair immune function, thereby increasing the host's susceptibility to infection . The purpose of this study was to assess the effect of acute ETOH intoxication on PKC activity and its intracellular distribution in nonparenchymal liver cells following an E . coli lipopolysaccharide (LPS) challenge . The liver was chosen for the study because it is the primary site both for metabolism of ETOH and detoxification of gut derived bacterial products . Catheterized conscious rats were administered saline or ETOH (175 mg/100 g body weight as a bolus followed by a continuous, 7 hr infusion of 28 mg/100 body weight/hr) . LPS was injected intravenously (100 micrograms/100 g body weight) 3 hr before the end of the saline or ETOH infusion . Kupffer and endothelial cells were isolated by collagenase-pronase digestion followed by centrifugal elutriation . PKC was assayed after extraction with digitonin containing buffer and partial purification on DE-52 cellulose minicolumns . LPS decreased PKC activity by 69% from control values . Although ETOH infusion alone did not affect PKC activity in Kupffer cells, it completely abrogated the LPS effect . A similar trend was observed for the endothelial cells . No significant differences were observed between groups with respect to the intracellular distribution of PKC . The down-regulation of PKC by LPS may represent a mechanism of functional adaptation of the immunocompetent cells to one of the cytokines, i.e., TNF, whose receptors are down regulated by activation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Microbiol, 1992 Feb, 30(2-3), 179 - 90
Characterization of Escherichia coli strains isolated from pigs with edema disease; Wasteson Y et al.; Escherichia coli strains belonging to serogroups O 138 and O 139 isolated from pigs with edema disease, were characterized with respect to the presence of genes encoding Shiga-like toxin I, Shiga-like toxin II and Shiga-like toxin IIv (SLT I, SLT II and SLT IIv) . Genes coding for the heat-stable and heat-labile enterotoxins (ST I and LT I) were also detected . Plasmid profiling, restriction enzyme digestion of total DNA, and ribotyping were performed for further characterization of the strains . The oligonucleotide probes applied in this study appeared to be useful tools for detecting genes coding cytotoxins and enterotoxins . DNA from 12 of 16 strains hybridized with two SLT II probes, and DNA from two SLT IIv encoding strains also hybridized with the ST I probe . DNA from one SLT IIv negative strain hybridized with the LT I probe . The results from plasmid profiling, restriction enzyme digestion, and ribotyping were compared with serogrouping in attempts to distinguish between the different E . coli edema disease isolates . Fourteen different plasmid profiles were identified, and as restriction patterns barely did, and ribotyping patterns did not, reveal any information useful for differentiation of the strains beyond serogroup level, plasmid profiling seemed to be the most suitable method for discrimination between the edema disease strains investigated here.

Clin Exp Pharmacol Physiol, 1992 Feb, 19(2), 113 - 8
Plasma phospholipase A2 activity in clinical acute myocardial infarction; Leong LL et al.; 1 . Phospholipase A2 (PLA2) cleaves phospholipids to produce a lyso-phospholipid and free fatty acid and, in view of the biological activity of the products, PLA2 may play a role in many disease states . Lyso-phospholipids and free arachidonic acid increase in ischaemic myocardium, indicating that ischaemia activates the enzyme . 2 . Plasma PLA2 activity was measured in patients with acute myocardial infarction, based on the release of labelled arachidonic acid from Escherichia coli cell membrane . Fourteen males (peak serum creatine phosphokinase (CK) above twice upper normal) were studied on day 1 (within 6 h of chest pain onset), days 2-4, and days 6-9 . Normal age matched males (n = 13) were also studied . 3 . Plasma PLA2 in patients with uncomplicated myocardial infarction (n = 12) was, initially, 1.14 +/- 0.10 (s.e.m.) nmol/min per mL plasma, similar to that in the normal group (1.52 +/- 0.14) . On days 2-4, PLA2 activity increased to 1.94 +/- 0.18 (P less than 0.001) and this activity was correlated with the earlier peak CK level (P less than 0.02) . On days 6-9, PLA2 activity was 1.49 +/- 0.13 while in two patients who developed complications and underwent open-heart surgery between the last two measurements, there were further increases to 4.22 and 4.04 nmol/min per mL . 4 . The increase in plasma PLA2 in uncomplicated myocardial infarction is likely to be due to release from the damaged myocardium; whether it contributes to pathophysiology is uncertain.

Mol Microbiol, 1992 Feb, 6(3), 371 - 7
Efficient repression by a heterodimeric repressor in Escherichia coli; Webster C et al.; Previous studies with purified variants of the 434 repressor having different operator-binding specificities have demonstrated the interactions of a heterodimeric repressor with a hybrid operator site . The present study investigates the interactions between the 434 repressor and its operator site . The optimum 434 operator half-site is used with a P22 operator half-site to create a hybrid 434/P22 operator . We show that this hybrid operator can be efficiently bound by a heterodimeric repressor, consisting of one wild-type 434 repressor monomer and one 434 repressor monomer with the binding specificity of the P22 repressor, to bring about repression in Escherichia coli.

Mol Microbiol, 1992 Feb, 6(3), 283 - 91
D-E-A-D protein family of putative RNA helicases; Schmid SR et al.; RNA metabolism plays a central role in cell growth . It is essential to regulate RNA synthesis, processing, stability and degradation . Conformational changes in RNA are key elements in regulating cellular processes . Recently, an increasing number of putative RNA helicases from different organisms ranging from Escherichia coli to humans and viruses have been identified . They are involved in diverse cellular functions such as RNA splicing, ribosome assembly, initiation of translation, spermatogenesis, embryogenesis, and cell growth and division . Based on sequence homologies these proteins were grouped in a family, the D-E-A-D box protein family (D-E-A-D = Asp-Glu-Ala-Asp) . Some of the better characterized members have been shown to possess ATP-binding and hydrolysing activities as well as ATP-dependent RNA helicase activities . Most of the genes encoding such proteins have been isolated from yeast, on which we will focus in this review . From sequence data, three of the members form a subfamily, the D-E-A-H subfamily.

Oncogene, 1992 Feb, 7(2), 289 - 93
Molecular cloning of the human cDNA for a stimulatory GDP/GTP exchange protein for c-Ki-ras p21 and smg p21; Kikuchi A et al.; We have previously purified smg GDP dissociation stimulator (GDS) from bovine brain and isolated its cDNA from a bovine brain cDNA library . smg GDS stimulates the GDP/GTP exchange reaction of a group of small GTP-binding proteins (G proteins), including at least c-Ki-ras p21, smg p21A, smg p21B, rhoA p21 and rhoB p21, by stimulating the dissociation of GDP from and the subsequent binding of GTP to each small G protein . In this study, we have isolated and sequenced the cDNA of smg GDS from a human brain cDNA library using the cloned bovine smg GDS cDNA . The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,122 . Human smg GDS shares 93% nucleotide and 96% amino acid sequence homologies with bovine smg GDS . The isolated cDNA is expressed in Escherichia coli, and the encoded protein shows the physical and functional properties similar to those of bovine smg GDS.

Gene, 1992 Feb 1, 111(1), 99 - 104
Single-step purification of bacterially expressed polypeptides containing an oligo-histidine domain; Van Dyke MW et al.; Plasmid expression vectors have been constructed that direct the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at either the N or C terminus . This oligo-histidine domain allows the single-step purification of the fusion proteins, under nondenaturing conditions, by immobilized metal affinity chromatography on Ni2+ bound to iminodiacetic acid-agarose . Several eukaryotic transcription factors (e.g., the upstream stimulatory factor for the adenovirus major late promoter) have been successfully purified, in an active state, by this method.

Gene, 1992 Feb 1, 111(1), 61 - 8
Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector; MacNeil DJ et al.; An integration vector for gene analysis in Streptomyces has been constructed . This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome . To overcome methylation-specific restriction barriers, an E . coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids . The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA . Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S . avermitilis DNA, were used in complementation analyses of seven S . avermitilis mutants defective in glycosylation of avermectin (Av) . Three complementation groups, located in a 7-kb region, were identified . Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer . Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.

Gene, 1992 Feb 1, 111(1), 145 - 6
T41 mutation in lac repressor is Tyr282----Asp; Chen J et al.; A monomeric mutant of the lac repressor protein, designated T41, produced originally by the in vivo MutT mutagenesis exhibited behavior similar to mutants identified as Tyr282----Ser or Tyr282----Gln {Schmitz et al., J . Biol . Chem . 251 (1976) 3359-3366} . The T41 gene, encoded within a phage lambda prophage in the Escherichia coli genome, was amplified by polymerase chain reaction and sequenced . The only mutation found in the nucleotide sequence corresponded to position 282 in the protein, and the amino acid encoded was Asp . The Tyr282----Asp mutant protein produced by site-specific methods was isolated and shares the oligomeric and inducer binding characteristics previously determined for the T41 mutation.

Gene, 1992 Feb 1, 111(1), 115 - 8
A versatile shuttle cosmid vector for use in Escherichia coli and actinomycetes; Denis F et al.; A shuttle cosmid vector has been constructed for Escherichia coli and actinomycetes . This vector, pFD666, utilizes the origin of replication (ori) of the broad-host-range plasmid, pJV1, from Streptomyces phaeochromogenes, for replication in actinomycetes and is compatible with vectors derived from pIJ101 . The pFD666 vector employs the neomycin phosphotransferase-encoding gene (neo) from transposon Tn5 as the selective marker . To achieve this, the native promoter of neo was replaced by one optimized for expression in both hosts . The polylinker used for cloning has nine unique sites flanked by the promoters for T7 and SP6 RNA polymerase for the production of specific RNA probes . Terminators on both sides of the polylinker protect the vector from transcription originating from cloned inserts . An M13 ori allows the production of single-stranded DNA.

Exp Mol Pathol, 1992 Feb, 56(1), 70 - 5
A study of endotoxin-associated hepatotoxicity on proliferating hepatocytes; Shibayama Y et al.; It is thought that regeneration of the liver provides a state of preparedness for the Shwartzman reaction and contributes to the development of endotoxin-associated massive hepatic necrosis following partial hepatectomy . Therefore we examined endotoxin hepatotoxicity in rats with hepatic regeneration after 35% hepatectomy and in rats with liver cell proliferation induced by lead nitrate . Biochemical and histopathological studies showed no enhanced endotoxin hepatotoxicity in either partially hepatectomized rats or in rats with lead nitrate-induced liver cell proliferation . These results indicate that the development of endotoxin-associated hepatic damage after partial hepatectomy may not relate to regeneration and proliferation of the liver.

Mol Biol Rep, 1992 Feb, 16(1), 49 - 59
Molecular cloning of a major CENP-B epitope and its use for the detection of anticentromere autoantibodies; Verheijen R et al.; An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of anticentromere autoantibodies in sera of patients with suspected or manifest rheumatic diseases . The antigen source used in this assay consists of the recombinant protein of glutathione S-transferase (GST) fused to the last 60 C-terminal amino acid residues of the major centromere protein CENP-B . Although this CENP-B segment is only a small part of the complete polypeptide, we show that it constitutes an important autoimmune antigenic domain which is recognized by all patient sera in which ACA can be detected using the immunoblotting technique with a HeLa S3 nuclear protein extract as antigen source.

Anticancer Drug Des, 1992 Feb, 7(1), 3 - 14
Studies on DNA damage and induction of SOS repair by novel multifunctional bioreducible compounds . II . A metronidazole adduct of a ruthenium-arene compound; Dale LD et al.; A new transition metal complex of the 5-nitroimidazole, metronidazole (1-beta-hydroxyethyl-2-methyl-5-nitroimidazole), has been prepared and its potential use as a hypoxic cell cytotoxic agent examined . The preparation of the complex {(eta6-C6H6)RuCl2(metronidazole)} is described together with its characterization using standard spectroscopic techniques . Electrochemical investigations showed that coordination to the metal centre had not altered the electron affinity of the metronidazole, but kinetic studies using the cyclic voltametric mode demonstrated that the one-electron addition product, the nitro radical anion, had a decreased lifetime, with a half-life of 7.75 and 11.9 s for the coordinated and free metronidazole ligand respectively . Biological studies employed viscosity measurements, DNA SOS repair capacity and a transfection assay to examine the effect on DNA . Conductance studies were also employed to determine the influence on intact Escherichia coli growth rates . The ruthenium-metronidazole complex showed greater activity than metronidazole aerobically, but a higher differential activity under hypoxic reduction conditions, due to activation of the NO2 group . Results with intact cells suggested a greater selective cytotoxicity with metronidazole coordinated to ruthenium than attained with the free ligand.

AIDS Res Hum Retroviruses, 1992 Feb, 8(2), 297 - 304
Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein; Woerner AM et al.; Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is the viral protein required for integration of the HIV-1 genome into host cell DNA . A series of clones expressing portions of IN as lambda cII fusion proteins has been constructed in an Escherichia coli expression system; a Southwestern procedure was used to examine binding of the expressed proteins to DNA oligonucleotides . Proteins expressed by clone pHIP106, encoding the entire IN protein but no other pol sequence, and pKNA101, which expresses an IN fusion protein containing 23 amino acids of HIV-1 reverse transcriptase at its amino terminus, exhibited similar levels of oligonucleotide binding . Little DNA sequence specificity was associated with binding activity and there was a preference for Mn2+ over Mg2+ and Ca2+ . Interestingly, the protein expressed by an N-terminal clone containing nucleotides coding for IN amino acids 1-141 (including a conserved His-Cys box) was unable to bind oligonucleotide, whereas the protein expressed by a C-terminal clone containing nucleotides coding for amino acids 142-288 exhibited binding equivalent to that of full-length IN . The C-terminal protein was unreactive with a MAb to the lambda cII leader peptide and with an antipeptide serum directed against amino acids 141-158 . These results are consistent with the previously reported internal initiation of IN protein synthesis in E . coli at met 154, and indicate that the C-terminal clone does not express IN amino acids 142-153 . These amino acids represent part of a conserved region termed D(35)E, containing amino acids 116-152, which has been implicated in IN DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Physiol, 1992 Feb, 262(2 Pt 1), L198 - 207
Cloning and expression of an airway epithelial 12-lipoxygenase; De Marzo N et al.; Arachidonate 12-lipoxygenase generates metabolites that may regulate airway function . To further characterize this enzyme, we isolated a cDNA corresponding to 12-lipoxygenase from a bovine tracheal epithelium cDNA library using human reticulocyte 15-lipoxygenase cDNA as a probe . The resulting 2.9-kb cDNA, the identity of which was confirmed by expression of active catalytic function in Escherichia coli has a 2.0-kb open reading frame encoding a protein of 75,000 kDa and includes 5 bp of 5'-untranslated region and 0.9 kb of 3'-untranslated region . On Northern blots, the 12-lipoxygenase cDNA hybridized to one band (3.5 kb) of bovine tracheal epithelium RNA . Polyclonal antibodies that recognize human tracheal 15-lipoxygenase cross-reacted on immunoblots to the expressed bovine tracheal 12-lipoxygenase . Further, the deduced amino acid sequence is 86% identical (93% similar) to human 15-lipoxygenase but 64% identical to human platelet 12-lipoxygenase, suggesting that the bovine tracheal enzyme is the homologue of the human 15-lipoxygenase . This is the first sequence of an epithelial lipoxygenase from any species . A comparison of the bovine sequence with other lipoxygenase sequences shows that there are only four amino acids which are conserved differences between a 12-lipoxygenase and a 15-lipoxygenase . We hypothesize that these four amino acids may be responsible for the positional specificity of the enzyme.

Toxicol Appl Pharmacol, 1992 Feb, 112(2), 222 - 8
Pentamidine blocks the pathophysiologic effects of endotoxemia through inhibition of cytokine release; Rosenthal GJ et al.; Pentamidine isethionate, an antiprotozoal agent with therapeutic value against Pneumocystis carinii pneumonia, has been used for over 30 years without a precise understanding of its mechanism of pharmacologic action . We have previously reported that pentamidine has the capacity to inhibit the release of cytokines from macrophages through a post-translational processing event . The present studies were undertaken to assess the ability of pentamidine to modulate the detrimental effects of murine endotoxemia, a disease with a pathophysiology clearly linked to host-produced cytokines . Under conditions where normal B6C3F1 mice succumbed to the lethal effects of endotoxin, mice pretreated with pentamidine were significantly protected from both mortality and loss of thermoregulatory control . The EC50 for protection from mortality by pentamidine was approximately 11.4 mg/kg . These observations correlated with decreased serum levels of tumor necrosis factor (TNF) and interleukin 6 . Inhibition of cytokines was not manifested as part of a generalized inhibition of protein synthesis as demonstrated by the lack of significant modulation of serum albumin in pentamidine-treated animals . In addition to decreased serum concentrations of cytokines, lungs isolated from mice treated with both pentamidine and endotoxin exhibited a decreased release of TNF compared to lungs isolated from mice treated with vehicle and endotoxin . The lower levels of TNF released from lung tissue in pentamidine-treated mice correlated with a lesser degree of alveolar deterioration than was observed in vehicle-treated mice . These data indicate that following endotoxin administration, pentamidine has a protective and antiinflammatory role both systemically and in the lung and suggest that inhibition of inflammatory cytokines may be one mechanism operable in the therapeutic activity of the drug against P carinii pneumonia.

J Gen Virol, 1992 Feb, 73 ( Pt 2), 223 - 7
Cooperative binding of the red clover necrotic mosaic virus movement protein to single-stranded nucleic acids; Osman TA et al.; The movement protein of red clover necrotic mosaic dianthovirus was produced in Escherichia coli using an expression vector . Gel retardation analysis and u.v . cross-linking studies showed that the movement protein bound cooperatively to ssRNA and ssDNA, but not to dsDNA . Binding competition experiments established that the movement protein bound to ssRNA and ssDNA with similar affinities and that the binding was not sequence-specific in the experimental conditions employed . A truncated movement protein lacking the C-terminal 88 amino acids was also shown to bind to ssRNA.

EMBO J, 1992 Feb, 11(2), 667 - 72
The apical localization of transcribing RNA polymerases on supercoiled DNA prevents their rotation around the template; ten Heggeler-Bordier B et al.; The interaction of Escherichia coli RNA polymerase with supercoiled DNA was visualized by cryo-electron microscopy of vitrified samples and by classical electron microscopy methods . We observed that when E . coli RNA polymerase binds to a promoter on supercoiled DNA, this promoter becomes located at an apical loop of the interwound DNA molecule . During transcription RNA polymerase shifts the apical loop along the DNA, always remaining at the top of the moving loop . This relationship between RNA polymerase and the supercoiled template precludes circling of the RNA polymerase around the DNA and prevents the growing RNA transcript from becoming entangled with the template DNA.

EMBO J, 1992 Feb, 11(2), 559 - 67
SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma; Rotin D et al.; Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain . SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma) . Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma . Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity . For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins . Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity . The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor . Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2 . This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1992 Feb, 11(2), 441 - 7
Colicin A unfolds during its translocation in Escherichia coli cells and spans the whole cell envelope when its pore has formed; Benedetti H et al.; The addition of the pore forming colicin A to Escherichia coli cells results in an efflux of cytoplasmic potassium . This efflux is preceded by a lag time which is related to the time needed for the translocation of the toxin through the envelope . Denaturing the colicin A with urea, before adding it to the cells, did not affect the properties of the pore but decreased the lag time . After renaturation, the lag time was similar to that of the native colicin . This suggests that the unfolding of colicin A accelerates its translocation . The addition of trypsin, which has access neither to the periplasmic space nor to the cytoplasmic membrane, resulted in an immediate arrest of the potassium efflux induced by colicins A and B . The possibility that trypsin may act on a bacterial component required for colicin reception and/or translocation was ruled out . It is thus likely that the arrest of the efflux corresponds to a closing of the pores . This long distance effect of trypsin suggests that part of the polypeptide chain of the colicins may still be in contact with the external medium even when the pore has formed in the inner membrane.

Plant Mol Biol, 1992 Feb, 18(3), 489 - 503
Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants; Fliegmann J et al.; Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes . Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes . It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type . We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli . The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin) . The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA . The protein deviated in 47 positions from the CHS consensus . It had 73.2% identity with the CHS from P . sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea) . We also investigated the regulation of both enzyme types in P . sylvestris plantlets exposed to stress . CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h . STS type activities were regulated differently and were absent in non-stressed plantlets . Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h . The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.

Biochem J, 1992 Feb 1, 281 ( Pt 3), 785 - 93
Purification, characterization and function of bacterioferritin from the cyanobacterium Synechocystis P.C.C . 6803; Laulhere JP et al.; Storage and buffering of iron is achieved by a class of proteins, the ferritins, widely distributed throughout the living kingdoms . All ferritins have in common their three-dimensional structure and their ability to store large amounts of iron in their central cavity . However, eukaryotic ferritins from plants and animals and bacterioferritins have no sequence similarity, and besides non-haem iron bacterioferritins contain haem residues whereas eukaryotic ferritins do not . In this paper we report the first purification and characterization of a bacterioferritin from a cyanobacterium . It has a molecular mass of 400 kDa and is built up from 19 kDa subunits . Its N-terminal sequence shows 73% identity with that of the Escherichia coli bacterioferritin subunit . It contains 2300 atoms of iron and 1500 molecules of phosphate per ferritin molecule and 0.25 haem residue per subunit; the alpha-peak of the cytochrome has its maximum at 559 nm . In contrast with what is known for eukaryotic ferritins, we found that bacterioferritin from Synechocystis is not inducible by iron under the conditions that we have tested and that it has a constant concentration whatever the iron status of the cells, even at very low iron concentration . Bacterioferritin from Synechocystis P.C.C . 6803 is fully assembled in vivo and it is shown by labelling with 59Fe that it is able to load iron in vitro as well as in vivo . Bacterioferritin from Synechocystis is shown to have an iron-buffering function while the bulk of cellular iron is found associated with a pool of low-molecular-mass electronegative molecules . The role of Synechocystis bacterioferritin in iron metabolism is discussed.

FEMS Microbiol Lett, 1992 Feb 1, 70(1), 37 - 41
Characterization of the genes coding for the F1F0 subunits of the sodium dependent ATPase of Propionigenium modestum; Krumholz LR et al.; The DNA coding for the eight structural genes and uncI of the sodium dependent ATPase of Propionigenium modestum has been cloned and sequenced . Based on sequence homology, the genes were determined to appear in the order uncBEFHAGDC as in several other bacterial species . Minicell experiments revealed that plasmids containing the P . modestum DNA expressed those ATPase polypeptides in Escherichia coli . These were very similar in molecular mass to those obtained from the purified ATPase of P . modestum . No membrane-bound ATPase activity was observed in E . coli unc deletion strains containing the P . modestum ATPase genes . Amino acid alignments which were done with the Fo subunits revealed only a few conservative changes in the highly conserved regions of the polypeptides.

Hum Gene Ther, 1992 Feb, 3(1), 21 - 33
Direct gene transfer into nonhuman primate myofibers in vivo; Jiao S et al.; Previously, we showed that rodent muscle has the ability to take up and express plasmid genes injected intramuscularly . This study now demonstrates that nonhuman primate muscle also has this ability to express injected plasmids . A scaled-up version of the standard large preparation of plasmid DNA allowed several tens of milligrams of CCC plasmid DNA to be relatively easily produced and administered to monkeys . After the injection of the E . coli beta-galactosidase reporter gene in pRSVLac-Z, foreign gene expression was localized to both type I and type II myofibers . The luciferase reporter gene in pRSVL was used to quantify the amount of expression . The multiple implantation of plasmid DNA pellets was more efficient in expressing luciferase than the injection of DNA in normal saline . Luciferase activity persisted for at least 4 months after injection . However, the luciferase expression was considerably less than that in rodents . Preliminary studies explored why expression was less in monkeys . Of particular interest was the increased thickness of the perimysium of monkeys as compared to that in rodents . This increased connective tissue may decrease delivery of the plasmid DNA to the myofibers . Anti-nuclear or anti-DNA antibodies were not observed, even after repetitive DNA administrations, and no adverse effects were observed in any of the monkeys.

Gene, 1992 Feb 1, 111(1), 69 - 76
Cloning and high-level expression of chitinase-encoding gene of Streptomyces plicatus; Robbins PW et al.; A chitinase (Cht)-encoding gene from Streptomyces plicatus was previously cloned and expressed in Escherichia coli {Robbins et al., J . Biol . Chem., 263 (1988) 442-447} . We have sequenced this gene, compared its sequence with other genes encoding Cht and have explored its expression and regulation when reintroduced into Streptomyces lividans on multicopy plasmids . We have also cloned two other Streptomyces Cht-encoding genes and a beta-hexosaminidase-encoding gene in E . coli by expression in the lambda ZAP-Bluescript vector . The hexosaminidase and one of the Chts were expressed directly from the genomic library in E . coli at a high level as chimeric fusions with the beta-galactosidase alpha-complementing peptide encoded by the vector . Direct cloning and high-level expression of such chimeric proteins, which overcomes the difficulties associated with expressing Streptomyces genes in E . coli, should generally be possible wherever large numbers of transformants can be conveniently screened.

EMBO J, 1992 Feb, 11(2), 673 - 82
GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast; Girard JP et al.; Among the few proteins of the eukaryotic nucleolus that have been characterized, four proteins, nucleolin, fibrillarin, SSB1 and NSR1, possess a common structural motif, the GAR domain, which is rich in glycine and arginine residues . In order to examine whether the presence of this domain is characteristic of a family of nucleolar proteins, we investigated whether other yeast genes encode proteins containing GAR domains . We report here the sequence and the characterization of a new yeast gene, GAR1, which encodes a protein of 205 residues containing two GAR domains . GAR1 is a non-ribosomal protein, localized in the yeast nucleolus, which is essential for cell growth . Immunoprecipitation with anti-GAR1 antibodies shows that GAR1 is associated with a subset of snoRNAs, including snR10 and snR30 . Depletion of GAR1 by expression under the control of a regulated GAL promoter, impairs processing of the 35S primary transcript of pre-rRNA and prevents synthesis of 18S rRNA . GAR1 is thus the fifth member of a family of nucleolar proteins containing GAR domains, and is involved in rRNA metabolism.

Genes Dev, 1992 Feb, 6(2), 177 - 85
The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F; Hiebert SW et al.; Recent experiments have shown that the E2F transcription factor is in a complex with the RB1 gene product . The E2F-pRB complex can be reconstituted in an in vitro assay using a GST-RB fusion protein isolated from Escherichia coli . This interaction is dependent on pRB sequences involved in E1A/T-antigen binding as well as carboxy-terminal pRB sequences that are not necessary for E1A/T binding . Moreover, reconstitution assays reveal a requirement for an accessory factor, in addition to E2F and pRB, for formation of the E2F-pRB complex . Assays of transcription from the adenovirus E2 promoter in transfection experiments demonstrate that formation of the complex containing pRB and E2F coincides with an inhibition of E2F-dependent transcriptional activity . A mutant pRB protein that does not associate with E2F does not inhibit transcription . We conclude that as a consequence of its interaction with E2F, pRB may regulate the transcriptional function of the E2F factor.

J Bacteriol, 1992 Feb, 174(4), 1222 - 8
Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12; Gibson FP et al.; The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated . Mutations that inactivate sbcC improve the ability to propagate lambda red gam phage that carry a palindromic sequence in their DNA . They also act with sbcB mutations as cosuppressors of the defects in recombination, DNA repair, and cell viability associated with recBC mutations . A 1,282-bp cassette encoding resistance to kanamycin was used to disrupt orf-45 . The mutation, which has a polar effect on the expression of sbcC, allowed stable propagation of palindromic lambda phage even when the sbcC gene product was provided in trans . Additional nonpolar mutations in orf-45 were isolated on the basis of their ability to improve the growth of recBC sbcB strains . These mutations also confer resistance to mitomycin C, allow efficient recombination in Hfr crosses, and facilitate stable propagation of palindromic phage . It is concluded that the products of orf-45 and sbcC are functionally related . The orf-45 gene is therefore renamed sbcD.

Mol Cell Biol, 1992 Feb, 12(2), 499 - 511
The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing; Kamper U et al.; The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by the nuclear gene cyt-18, functions not only in aminoacylation but also in the splicing of group I introns . Here, we isolated the cognate Podospora anserina mt tyrRS gene, designated yts1, by using the N . crassa cyt-18 gene as a hybridization probe . DNA sequencing of the P . anserina gene revealed an open reading frame (ORF) of 641 amino acids which has significant similarity to other tyrRSs . The yts1 ORF is interrupted by two introns, one near its N terminus at the same position as the single intron in the cyt-18 gene and the other downstream in a region corresponding to the nucleotide-binding fold . The P . anserina yts1+ gene transformed the N . crassa cyt-18-2 mutant at a high frequency and rescued both the splicing and protein synthesis defects . Furthermore, the YTS1 protein synthesized in Escherichia coli was capable of splicing the N . crassa mt large rRNA intron in vitro . Together, these results indicate that YTS1 is a bifunctional protein active in both splicing and protein synthesis . The P . anserina YTS1 and N . crassa CYT-18 proteins share three blocks of amino acids that are not conserved in bacterial or yeast mt tyrRSs which do not function in splicing . One of these blocks corresponds to the idiosyncratic N-terminal domain shown previously to be required for splicing activity of the CYT-18 protein . The other two are located in the putative tRNA-binding domain toward the C terminus of the protein and also appear to be required for splicing . Since the E . coli and yeast mt tyrRSs do not function in splicing, the adaptation of the Neurospora and Podospora spp . mt tyrRSs to function in splicing most likely occurred after the divergence of their common ancestor from yeast.

J Bacteriol, 1992 Feb, 174(3), 659 - 63
Targeted insertion of selenocysteine into the alpha subunit of formate dehydrogenase from Methanobacterium formicicum; Heider J et al.; Selenocysteine incorporation into proteins is directed by an opal (UGA) codon and requires the existence of a stem-loop structure in the mRNA flanking the UGA at its 3' side . To analyze the sequence and secondary-structure requirements for UGA decoding, we have introduced mutations into the fdhA gene from Methanobacterium formicicum, which codes for the alpha subunit of the F420-reducing formate dehydrogenase . The M . formicicum enzyme contains a cysteine residue at the position where the Escherichia coli formate dehydrogenase H carries a selenocysteine moiety . The codon (UGC) for this cysteine residue was changed into a UGA codon, and mutations were successively introduced at the 5' and 3' sides to generate a stable secondary structure of the mRNA and to approximate the sequence of the predicted E . coli fdhF mRNA hairpin structure . It was found that introduction of the UGA and generation of a stable putative stem-loop structure were not sufficient for decoding with selenocysteine . Efficient selenocysteine incorporation, however, was obtained when the loop and the immediately adjacent portion of the putative stem had a sequence identical to that present in the E . coli fdhF mRNA structure.

Arch Biochem Biophys, 1992 Feb 1, 292(2), 376 - 81
Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector; Jounouchi M et al.; The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants . The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo . The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm . Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable . Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones . The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s) . Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit . These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.

J Leukoc Biol, 1992 Feb, 51(2), 181 - 7
Transcriptional inhibition of endotoxin-induced monokine synthesis following heat shock in murine peritoneal macrophages; Snyder YM et al.; The role of heat shock proteins (HSP) during the inflammatory response has been controversial . The effect of heat shock (HS) on the synthesis of the monokines tumor necrosis factor (TNF) and interleukin 1 (IL-1) by endotoxin-stimulated thioglycollate-elicited peritoneal macrophages was investigated . HS was deemed to have affected macrophages if a 70 kD HSP appeared on SDS gels; identity of this protein as the highly conserved HSP70 was then confirmed by immunoprecipitation . Maximal increases in HSP70 were apparent 2-5 h after HS at 45 degrees C for 12 min . Synthesis of HSP70 was no longer detected 24 h post HS . A reciprocal relationship between HSP70 and TNF was apparent in kinetic studies . TNF was not detected in culture supernatants if macrophages were endotoxin-stimulated 2 to 6 h after HS; however, the same stimulation 24 h later induced significant TNF secretion . RNA analysis of HS and non-HS macrophage cultures demonstrated a 60-fold reduction in TNF message in the HS macrophages 1 h after endotoxin stimulation . TNF mRNA levels remained depressed at 6 h while the HSP70 message had increased 30-fold . The ability of HS macrophages to ingest antibody-coated erythrocytes was not significantly affected following heat treatment . Macrophage response to HS can be said to inhibit transcription of inducible monokines while retaining other macrophage functions.

Protein Expr Purif, 1992 Feb, 3(1), 8 - 17
The adenovirus E1A 243R protein purified from Escherichia coli under nondenaturing conditions is found in association with dnaK; Wang DM et al.; The adenovirus E1A 243R protein immortalizes primary cells in culture and induces part of the phenotypes required for transformation . It has also been shown to interact with a number of cellular polypeptides, including the product of the retinoblastoma gene . To understand more fully the molecular activities of the E1A 243R protein in association with these proteins as well as its role in the processes of cellular growth, we have developed a method for rapidly purifying this protein from genetically engineered Escherichia coli under nondenaturing conditions . The plasmid-encoded E1A protein, when expressed in a protease-deficient mutant, is found to have the same length and amino acid sequence as that which is produced in a mammalian cell . The procedure for purifying the E1A 243R protein from bacteria relies primarily upon immunoaffinity chromatography and the use of a peptide comprising the epitope recognized by an E1A-specific antibody . Elution of the E1A protein under this condition allows for gentle isolation and a purity that ranges from 90 to 96% . However, without the addition of micromolar amounts of ATP prior to its elution from the antibody column, the E1A protein is found in association with an E . coli protein of 70 kDa . Immunoblot analysis with a specific antibody showed that this bacterial protein was the heat shock protein dnaK, which is known to have extensive homology with the hsp-hsc70 family of proteins in mammalian cells . Recognition of E1A by the dnaK protein may very well reflect a situation that also occurs between the mammalian heat shock proteins and the E1A 243R protein after adenovirus infection.

Res Microbiol, 1992 Feb, 143(2), 173 - 81
Catabolic pools in Escherichia coli; Pai SR et al.; Methods are described for measuring soluble pool magnitudes in steady-state or exponentially growing cultures, and for distinguishing between anabolic, catabolic and total metabolic pools within cells . These methods were applied to the measurement of pool magnitudes for several amino acids and other precursors in Escherichia coli THU . Our results support the independence of the magnitudes of total metabolic pools and growth rate in steady-state cultures . Our results also show that the total metabolic pool size is much larger than previously published estimates, which failed to include the contribution of catabolic pools . The average value of the total soluble material in exponential-phase cells is estimated to be 8 to 9% of the cell dry mass; pool values could be almost twice as large during midcycle because of the known increase in the magnitudes of protein and RNA precursor pool during the cell cycle.

Res Microbiol, 1992 Feb, 143(2), 139 - 49
Structure/function relationships in the Escherichia coli mannitol permease: identification of regions important for membrane insertion, substrate binding and oligomerization; Briggs CE et al.; The Escherichia coli mannitol permease (EIIMtl) of the phosphoenolpyruvate-dependent phosphotransferase system is a 68-kDa membrane protein that carries out the concomitant transport and phosphorylation of D-mannitol . Previous studies indicated that there are ca . 6 membrane-spanning helices within the N-terminal half of the protein, while the hydrophilic C-terminal half was shown to be exposed in the cytoplasm . In the present study, an analysis of C-terminally truncated EIIMtl mutants showed that proteins from which only the cytoplasmic domain has been deleted were present in the membrane at > or = 50% the amount of the intact protein . However, deletion proteins smaller than ca . 34 kDa were present in the membrane at only about 20% the amount of the intact protein . We also constructed a plasmid that encodes the first 43 amino acid residues of ELLMtl fused to residues 378 to 637 (the C-terminal domain) . The corresponding protein was associated with the cytoplasmic membrane . These results show that the first 43 amino acid residues of the N terminus are sufficient for membrane localization, although the region comprising the last 2 membrane-spanning helices appears to be important for maximum stability and/or efficient membrane insertion of the complete N-terminal domain . Further studies of these deletion proteins showed that binding of mannitol to the permease occurs even if the entire cytoplasmic domain is absent, but is abolished if the last putative membrane-spanning region is removed . Finally, regions of the protein within the membrane-bound domain were identified that influence the oligomerization state of the protein . These results further define domains of this multifunctional transport protein that are important for membrane insertion, stability, substrate binding and oligomerization.

Zhonghua Wai Ke Za Zhi, 1992 Feb, 30(2), 84 - 7, 124
{Correlativity of plasma endotoxin, plasma fibronectin and common bile duct pressure in severe acute cholangitis}; Zhang LM; On the basis of common bile duct pressure measurement (CBDP) in 18 patients of severe acute cholangitis, plasma endotoxin (ET) was determined by modified synthetic chromogenic limulus amebocyte lysate assay and plasma fibronectin (FN) was detected with Laurell's rocket immunoelectrophoresis . CBDP was 2.23 +/- 0.49 KPa in patient group . There was striking positive correlation between ET and CBDP . Preoperative ET was 202.73 +/- 88.57 ng/L in patient group which was much higher than 15.47 +/- 7.38 ng/L in the control (P less than 0.001) . Preoperative FN was 141.77 +/- 82.37 mg/L in the patient group which was lower than 317.21 +/- 12.57 mg/L in the control (p less than 0.001) . Statistical differences could be noticed in postoperative ET and FN between the survivor and the dead . The study suggested that plasma ET levels are greatly influenced by pressure gradient of bile duct, dynamic observations of ET and FN levels are helpful to monitor disease course and predict prognosis.

Glycoconj J, 1992 Feb, 9(1), 21 - 6
Detection of the ganglioside N-glycolyl-neuraminyl-lactosyl-ceramide by biotinylated Escherichia coli K99 lectin; Ouadia A et al.; K99 lectin from Escherichia coli was purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide . Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity . It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent . Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognize N-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay.

Protein Expr Purif, 1992 Feb, 3(1), 71 - 4
Higher specific activity of the Escherichia coli glutamyl-tRNA synthetase purified to homogeneity by a six-hour procedure; Lin SX et al.; The glutamyl-tRNA synthetase (EC 6.1.1.17) of Escherichia coli was purified to homogeneity from the overproducing strain DH5 alpha(pLQ7612) by a two-step procedure that takes only about 6 h and yields 10 mg of enzyme per gram of wet cells . The process consists of a two-phase polyethylene glycol-dextran partition, the top phase of which is diluted and directly applied to an anion-exchange FPLC MonoQ column . The purified enzyme has a specific activity about twice that of the same enzyme purified to homogeneity by the lengthy conventional procedure from either a normal strain or this overproducing strain . This difference is discussed in relation to the generation of microheterogeneity in proteins during their purification.

Protein Expr Purif, 1992 Feb, 3(1), 18 - 26
On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli; Vosters AF et al.; Recently we reported (D . B . Evans, W . G . Tarpley, and S . K . Sharma, 1991, Protein Expression Purif . 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site . Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC) . In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase . These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole . When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole . The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin . It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step . Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.

Biosci Biotechnol Biochem, 1992 Feb, 56(2), 207 - 10
Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A; Nagashima T et al.; The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering . The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively . Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene . All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody . The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no alpha-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.

Mol Microbiol, 1992 Feb, 6(3), 355 - 61
Sequence diversity of the 1.3 kb retron (retron-Ec107) among three distinct phylogenetic groups of Escherichia coli; Kawaguchi T et al.; In the preceding paper, we showed that a new 1.3 kb retron (retron-Ec107) in Escherichia coli is responsible for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec107) . Here, we show that this retron occurs in strains from different branches, A, B1, and D of a well-defined phylogenetic tree of a collection of wild E . coli . Sequence comparisons of the retrons from these three branches were carried out . Sequence homology was well conserved among the strains within the same branch and the retron sequence from branch A was exactly the same with that from branch D, while there were 18 base substitutions between the retrons from branch B1 and A or D, resulting in seven amino acid substitutions in reverse transcriptase . No substitutions were found in the msDNA- and msdRNA-coding regions, and there was no difference in the ability of msDNA production between them . These results suggest that the retron has probably been integrated into at least one of the three branches at an early stage of evolution and subsequently transferred to the other two branches, and also that the msDNA-producing system has been conserved during evolution with some mutations in the retron.

Mol Microbiol, 1992 Feb, 6(3), 345 - 54
Retron-Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence; Herzer PJ et al.; Some natural isolates of Escherichia coli have been shown to produce a unique branched RNA-linked single-stranded DNA called msDNA . These bacteria contain a retro-element called retron consisting of the msr-msd region and the gene for reverse transcriptase (RT) . All three E . coli retrons characterized to date have been shown to be integrated into a prophage or to be associated with phage-related genes . In this report, we identified a new msDNA from an E . coli wild strain . Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined . The retron was found to consist of a 1.3kb DNA fragment, making it the smallest retron isolated to date . The msDNA produced from the retron consists of a 107 base single-stranded DNA, which is considered to be branched out from the 18th G residue of a 75-base RNA molecule by a 2',5'-phosphodiester linkage . Thus, the msDNA and the retron were designated msDNA-Ec107 and retron-Ec107, respectively . Most significantly, retron-Ec107 was inserted into the E . coli genome by replacing a 34bp intergenic sequence between the pyrE and ttk genes located at 82 min on the E . coli chromosome . Interestingly, the retron contains palindromic structures at both ends and the E . coli 34bp intergenic sequence also contains a 10bp inverted repeat structure . These palindromic structures might have played a role in the integration of retron-Ec107 into the E . coli genome.

Jpn Circ J, 1992 Feb, 56(2), 192 - 8
Endothelial function in thrombosis and thrombolysis; Sueishi K et al.; The localization of tissue factor (TF) in atherosclerotic plaques of human aortas was immunohistochemically examined using rabbit anti-IgG against recombinant TF, which was expressed in E . coli . TF, the initiator of the extrinsic coagulation pathway, was ubiquitously present in atherosclerotic intima, and was expressed mainly by macrophages, but not by endothelial cells . It has been suggested that some macrophages in atherosclerotic intima co-express both molecules of TF and platelet-derived growth factor-B chain . We have developed a morphometrically quantitative in vitro assay for angiogenesis, using endothelial cultures on collagen gel incorporating plasminogen . With this method, we have obtained findings suggesting that plasminogen and plasminogen activators (PAs), especially urokinase-type PA (uPA) derived from endothelial cells, enhance angiogenic activity, probably by increasing endothelial migration . uPA was immunohistochemically observed to be primarily cell-associated on the focal contract areas, probably via its receptors on endothelial cells . These findings may support the hypothesis that the activation and regulation of the pericellular fibrinolysis system is closely related to angiogenesis.

Biochem J, 1992 Feb 1, 281 ( Pt 3), 865 - 70
Expression of Escherichia coli homoserine kinase in mouse 3T3 cells; Rees WD et al.; The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene . Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity . It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine . In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E . coli enzyme . These experiments demonstrate that E . coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 927 - 31
Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase; Ben-Artzi H et al.; Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA . This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities . In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme . The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription . The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex . This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different . Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex . We refer to this unusual activity as RNase D . Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT . The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column . Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity . Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3' . The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.

Mutat Res, 1992 Feb, 281(2), 137 - 41
SOS system induction in Escherichia coli cells with distinct levels of ribonucleotide reductase activity; Villaverde A et al.; The UV-mediated induction of recA and sfiA genes in Escherichia coli cells with distinct levels of dATP has been studied . Low levels of dATP were obtained by using either a temperature-sensitive ribonucleotide (RDP) reductase-deficient (nrdA) mutant or a wild-type strain treated with hydroxyurea . High pools of dATP were achieved by using a plasmid overproducing RDP reductase . The results obtained show that expression of the recA and sfiA genes was inhibited neither in the UV-irradiated nrdA mutant at 42 degrees C nor in the wild-type strain in the presence of hydroxyurea . Likewise, the increase of the dATP pool did not enhance recA and sfiA gene expression after UV irradiation . All these data suggest that the basal level of dATP is not a limiting factor in the process of induction of the SOS system in Escherichia coli.

Mutat Res, 1992 Feb, 281(2), 123 - 7
Overproduction of the RecD polypeptide sensitizes Escherichia coli cells to gamma-radiation; Brcic-Kostic K et al.; We investigated DNA metabolism in Escherichia coli cells carrying the multicopy recD+ plasmid (pKI13) . In the presence of pKI13, the cellular level of the recD gene product (RecD polypeptide) is amplified at least 60-fold . Overproduction of the RecD polypeptide has no effect on UV repair and conjugational recombination . In contrast, high cellular levels of this polypeptide sensitize wild-type cells to gamma-radiation; also, they increase the rate of radiation-induced DNA degradation . A possible mechanism for the enhancement of gamma-ray-induced killing by large amounts of the RecD polypeptide is discussed.

Mutat Res, 1992 Feb, 265(2), 155 - 63
Chlorpromazine-induced damage on nucleic acids: a combined cytogenetic and biochemical study; Lialiaris T et al.; Chlorpromazine is now emerging as an adjuvant chemotherapeutic agent for the treatment of neoplasia . This was further supported in the present study by the following lines of evidence: it was shown that chlorpromazine causes damage in a series of native nucleic acids, though at somewhat high concentrations . Furthermore, chlorpromazine and caffeine were shown to act synergistically to potentiate the cytogenetic effect of adriamycin on human lymphocytes in vitro and on Ehrlich ascites tumour (EAT) cells in vivo . It is suggested that chlorpromazine alone or in combination with caffeine may exert its cytotoxic effect on normal and neoplastic cells not only indirectly, i.e . by facilitating the intracellular retention of adriamycin, but also directly by intercalating into nucleic acids.

J Virol, 1992 Feb, 66(2), 615 - 22
Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H; Telesnitsky A et al.; We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT . This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H . This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay . However, proviruses reconstructed to include this deletion were noninfectious . Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT . The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.

Biotechnology (N Y), 1992 Feb, 10(2), 163 - 7
High level Escherichia coli expression and production of a bivalent humanized antibody fragment; Carter P et al.; Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized . We therefore attempted to generate humanized F(ab')2 fragments by secretion from E . coli . Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA . Surprisingly, this high expression level of Fab' in the periplasmic space of E . coli does not drive dimerization . However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro . The E . coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2 . This system makes E . coli expression of bivalent antibody fragments for human therapy (or other uses) practical.

Appl Microbiol Biotechnol, 1992 Feb, 36(5), 655 - 8
Enhanced expression of heterologous proteins by the use of a superinducible vector in budding yeast; Porro D et al.; We report the effects of a strong overexpression of the GAL4 activator protein on the expression of UASGAL regulated genes, obtained by cloning the GAL4 gene and the GAL1-10 upstream activating sequence (UASGAL)-lacZ fusion in the same high copy number plasmid . Comparable amounts of active enzyme were obtained by host strains usually producing different levels of cloned proteins due to their different genetic background . The transformed cells constitutively produced low levels of beta-galactosidase (1-2% of total proteins) both in glucose and in raffinose minimal media . Nevertheless, expression was still inducible and a tenfold induction could be rapidly obtained by the addition of 0.5% (w/v) galactose to the culture, even when glucose was still present in the medium.

J Biotechnol, 1992 Feb, 22(3), 299 - 310
Cloning and high level expression of a synthetic gene for human basic fibroblast growth factor; Milev PV et al.; A gene encoding human basic fibroblast growth factor has been chemically synthesized, cloned and expressed in Escherichia coli as a biologically active protein . The 465 bp gene was assembled by enzymatic ligation of 6 pairs of oligonucleotides and cloned in the expression vector pLCII downstream from the strong PL promoter . This promoter directed the synthesis of a fusion protein between a 31 amino acids fragment of the lambda phage cII protein and bFGF . A four amino acid recognition sequence for the site-specific protease fXa was introduced in the plasmid construct and this allowed cleavage of the fusion protein at the boundary between cII and bFGF . bFGF was purified close to homogeneity using a Heparin-Sepharose column and Mono S cation exchange chromatography . The use of the pLCII expression system resulted in the accumulation of 20 to 25 mg of purified bFGF per l of bacterial culture . The recombinant bFGF was mitogenic for mouse 3T3 fibroblasts and the dose-response curve was similar to the one for native bFGF.

Lett Appl Microbiol, 1992 Feb, 14(2), 47 - 50
Comparison of a competitive enzyme immunoassay kit and the infant mouse assay for detecting Escherichia coli heat-stable enterotoxin; Stavric S et al.; A commercial competitive enzyme immunoassay kit, Escherichia coli ST EIA, was compared with the conventional infant mouse assay for sensitivity and specificity in detecting E . coli heat-stable enterotoxin . Thirty-one of 46 strains of E . coli tested were positive by both assays, while 15 strains were negative . The sensitivity of the ST EIA kit was up to 64-fold lower than the infant mouse assay.

Rinsho Shinkeigaku, 1992 Feb, 32(2), 203 - 8
{An autopsy case of spinal subdural abscess in the aged--comparative study with neuroradiological findings}; Fukui T et al.; An 82-year-old woman without previous medical problem noticed vague back pain on December 31, 1989, and was admitted to a hospital because she developed a fever, a rapidly progressive weakness followed by anesthesia of the lower extremities and sphincter disturbance . On myelography and myelo-CT, the spinal cord appeared to be displaced by an extramedullary mass which partially blocked the subdural space at the level of T-9 to L-1 . When transferred to our hospital on January 8, 1990, she was febrile and complaining of headache with meningeal signs . Percussion tenderness was present at T-8 to L-1 spinal spinous process . Neurological examination revealed that the patient had mild consciousness clouding, total paraplegia in the legs, sensory disturbance of a partial degree at L-1 to L-3 and totally below L-3, brisk but equal tendon reflexes in the upper extremities, areflexia in the legs with positive bilateral Babinski signs and sphincter disturbance . Otherwise she was neurologically unremarkable . Acute inflammatory reactions were prominent among the laboratory findings on admission . A lumbar tap yielded purulent fluid with more than 170,000 cells/mm3, 5,000 mg/dl of protein, 44 mg/dl of glucose and culture of the fluid isolated Escherichia coli . T1-weighted sagittal MRI disclosed an ill defined mass which showed the same or locally higher with gadopentetate dimeglumine (Gd-DTPA) signal intensity as soft tissue, compressing the spinal cord anteriorly from T-7 to L-3 . The lesion was noticed to have a more extensive rostral-caudal extent than was inferred from myelography and myelo-CT.(ABSTRACT TRUNCATED AT 250 WORDS)

Biull Eksp Biol Med, 1992 Feb, 113(2), 208 - 9
{Morphobiochemical study of the liver in systemic endotoxinemia}; Sergeeva NA et al.; Dog liver in systemic endotoxemia induced by intravenous injection of E . coli lipopolysaccharide in 2 mg/kg dosage has been studied . Morpho- and pathogenetic characteristics of developing intoxication have been specified histologically and biochemically.

Eur J Surg, 1992 Feb, 158(2), 95 - 103
On the value of giving a combination of drugs for the treatment of endotoxaemia in pigs; Naess F et al.; OBJECTIVE: To see if treatment with a combination of drugs each directed at a different mediator was successful in preventing activation of those mediators in experimental endotoxic shock . DESIGN: Controlled study . MATERIAL: 40 juvenile pigs . INTERVENTIONS: 33 animals received 0.01 mg/kg endotoxin infusion, the rest being given the same volume of saline; 10 of the 33 received no treatment . Of the remaining 23, 5 were given combination treatment with methylprednisolone, naloxone, ketanserin, promethazine, C1 esterase inhibitor, antithrombin III and aprotinin; 7 were given methylprednisolone only; 6 were given the three protease inhibitors (C1 esterase inhibitor, antithrombin III and aprotinin); and 5 were given naloxone, ketanserin and promethazine . MAIN OUTCOME MEASURES: Assessment of haemodynamic, proteolytic, and cellular effects of endotoxaemia . RESULTS: Only the combination treatment totally blocked all the effects of the infusion of endotoxin . CONCLUSION: As endotoxin affects several mediators, combination treatment is necessary to block all its deleterious effects in pigs.

Eur J Surg, 1992 Feb, 158(2), 89 - 93
Septic shock in rats treated with terbutaline alone and in combination with chemotherapeutics, dexamethasone, and infusion of 3% albumin; Ottosson J et al.; The effects of a beta 2-receptor agonist, terbutaline, on haematocrit and survival were studied in rats in which septic shock had been induced by intraperitoneal injection of a mean (SD) dose of 6.0 (4.5) x 10(8) live E . coli . Untreated septic animals developed haemoconcentration, the mean (SD) haematocrit increasing from 47.5 (1.4) to 53.1 (2.2) . Mean (SD) survival time was 8.9 (0.6) hours, and no animal survived for 24 hours . Terbutaline given as the only treatment in doses of 0.1, 0.5, and 2.5 mg/kg before injection of E . coli significantly reduced the haemoconcentration, with haematocrit of 51.9, 46.6 and 47.9, respectively, at 4 hours . Survival was not significantly prolonged . When terbutaline was started 5.5 hours after injection of E . coli and given in addition to a chemotherapeutic drug (trimethoprim + sulphamethoxazole) and dexamethasone, haematocrit were reduced, 24 hour survival improved from 44% to 68%, and 7 day survival improved from 20% to 48% . We conclude that terbutaline given alone counteracts the loss of plasma volume during septicaemia and, when combined with a chemotherapeutic and dexamethasone, significantly improves long term survival.

Mol Microbiol, 1992 Feb, 6(3), 293 - 300
CooB is required for assembly but not transport of CS1 pilin; Scott JR et al.; CS1 pili are filamentous proteinaceous appendages found on many enterotoxigenic Escherichia coli (ETEC) strains isolated from human diarrhoeal disease . They are thought to effect colonization of the upper intestine by facilitating binding to human ileal epithelial cells . We have identified a gene, cooB, which lies directly upstream of cooA, the gene that encodes the major structural CS1 protein . When translated in vitro, the protein product of cooB migrates in sodium dodecyl sulphate/polyacrylamide gel with an apparent molecular mass of 26 kDa, which is consistent with that predicted from its DNA sequence . We constructed a mutant allele (cooB-1) by insertion of the omega fragment, which inhibits transcription and translation, into the cooB gene in vitro . In a derivative of an ETEC strain with the cooB-1 mutation (JEF100) and a plasmid that encodes Rns (pEU2030), the positive regulator required for CS1 expression, no cooB and a greatly reduced level of cooA product was detectable in total cell extracts . The reduction of cooA in this strain appears to result from polarity of the cooB mutation because introduction of the wild-type cooA gene in trans causes production of CooA protein, which is found in cell pellet extracts, in extracts containing only surface proteins and in the culture supernatant . Therefore, in the absence of CooB, CooA is stable and it is transported through both inner and outer membranes . However, the cooB-1 strain with cooA in trans does not cause haemagglutination of bovine erythrocytes (the model system used to assay adherence mediated by coli surface antigen 1 (CS1) pili).(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1992 Feb, 174(4), 1109 - 18
Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli; Ernsting BR et al.; The leucine-responsive regulatory protein (Lrp) has been shown to regulate, either positively or negatively, the transcription of several Escherichia coli genes in response to leucine . We have used two-dimensional gel electrophoresis to analyze the patterns of polypeptide expression in isogenic lrp+ and lrp mutant strains in the presence or absence of leucine . The absence of a functional Lrp protein alters the expression of at least 30 polypeptides . The expression of the majority of these polypeptides is not affected by the presence or absence of 10 mM exogenous leucine . Outer membrane porins OmpC and OmpF, glutamine synthetase (GlnA), the small subunit of glutamate synthase (GltD), lysyl-tRNA synthetase form II (LysU), a high-affinity periplasmic binding protein specific for branched-chain amino acids (LivJ), W protein, and the enzymes of the pathway converting threonine to glycine, namely, threonine dehydrogenase (Tdh) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), were identified as members of the Lrp regulon by electrophoretic analysis . We have shown that Lrp is a positive regulator of glutamate synthase and glutamine synthetase and that exogenous leucine has little or no effect on the expression of these proteins . In strains carrying a glnL deletion and in strains carrying the glnL2302 allele, which directs the synthesis of a GlnL protein that is constitutively active, expression of glutamine synthetase is no longer regulated by Lrp, demonstrating that the effect of Lrp on glutamine synthetase levels is indirect and requires an intact glnL gene . lrp::Tn10 strains grow poorly when arginine or ornithine is present as the sole nitrogen source in the medium . On the bases of present studies and previous research, we propose that Lrp is involved in the adaptation of E . coli cells to major shifts in environment, such as those which occur when E . coli leaves the intestinal tract of its animal host . Several genes required for amino acid and peptide transport and catabolism are negatively regulated by Lrp, and other genes required for amino acid biosynthesis and ammonia assimilation in a nitrogen-poor environment are positively regulated by Lrp.

J Biochem (Tokyo), 1992 Feb, 111(2), 168 - 74
Cloning of a promoter-like soybean DNA sequence responding to IAA induction in Escherichia coli K12; Kline EL et al.; We have constructed a soybean genomic DNA library in Escherichia coli K12 strain KC13 using plasmid pPV33, which consists of a promoter-less tetracycline resistance (Tcr) gene . A recombinant clone, KC13(pAU-SB1)+, was obtained by selecting for resistance to tetracycline in the presence of indole-3-acetic acid (IAA) . Restriction enzyme cleavage and Southern hybridization analysis revealed that the pAU-SB1 plasmid has a 250 bp soybean DNA insert fused with the Tcr gene . In the presence of a selected group of auxins, induction of the Tcr phenotype and mRNA synthesis of the Tcr gene are observed only in KC13(pAU-SB1)+ cultures . On the other hand, induction of the Tcr phenotype and mRNA synthesis of the Tcr gene are absent in cells harboring the cloning vector pPV33 or a recombinant plasmid containing the 250 bp insert in the reverse orientation, pAU-SB1ro . This demonstrated a need for the insertion of the 250 bp soybean DNA and the specificity of its orientation in response to IAA induction . The start point of mRNA transcription in response to IAA, IBA, IPA, 2,4,5-T, and a-NAP is at base pair -96 or -95 upstream of the translational start site of the Tcr gene and base pair -98 with 2,4-D.

PCR Methods Appl, 1992 Feb, 1(3), 187 - 92
Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR; Subramanian PS et al.; We describe a novel method to map chromosomal Escherichia coli::Tn5 insertion mutations rapidly . This method utilizes the ends of Tn5 and the E . coli REP sequence as primer binding sites for the polymerase chain reaction (PCR) . The unique E . coli chromosomal sequence located between these primer binding sites is amplified by PCR and used as a probe to identify the recombinant clones from the Kohara phage ordered E . coli miniset bank that contains the Tn5 mutated loci . We used this approach to map two Tn5 insertion mutations previously identified by their effect on glycerol metabolism . The insertion mutations mapped to glpD, the aerobic sn-glycerol-3-phosphate dehydrogenase gene . Phenotypic analysis of the mutant strains revealed one with partial GlpD activity, suggesting transposon-mediated alteration of promoter activity . This mapping method should be applicable to the rapid physical mapping of any insertion mutation in the E . coli chromosome.

Protein Expr Purif, 1992 Feb, 3(1), 80 - 4
Heterologous expression of recombinant human glutathione transferase A1-1 from a hepatoma cell line; Stenberg G et al.; A cDNA clone, lambda GTHA1, encoding human glutathione transferase A1-1 has been isolated from a hepatoma HepG2 cDNA library . At the nucleotide level, the new clone showed minor differences from cDNA deriving from normal liver, but the deduced amino acid sequence was identical to the structure previously described . The protein was expressed from a plasmid, pKHA1, and isolated by a single-step affinity purification on an S-hexylglutathione Sepharose matrix . The yield of the recombinant protein was 165 mg from a 3-liter culture of bacteria.

Zhonghua Jie He He Hu Xi Za Zhi, 1992 Feb, 15(1), 23 - 4, 61-2
{The changes in serum angiotensin-converting enzyme and their relationships to pulmonary failure in experimental model of multiple organ failure}; Xue LB; The correlation of serum angiotensin-converting enzyme (SACE), PaO2 and pulmonary coefficient in rabbit model of multiple system organ failure (MOF) . 16 rabbits were fed E coli (0.5-3.0 x 10(11)/kg B.W.) by gastrogavage, were induced hemorrhagic shock (5.33 kPa arterial pressure for an hour) and were rapidly reinfused with shed blood and dextran . According to the criteria of pulmonary failure, that is respiratory rates greater than 100/min, PaO2 less than 8.00 kPa, pulmonary coefficient greater than 6.0, 8 rabbits developed pulmonary failure . They showed a significantly low SACE values, decreased PaO2 values and increased pulmonary coefficients, while in other 8 control animals the values of SACE and PaO2 did not significantly change.

FEMS Microbiol Lett, 1992 Feb 1, 70(1), 31 - 6
Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli; Cotter PA et al.; The individual and the combined effect of the fnr and arcA regulatory gene products on cytochrome o oxidase and cytochrome d oxidase gene expression in Escherichia coli were evaluated using lacZ reporter fusions to the cyo-ABCDE and cydAB operons . Fnr repressed cyo-lacZ and cyd-lacZ expression during anaerobic growth but not during aerobic growth conditions . ArcA functioned as an anaerobic repressor of cyo-lacZ expression while, in contrast, it activated cydAB expression during both aerobic and anaerobic growth . ArcA and Fnr appear to function independently of each other to control cyo-ABCDE operon expression . In contrast, FNR repression of cydAB expression was dependent on arcA+, as indicated by the inability of fnr+ plasmids to repress cyd-lacZ expression in an arcA strain . Under no conditions tested did Fnr activate cydAB expression . Most, but not all, of the observed aerobic/anaerobic regulation of cyo and cyd was accounted for by the two transcriptional regulators . These data suggest the existence of additional levels of anaerobic gene control in E . coli . Additionally, the expression of the fnr regulatory gene, and regulation of the anaerobic respiratory genes, narGHJI, dmsABC and frdABCD, was found to be independent of ArcA.

Zentralbl Bakteriol, 1992 Feb, 276(3), 313 - 22
Radioiodination of S-type lipopolysaccharide . Possibilities and limitations of labelling of the carbohydrate chain by periodate oxidation; Lachmann U et al.; S-type Lipopolysaccharide (LPS) from E . coli O 139:K 82 (B) was radiolabelled by coupling 125I-tyramine to the carbohydrate chain of LPS after oxidation of sugar residues carrying vicinal hydroxy groups, with sodium metaperiodate . The specific activity amounted to 1.3 kBq/microgram LPS . As LPS is a mixture of molecules with different carbohydrate chain lengths that are associated to large micelles of high kinetic stability, the preparation and the properties of the tracer exhibit some peculiarities . Most important, partial cleavage of the carbohydrate chains of LPS is caused on radioiodination by the procedure described . As a consequence, the tracer differs markedly from the starting LPS with respect to carbohydrate chain length distribution . Another feature of special interest is the variation of the specific activity (radioactivity/unit mass) for LPS molecules of different carbohydrate chain lengths . Because of the kinetic stability of LPS micelles, the equilibration of radioiodinated and non-radioactive LPS aggregates requires their solubilization by a detergent and its subsequent removal by dialysis . It could be demonstrated that short-chain LPS molecules predominantly associate to larger micelles than long-chain molecules . Regardless of these restrictions, the 125I-LPS proved to be useful for certain analytical purposes.

Vet Microbiol, 1992 Feb, 30(2-3), 191 - 202
Reduction in morbidity due to diarrhea in nursing beef calves by use of an inactivated oil-adjuvanted rotavirus-Escherichia coli vaccine in the dam; Cornaglia EM et al.; An outbreak of neonatal diarrhea occurred among beef calves (2000 animals) from one large Argentinian farm in 1985 . Rotavirus was detected in 78% (106/136) and enterotoxigenic Escherichia coli in 1.5% of the samples (2/136) obtained from sick calves . In comparison rotavirus was identified in only 1.6% (1/63) of the samples from clinically healthy calves . The rotavirus strain responsible for the outbreak was characterized as serotype 6 belonging to group A . In the following three years the protective capacity of a combined rotavirus-E . coli inactivated vaccine administered to the dams during the last third of the gestation period was evaluated on this farm by comparison of morbidity due to diarrhea in calves from vaccinated vs . placebo cows within the same year . The morbidity due to diarrhea among calves from dams in the vaccinated and placebo groups was 34% and 77%, respectively in 1986; 23% and 47% in 1987, and 15% and 34%, in 1988 . In 1987 morbidity of diarrhea in calves born from vaccinated heifers was 54% and 74% in calves from placebo heifers . In 1988 morbidity from diarrhea was 41% and 54%, respectively among calves in these two groups . In all experiments, calves from heifers showed significantly greater morbidity than calves from cows . Differences in diarrhea morbidity between the vaccinated and placebo groups were statistically significant (P less than 0.05) . Additional studies showed that the diarrhea had a significant influence (P less than 0.05) on the average live weight of the calves at weaning (5 to 7 months) with an average weight loss of 7.8 kg per calf among the calves affected with diarrhea.

Gene, 1992 Feb 1, 111(1), 11 - 20
Complete nucleotide sequence of Tn1721: gene organization and a novel gene product with features of a chemotaxis protein; Allmeier H et al.; The complete 11,139-nucleotide sequence of transposon Tn1721 has been determined . It contains three 38-bp inverted repeats, and (in this order) a new orfI, a resolution site (res), genes encoding resolvase (tnpR), transposase (tnpA), tetracycline-resistance (TcR) repressor (tetR), TcR (tetA) and a truncated transposase gene (tnpA') . The modulator origin of Tn1721 from at least three separate sources is supported by the distinctive codon usages of orfI, tnpR/tnpA and tetR/tetA, and by sequence similarities with Tn501 (tnpR/tnpA) and RP1 (tetR/tetA) . The ORFI-encoded 56-kDa polypeptide exhibits features of a methyl-accepting chemotaxis protein (MCP) with a conserved signal domain and a potential transmembrane domain; this polypeptide cross-reacts with anti-MCP antiserum . Like chemotaxis genes, orfI is transcribed from a sigma 28-like promoter . The overexpressed orfI gene product interferes with MCP-dependent chemotaxis suggesting that it completes for soluble transducer protein(s) in the cell . The potential selective advantage of this novel transposon-borne gene is discussed.

J Mol Endocrinol, 1992 Feb, 8(1), 29 - 41
Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3, following their expression in Escherichia coli as fusion proteins; King R et al.; The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described . The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ({Met1}-pGH) followed by the dipeptide Val-Asn . The latter two residues provide a unique hydroxylamine-sensitive link between {Met1}-pGH(1-46) and the N-terminal Gly of IGF-I . Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity . This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give {Gly3}-IGF-I and {Arg3}-IGF-I respectively . Production of milligram quantities of IGF-I peptide was readily achieved . The purity of the IGF-I, {Gly3}-IGF-I and {Arg3}-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis . {Gly3}-IGF-I and {Arg3}-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts . Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts . We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.

Genetics, 1992 Feb, 130(2), 241 - 9
Genetic changes accompanying increased fitness in evolving populations of Escherichia coli; Modi RI et al.; Two populations of Escherichia coli, each initiated with a single clone containing a derivative of the plasmid pBR322, were maintained for long periods in glucose-limited continuous culture . In both populations, after an extensive number of generations had elapsed, clones were isolated in which the transposon Tn3 from the plasmid had integrated into the bacterial chromosome . In both cases examined, the transpositions were shown to increase relative fitness approximately 6-7%, in the environment in which the populations were maintained . The loci of integration were mapped to approximately 13.2 min (population 1) and approximately 32.8 min (population 2).

EMBO J, 1992 Feb, 11(2), 769 - 76
Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen; Dornreiter I et al.; The purified human single-stranded DNA binding protein, replication protein A (RP-A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha-primase, as shown by ELISA and a modified immunoblotting technique . RP-A associated efficiently with the isolated primase, as well as with intact polymerase alpha-primase . The 70 kDa subunit of RP-A was sufficient for association with polymerase alpha-primase . Purified SV40 large T antigen bound to intact RP-A and to polymerase-primase, but not to any of the separated subunits of RP-A or to the isolated primase . These results suggest that the specific protein-protein interactions between RP-A, polymerase-primase and T antigen may play a role in the initiating of SV40 DNA replication.

EMBO J, 1992 Feb, 11(2), 741 - 50
IS10 transposase mutations that specifically alter target site recognition; Bender J et al.; IS10 inserts preferentially into particular hotspots . We describe here mutations of IS10 transposase, called 'ATS' that confer Altered Target Specificity . These mutations yield a general relaxation in target specificity but do not affect other aspects of transposition . Thus, the preference for specific nucleotide sequences at the target site can be cleanly separated from other steps of the transposition reaction . Eleven ATS mutations identified in a genetic screen occur at only two codons in transposase, one in each of two regions of the protein previously implicated in target site interactions (Patch I and Patch II) . Genetic analysis suggests that mutations at the two ATS codons affect the same specific function of transposase, thus raising the possibility that Patch I and Patch II interact . For wild-type IS10, insertion specificity is determined in part by a specific 6 bp consensus sequence and in part by the immediately adjacent sequence context of the target DNA . The ATS mutations do not qualitatively alter the hierarchy with which base pairs are recognized in the consensus sequence; instead, sites selected by ATS transposase exhibit a reduction in the degree to which certain base pairs are preferred over others . Models for the basis of this phenotype are discussed.

Neuron, 1992 Feb, 8(2), 387 - 97
A regulatory subunit of the cAMP-dependent protein kinase down-regulated in aplysia sensory neurons during long-term sensitization; Bergold PJ et al.; Binding of cAMP by the five neuronal isoforms (N1-5) of the regulatory (R) subunit of the Aplysia cAMP-dependent protein kinase is diminished in sensory neurons stimulated to produce long-term presynaptic facilitation . To determine how the cAMP-binding activity of the R subunits is lost, we isolated cDNAs encoding N4, which is a homolog of mammalian RI . Immunoblots with antisera raised against the R protein overexpressed in E . coli show that the diminished binding activity, which occurs in long-term facilitation, results from coordinate loss of R protein isoforms . No change was detected in the amount of transcripts for R subunits, suggesting that the down-regulation results from enhanced proteolytic turnover.

J Pharmacol Exp Ther, 1992 Feb, 260(2), 581 - 6
Chronic morphine administration impairs cell-mediated immune responses in swine; Molitor TW et al.; Swine were used as an experimental animal model to evaluate immunomodulating effects of the opiate drug, morphine, on antigen-specific humoral and cell-mediated immune responses . Morphine free base in peanut oil was administered at 4-day intervals for up to 42 days to maintain drug levels at or above 100 ng/ml . The effect of morphine administration on humoral immune responses was evaluated by immunization on day 7 of morphine treatment with a battery of antigens, including swine herpes virus (also known as pseudorabies virus, PRV), Brucella abortus and the Escherichia coli pilus antigens K88, K99, 987P and F41 . Fourteen days later, swine were reimmunized with B . abortus and E . coli pilus antigens . Antibody titers to these antigens were evaluated on a weekly basis . Cell-mediated immunity was evaluated by measuring skin immune responses to the antigen 2,4-dintroflurobenozene (DNFB) . In one experiment, swine were sensitized with DNFB on day 7 of morphine treatment at the same time as immunization with the other antigens . In a second series of experiments, swine were sensitized either 7 days before or 7 days after initiation of morphine treatment . Pigs were challenged with DNFB administered 27 days after the initiation of morphine treatment and skin responses were evaluated 24 h later . The ability of swine to resist PRV infection was evaluated by exposure to virulent virus on day 28 of morphine treatment . Chronic morphine administration did not impair the induction of the humoral immune responses to bacterial or viral antigens . In addition, morphine treatment did not alter the resistance of immunized swine to PRV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 996 - 1000
The free radical in pyruvate formate-lyase is located on glycine-734; Wagner AF et al.; Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure . By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue . Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models . N-coupling was verified through uniform 15N-labeling . The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable . Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain . The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position . The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.

Proc Natl Acad Sci U S A, 1992 Feb 1, 89(3), 982 - 6
Metabolism of glyceryl trinitrate to nitric oxide by endothelial cells and smooth muscle cells and its induction by Escherichia coli lipopolysaccharide; Salvemini D et al.; Here, we demonstrate that the metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) occurs not only in bovine aortic smooth muscle cells (SMCs) but also in endothelial cells (ECs) and that this biotransformation is enhanced by pretreatment with Escherichia coli lipopolysaccharide (LPS) . Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in SMCs or ECs . In addition, NO produced from GTN by cells was measured as nitrite (NO2-), one of its breakdown products . Indomethacin (10 microM)-treated SMCs or ECs enhanced the platelet inhibitory activity of GTN . This effect was abrogated by coincubation with oxyhemoglobin (oxyHb; 10 microM), indicating release of NO from GTN . LPS (0.5 microgram/ml; 18 h) enhanced at least 2- to 3-fold the capacity of SMCs or ECs to form NO from GTN, and this enhancement was attenuated when cycloheximide (10 micrograms/ml) was incubated together with LPS . Furthermore, when incubated with GTN (200 microM) SMCs or ECs treated with LPS (0.5 microgram/ml; 18 h) released more NO from GTN than nontreated cells as indicated by a much higher (8- to 9-fold) increase in the levels of cGMP . Exposure of SMCs to GTN (600 microM) for 30 min led to an increase in the levels of NO2- dependent on cell numbers, which was enhanced when SMCs were treated with LPS . Incubation of nontreated or LPS-treated cells with NG-monomethyl-L-arginine (300 microM; 60 min) did not influence the metabolism of GTN to NO . SMCs failed to enhance the antiplatelet activity of sodium nitroprusside . Anesthetized rats treated with an intraperitoneal injection of LPS (20 mg/kg) 18 h beforehand showed enhanced hypotensive responses to GTN (0.25-1 mg/kg) . These effects were blocked by methylene blue (10 mg/kg) but not by indomethacin (3 mg/kg) . LPS did not alter the hypotensive responses induced by phentolamine, verapamil, or SIN-1 . Thus, both in vitro and in vivo, LPS induces the enzyme(s) metabolizing GTN to NO.

J Bacteriol, 1992 Feb, 174(4), 1414 - 6
Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity; Ikeda TP et al.; Mutations in aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase, which selectively inhibit phosphatase activity have been isolated . These mutations yield amino acid substitutions within a 113-residue region of this 578-residue protein . These mutations may define a regulatory domain of this protein.

J Bacteriol, 1992 Feb, 174(4), 1229 - 39
Characterization of two hypertransposing Tn5 mutants; Wiegand TW et al.; Transposition of Tn5 in Escherichia coli is regulated by two transposon-encoded proteins: transposase (Tnp), promoting transposition preferentially in cis, and the trans-acting inhibitor (Inh) . Two separate transposase mutants were isolated that replace glutamate with lysine at position 110 (EK110) and at position 345 (EK345) . The EK transposase proteins increase the Tn5 transposition frequency 6- to 16-fold in cis and enhance the ability of transposase to act in trans . The purified mutant transposase proteins interact with transposon outside end DNA differently from the wild-type protein, resulting in the formation of a novel complex in gel retardation assays . During characterization of the transposase proteins in the absence of inhibitor, we found that wild-type transposase itself has a transposition-inhibiting function and that this inhibition is reduced for the mutant proteins . We present a model for the regulation of Tn5 transposition, which proposes the existence of two transposase species, one cis-activating and the other trans-inhibiting . The phenotype of the EK transposase mutants can be explained by a shift in the ratio of these two species.

J Bacteriol, 1992 Feb, 174(4), 1172 - 8
Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli; Rinken R et al.; Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation) . A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid . Inhibition of Chi activation was not accompanied by an overall decrease of recombination . A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition . Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not . It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.

J Cell Physiol, 1992 Feb, 150(2), 433 - 9
Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells; Watanabe K et al.; Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin . Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity . ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells . The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS . The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml . That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane . While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S . minnesota) were more potent inhibitors of ACE activity than others (E . coli 026:B6 and S . marcescens), all LPS serotypes tested were inhibitory . These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.

J Bacteriol, 1992 Feb, 174(3), 889 - 98
Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF (AppR)
Kaasen I, Falkenberg P, Styrvold OB, Strom AR.
It has been shown previously that the otsA and otsB mutations block osmoregulatory trehalose synthesis in Escherichia coli . We report that the transcription of these osmoregulated ots genes is dependent on KatF (AppR), a putative sigma factor for certain stationary phase- and starvation-induced genes . The transcription of the osmoregulated bet and proU genes was not katF dependent . Our genetic analysis showed that katF carries an amber mutation in E . coli K-12 and many of its derivatives but that katF has reverted to an active form in the much-used strain MC4100 . This amber mutation in katF leads to strain variations in trehalose synthesis and other katF-dependent functions of E . coli . We have performed a molecular cloning of the otsBA genes, and we present evidence that they constitute an operon encoding trehalose-6-phosphate phosphatase and trehalose-6-phosphate synthase . A cloning and restriction site analysis, performed by comparing the cloned fragments with the known physical map of the E . coli chromosome, revealed that the otsBA genes are situated on a 2.9-kb HindIII fragment located 8 to 11 kb clockwise of tar (41.6 min).

J Bacteriol, 1992 Feb, 174(3), 785 - 92
Propagation of pSC101 plasmids defective in binding of integration host factor; Biek DP et al.; Integration host factor (IHF), a multifunctional protein of E . coli, normally is required for the replication of plasmid pSC101 . T . T . Stenzel, P . Patel, and D . Bastia (Cell 49:709-717, 1987) have reported that IHF binds to a DNA locus near the pSC101 replication origin and enhances a static bend present in this region; mutation of the IHF binding site affects the plasmid's ability to replicate . We report here studies indicating that the requirement for IHF binding near the pSC101 replication origin is circumvented partially or completely by (i) mutation of the plasmid-encoded repA (replicase) gene or the chromosomally encoded topA gene, (ii) the presence on the plasmid of the pSC101 partition (par) locus, or (iii) replacement of the par locus by a strong transcriptional promoter . With the exception of the repA mutation, the factors that substitute for a functional origin region IHF binding site are known to alter plasmid topology by increasing negative DNA supercoiling, as does IHF itself . These results are consistent with the proposal that IHF binding near the pSC101 replication origin promotes plasmid replication by inducing a conformational change leading to formation of a repA-dependent DNA-protein complex . A variety of IHF-independent mechanisms can facilitate formation of the putative replication-initiation complex.

J Bacteriol, 1992 Feb, 174(3), 758 - 64
Influence of cyclic AMP, agmatine, and a novel protein encoded by a flanking gene on speB (agmatine ureohydrolase) in Escherichia coli; Szumanski MB et al.; The speB gene of Escherichia coli encodes agmatine ureohydrolase (AUH), a putrescine biosynthetic enzyme . The speB gene is transcribed either from its own promoter or as a polycistronic message from the promoter of the speA gene encoding arginine decarboxylase . Two open reading frames (ORF1 and ORF2) are present on the strand complementary to speB; approximately 90% of ORF2 overlaps the speB coding region . Analysis of transcriptional and translational fusions of ORF1 or ORF2 to lacZ revealed that ORF1 encoded a novel protein while ORF2 was not transcribed . Deletion of ORF1 from a plasmid containing ORF1, ORF2, and speB reduced the activity of AUH by 83% . In contrast, the presence of plasmid-encoded ORF1 caused an 86% increase in chromosomally encoded AUH activity . ORF1 did not stimulate alkaline phosphatase expressed from a phi(speB-phoA) transcriptional fusion encoded on the same plasmid . Western analysis (immunoblot) of a phi(ORF1-lacZ) translational fusion revealed that ORF1 encodes a 25.3-kDa protein . Agmatine induced transcription of phi(speB-phoA) but not phi(speA-phoA) fusions . Consequently, agmatine affects selection between the monocistronic and the polycistronic modes of speB transcription . In contrast, cyclic AMP (cAMP) repressed AUH activity of chromosomally encoded AUH but had no effect on plasmid-borne speB nor phi(speB-phoA) . It is concluded that ORF1 encodes a protein which is a posttranscriptional regulator of speB, agmatine induces speB independent of speA, and cAMP regulates speB indirectly.

Cancer Res, 1992 Feb 1, 52(3), 525 - 32
Overexpression of human topoisomerase I in baby hamster kidney cells: hypersensitivity of clonal isolates to camptothecin; Madden KR et al.; The 3645-base pair human topoisomerase I complementary DNA (cDNA) clone isolated by D'Arpa et al . (Proc . Natl . Acad . Sci . USA, 85:2543-2547, 1988) and a mutated version of the cDNA encoding a protein with phenylalanine instead of tyrosine at position 723 have been overexpressed 2- to 5-fold in stably transfected baby hamster kidney cells . The overexpressed proteins are the same size as the topoisomerase I present in Hela cells, indicating that the cDNA clone contains the complete topoisomerase I coding sequence . Some human colon carcinoma cells have increased levels of topoisomerase I and are hypersensitive to the drug camptothecin . The overexpressed wild-type topoisomerase I does not affect the cell growth or morphology of the baby hamster kidney cells, suggesting that elevated levels of topoisomerase I alone are not sufficient to cause cell transformation . However, the overexpressed wild-type protein is active, as shown by the hypersensitivity of clonal cell lines to camptothecin . The mutant form of topoisomerase I is enzymatically inactive by two criteria . First, extracts of Escherichia coli cells carrying the mutant cDNA contain no activity capable of relaxing superhelical DNA under conditions where activity is easily detectable in extracts from cells containing the wild-type cDNA . Second, baby hamster kidney cells stably transfected by the mutant cDNA are no more sensitive to camptothecin than control untransfected cells . These results indicate that tyrosine 723 is essential for enzyme activity and are consistent with predictions based on homology comparisons with the yeast enzymes, that this is the active-site tyrosine in the human topoisomerase I.

Arch Biochem Biophys, 1992 Feb 1, 292(2), 493 - 8
Stereochemistry of D-galactal and D-galacto-octenitol hydration by coffee bean alpha-galactosidase: insight into catalytic functioning of the enzyme; Weiser W et al.; Green coffee bean alpha-galactosidase was found to catalyze the hydration of D-galactal and (Z)-3,7-anhydro-1,2-dideoxy-D-galacto-oct-2-enitol (D-galacto-octenitol), each a known substrate for beta-galactosidase . The hydration of D-galactal by the alpha-galactosidase in D2O yielded 2-deoxy-2(S)-D-{2-2H}galactose; the hydration of D-{2-2H}galacto-octenitol in H2O yielded 1,2-dideoxy-2(R)-D-{2-2H}galactooct-3-ulose . Thus, the enzyme protonated each substrate from beneath the plane of the ring, as assumed for alpha-D-galactosides . These results provide an unequivocal assignment of the orientation of an acidic catalytic group to the alpha-galactosidase reaction center . In addition, they reveal a pattern of glycal/exocyclic enitol/glycoside protonation by the enzyme that differs from the pattern reported for beta-galactosidase and from that reported for alpha-glucosidases . Further findings show that D-galacto-octenitol is hydrated by the coffee bean alpha-galactosidase to form the alpha-anomer of 1,2-dideoxy-D-galactooctulose and by Escherichia coli beta-galactosidase to form the beta-anomer . That each enzyme converts this enolic substrate to a product whose de novo anomeric configuration matches that formed from its D-galactosidic substrates provides new evidence for the role of protein structure in controlling the steric outcome of reactions catalyzed by these and other glycosylases . The findings are discussed in light of the concept that catalysis by glycosidases involves a "plastic" protonation phase and a "conserved" product configuration phase.

Arch Biochem Biophys, 1992 Feb 1, 292(2), 419 - 26
Cytochrome c2 mutants of Rhodobacter capsulatus; Caffrey M et al.; Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c . Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point . In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis . We describe here overproduction of R . capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R . capsulatus and Rhodobacter sphaeroides . We demonstrate that R . capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R . sphaeroides . Further, we describe the generation, expression, and in vivo functionality properties of nine R . capsulatus site-directed mutants . We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo . In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo . These findings establish an effective system for the production of R . capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.

J Virol, 1992 Feb, 66(2), 623 - 31
The ICP4 binding sites in the herpes simplex virus type 1 glycoprotein D (gD) promoter are not essential for efficient gD transcription during virus infection; Smiley JR et al.; Activation of the early and late genes of herpes simplex virus type 1 during infection in tissue culture requires functional immediate-early regulatory protein ICP4 . ICP4 is a specific DNA-binding protein which recognizes a variety of DNA sequences, many of which contain the consensus ATCGTC . In general, mutations which impair the ability of ICP4 to bind to DNA also eliminate its ability to activate viral early and late promoters both in transfection assays and in the infected cell . However, the role of ICP4 binding sites in the viral genome is unclear; many early and late promoters do not contain consensus binding sites in their vicinity . The glycoprotein D (gD) gene contains two well-characterized ICP4 binding sites upstream of its promoter and a third downstream of the transcription start site . Multimerization of one of these sites has been shown to increase the response of the gD promoter to ICP4 in transfection assays, while their removal reduces stimulation of the gD promoter by ICP4 in vitro . To assess the role of these binding sites during virus infection, we have constructed a recombinant viral genome which has mutations affecting all three . Comparison of the amounts of gD RNA synthesized by the recombinant and wild-type viruses indicated that the mutations had little or no effect on the activity of the gD promoter . Therefore, either the sites have no essential role in gD promoter regulation in the presence of all of the herpes simplex virus type 1 IE polypeptides during a normal infection or they can be functionally substituted by other ICP4 binding sites elsewhere in the genome.

J Virol, 1992 Feb, 66(2), 1109 - 18
Sequential phosphorylation of the phosphoprotein of vesicular stomatitis virus by cellular and viral protein kinases is essential for transcription activation; Barik S et al.; The phosphoprotein (P) and the large protein (L) constitute the RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) . We show that phosphate-free P protein expressed in bacteria is transcriptionally inactive when reconstituted with L protein and viral N-RNA template free of cellular protein kinase . Phosphorylation of P protein by a cellular kinase(s) was essential for transcription as well as for further phosphorylation by an L-associated kinase, the two kinases acting in a sequential (cascade) manner . Phosphate groups introduced by cell kinase were stable, whereas those due to L kinase underwent a turnover which was coupled to ongoing transcription . We present a model for the phosphorylation pathway of P protein and propose that continued phosphorylation and dephosphorylation of P protein may represent a transcriptional regulatory (on-off) switch of nonsegmented negative-strand RNA viruses.

J Virol, 1992 Feb, 66(2), 1098 - 108
Characterization of human cytomegalovirus UL84 early gene and identification of its putative protein product; He YS et al.; The DNA sequence and transcription pattern of human cytomegalovirus early gene UL84 were analyzed . This gene was mapped within a 2.6-kb PstI fragment located between 0.534 and 0.545 map unit of the large unique segment of the human cytomegalovirus genome, which is adjacent to the pp65 and pp71 genes . A 2.0-kb mRNA was transcribed from this region in the same leftward direction as the mRNAs of the pp65 and pp71 genes . The message was first detected at 2.5 h postinfection and reached a maximal level between 72 and 96 h postinfection . The nucleotide sequences of the 2.6-kb PstI genomic DNA fragment and the cDNA derived from this region were determined . The resulting data revealed a polyadenylation signal (AATAAA) located 14 nucleotides upstream from the poly(A) tail of the cDNA and a 1,761-bp open reading frame capable of encoding a 65-kDa polypeptide . A potential leucine zipper was found in the N-terminal half of the peptide molecule between amino acids 114 and 135 . In addition, a different periodic leucine repeat with leucine at every eighth position was found between amino acids 325 and 373 . The transcriptional initiation site of this early gene was determined by primer extension analysis . A putative TATA box (TATTTAA) located 24 bp upstream of the cap site and several inverted repeats were found in the region further upstream of the TATA box . To test whether the open reading frame of this cDNA encodes a virus-specific protein, the cDNA was overexpressed in Escherichia coli as a fusion protein used to generate antibodies in rabbits . A protein with a molecular size of 65 kDa was detected in the infected-cell extracts harvested at 6 to 72 h postinfection, but not in purified virions, using immunoblot analysis . Both nuclear and cytoplasmic fluorescences were found at late stages of virus infection . From the results obtained, we postulate that UL84 may be a stable, virus-specific, nonstructural protein capable of forming a homo- or heterodimeric molecule.

Infect Immun, 1992 Feb, 60(2), 690 - 3
Molecular cloning of the recA gene and construction of a recA strain of Francisella novicida; Berg JM et al.; A gene locus that is functionally analogous to the recA gene of Escherichia coli was molecularly cloned from Francisella novicida . The cloned gene was found to suppress the sensitivity of an E . coli strain to DNA-damaging agents and to support genetic recombination in E . coli . After transposon mutagenesis, the recA-like gene locus was returned to F . novicida and a UV-sensitive F . novicida strain was isolated . In contrast to the wild-type strain, this UV-sensitive strain could not be transformed with chromosomal DNA.

Protein Sci, 1992 Feb, 1(2), 254 - 8
pH dependence of the reverse reaction catalyzed by phosphofructokinase I from Escherichia coli: implications for the role of Asp 127; Auzat I et al.; The kinetics of the reverse reaction catalyzed by Escherichia coli phosphofructokinase, i.e., the synthesis of ATP and fructose-6-phosphate from ADP and fructose-1,6-bisphosphate, have been studied at different pH values, from pH 6 to pH 9.2 . Hyperbolic saturations of the enzyme are observed for both substrates . The affinity for fructose-1,6-bisphosphate decreases with pH following the ionization of a group with a pK of 6.6, whereas the catalytic rate constant and perhaps the affinity for ADP are controlled by the ionization of a group with a pK of 6 . Several arguments show that the pK of 6.6 is probably that of the carboxyl group of Asp 127, whereas the pK of 6 is tentatively attributed to the carboxyl group of Asp 103 . The pK of 6.6 is assigned to the carboxyl group of Asp 127 in the free enzyme, and a simple model suggests that the same group would have an abnormally high pK, above 9.6, in the complex between phosphofructokinase and fructose-1,6-bisphosphate . It is proposed that the large pK shift of more than 3 pH units upon binding of fructose-1,6-bisphosphate is due to an electrostatic repulsion that could exist between the 1-phosphate group and the carboxyl group of Asp 127, which are close to each other in the crystal structure of phosphofructokinase (Shirakihara, Y . & Evans, P.R., 1988, J . Mol . Biol . 204, 973-994) . The same interpretation would also explain the much higher affinity of the enzyme for fructose-1,6-bisphosphate when Asp 127 is protonated.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1992 Jan 31, 255(5044), 594 - 7
Ordered self-assembly of polypeptide fragments to form nativelike dimeric trp repressor; Tasayco ML et al.; Subdomain-size proteolytic fragments of Escherichia coli trp repressor have been produced that assemble in defined order to regenerate fully native dimers . By characterization of the secondary and tertiary structures of isolated and recombined fragments, the structure of assembly intermediates can be correlated with the kinetic folding pathway of the intact repressor deduced from spectroscopic measurement of folding rates . The nativelike structure of these intermediates provides further evidence that protein folding pathways reflect the stabilities of secondary structural units and assemblies found in the native state . The proteolytic method should be generally useful in adding structural detail to spectroscopically determined folding mechanisms.

Biochem Biophys Res Commun, 1992 Jan 31, 182(2), 651 - 8
Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid; Cai XY et al.; The repression of MetE synthesis in Escherichia coli by vitamin B12 is known to require the MetH holoenzyme (B12-dependent methyltransferase) and the metF gene product . Experiments using trimethoprim, an inhibitor of dihydrofolate reductase, show that the MetF protein is not directly involved in the repression, but that N5-methyltetrahydrofolic acid (N5-methyl-H4-folate), the product of the MetF enzymatic reaction is required . Since the methyl group from N5-methyl-H4-folate is normally transferred to the MetH holoenzyme to form a methyl-B12 enzyme, the present results suggest that a methyl-B12 enzyme is involved in the vitamin B12 repression of metE expression . Other results argue against the possibility that a methyl-B12 enzyme functions in this repression solely by decreasing the cellular level of homocysteine, which is required for MetR activation of metE expression . Experiments with metJ mutants show that the MetJ protein mediates about 50% of the repression of metE expression by B12 but is totally responsible for the regulation of metF expression by vitamin B12.

Biochem Biophys Res Commun, 1992 Jan 31, 182(2), 617 - 23
Regulation of cytochrome P450 IID by acute phase mediators in C3H/HeJ mice; Trautwein C et al.; Cytochrome P450 IID6 is a drug metabolizing enzyme and the major target antigen in LKM-1 antibody positive chronic active hepatitis . The histological hallmark of chronic active hepatitis is a lymphocytic infiltrate in the liver . It is unknown whether and how cytokines produced and secreted by these tissue infiltrating mononuclear cells regulate the cellular expression of cytochrome P450 IID6 . To study the effect of interleukin 1, tumor necrosis factor and interleukin 6 on the hepatocellular RNA expression of cytochrome P450 IID, we injected each of the cytokines in C3H/HeJ mice . We found a time-dependent suppression of the cytochrome in the liver . Six hours after the intraperitoneal injection of 0.5 micrograms interleukin 1 beta the specific RNA-expression was reduced to 25% of the original level . A similar reduction was found after the injection of 2 micrograms tumor necrosis factor alpha . A mild suppression to 65% of the original level was seen six hours following the dose of 100 ng interleukin 6 . Our studies show how immune mediators can change the expression of an autoantigen . Further studies in the human system are necessary to estimate this regulation for the elimination of drugs and in LKM-1 antibody positive chronic active hepatitis.

Nature, 1992 Jan 30, 355(6359), 464 - 7
A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II; Finkelstein A et al.; RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II . The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region . RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex . A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74 . RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.

Biochim Biophys Acta, 1992 Jan 30, 1099(1), 102 - 10
Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophore; Lanyi JK et al.; The consequences of replacing Asp-85 with glutamate in bacteriorhodopsin, as expressed in Halobacterium sp . GRB, were investigated . Similarly to the in vitro mutated and in Escherichia coli expressed protein, the chromophore was found to exist as a mixture of blue (absorption maximum 615 nm) and red (532 nm) forms, depending on the pH . However, we found two widely separated pKa values (about 5.4 and 10.4 without added salt), arguing for two blue and two red forms in separate equilibria . Both blue and red forms of the protein are in the two-dimensional crystalline state . A single pKa, such as in the E . coli expressed protein, was observed only after solubilization with detergent . The photocycle of the blue forms was determined at pH 4.0 with 610 nm photoexcitation, and that of the red forms at pH 10.5 and with 520 nm photoexcitation, in the time-range of 100 ns to 1 s . The blue forms produced no M, but a K- and an L-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodopsin below pH 3 . The red forms produced a K-like intermediate, as well as M and N . Only the red forms transported protons . Specific perturbation of the neighborhood of the Schiff base by the replacement of Asp-85 with glutamate was suggested by (1) the shift and splitting of the pKa for what is presumably the protonation of residue 85, (2) a 36 nm blue-shift in the absorption of the all-trans red chromophore and a 25 nm red-shift of the 13-cis N chromophore, as compared to wild-type bacteriorhodopsin and its N intermediate, and (3) significant acceleration of the deprotonation of the Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.

Nature, 1992 Jan 30, 355(6359), 455 - 7
Different conformations for the same polypeptide bound to chaperones DnaK and GroEL; Landry SJ et al.; The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution . The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides . The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref . 3) and the intrinsic ATPase of DnaK . The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP . Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL . NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.

Biochemistry, 1992 Jan 28, 31(3), 806 - 10
Steady-state kinetics of the schistosomal hypoxanthine-guanine phosphoribosyltransferase; Yuan L et al.; Schistosomiasis is a trematode infection of some 200 million people . The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of the major etiologic agent, Schistosoma mansoni, has been proposed as a potential target for antischistosomal chemotherapy {Dovey, H . F., McKerrow, J . H., & Wang, C . C . (1984) Mol . Biochem . Parasitol, 11, 157-167} . The steady-state kinetic mechanism for the schistosomal HGPRTase has been determined by including both hypoxanthine and guanine in the forward and reverse reactions under identical conditions . Double-reciprocal plots of initial velocity versus the concentration of one substrate, at a series of fixed concentrations of the other, give groups of intersecting straight lines indicating a sequential mechanism for the schistosomal HGPRTase-catalyzed reactions . In product inhibition studies, the results show that magnesium pyrophosphate (MgPPi) is a noncompetitive inhibitor with respect to dimagnesium phosphoribose pyrophosphate (Mg2PRPP), hypoxanthine, and guanine . Also, magnesium inosine monophosphate (MgIMP) and magnesium guanosine monophosphate (MgGMP) are noncompetitive inhibitors with respect to hypoxanthine or guanine, respectively, but are competitive inhibitors to Mg2PRPP . Furthermore, Mg2PRPP is a competitive inhibitor with respect to MgIMP and MgGMP but is a non-competitive inhibitor to MgPPi . The minimum kinetic model which fits the experimental data is an ordered bi-bi mechanism, where the substrates bind to the enzyme in a defined order (first Mg2PRPP followed by the purine bases), while products are released in sequence (first MgPPi followed by MgIMP or MgGMP).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jan 28, 31(3), 799 - 805
Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli; Angeles TS et al.; The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies . Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant . The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates . In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates . This was unexpected since the beta-carboxyl group is the usual site of phosphorylation . The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy . These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor . Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway . This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway.

Biochemistry, 1992 Jan 28, 31(3), 792 - 8
Probing the regulatory site of Escherichia coli aspartate transcarbamoylase by site-specific mutagenesis; Zhang Y et al.; The effector binding site of Escherichia coli aspartate transcarbamoylase, composed of the triphosphate and ribose-base subsites, is located on the regulatory (r) chains of the enzyme . In order to probe the function of amino acid side chains at this nucleotide triphosphate site, site-specific mutagenesis was used to create three mutant versions of the enzyme . On the basis of the three-dimensional structure of the enzyme with CTP bound, three residues were selected . Specifically, Arg-96r was replaced with Gln, and His-20r and Tyr-89r were both replaced with Ala . Analyses of these mutant enzymes indicate that none of these substitutions significantly alter the catalytic properties of the enzyme . However, the mutations at His-20r and Tyr-89r produced altered response to the regulatory nucleotides . For the His-20r----Ala enzyme, the affinities of the enzyme for ATP and CTP are reduced 40-fold and 10-fold, respectively, when compared with the wild-type enzyme . Furthermore, CTP is able to inhibit the His-20r----Ala enzyme 40% more than the wild-type enzyme . In the case of the Tyr-89r----Ala enzyme . ATP can increase the mutant enzyme's activity 181% compared to 157% for the wild-type enzyme, while simultaneously the affinity of this enzyme for ATP decreases about 70% . These results suggest that Tyr-89r does have an indirect role in the discrimination between ATP and CTP . The His-20r----Ala enzyme shows no UTP synergistic inhibition in the presence of CTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jan 28, 31(3), 786 - 91
Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase; Lipman RS et al.; The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate {5,10-CH(+)-H4Pte(Glu)n} . Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10-CH(+)-H4folate) can be prepared in reconstitution experiments . Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism . The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10(-2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield {phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process} . With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jan 28, 31(3), 775 - 80
13C NMR investigation of the anomeric specificity of CMP-N-acetylneuraminic acid synthetase from Escherichia coli; Ambrose MG et al.; The anomeric specificity of Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was investigated by NMR using 13C-labeled N-acetylneuraminic acid (NeuAc) . Consumption of the beta-anomer of {2-13C}N-acetylneuraminic acid was observed upon addition of enzyme, with a concomitant appearance of an anomeric resonance for CMP-N-acetylneuraminic acid . Inhibition by substrate analogues the anomeric oxygen was determined in a similar manner using {2-13C,(50 atom %)18O}N-acetylneuraminic acid . An upfield shift of 1.5 Hz in the anomeric resonance of both the {13C}NeuAc substrate and CMP-{13C}NeuAc product was observed due to the 18O substitution . This result implies conservation of the NeuAc oxygen . Results of steady-state kinetic analysis suggest a sequential-type mechanism and therefore no covalent intermediate . Thus, CMP-beta-NeuAc is probably formed by a direct transfer of the anomeric oxygen of beta-NeuAc to the alpha-phosphate of CTP.

Biochemistry, 1992 Jan 28, 31(3), 725 - 32
Contribution of hydrogen bonding to the conformational stability of ribonuclease T1; Shirley BA et al.; For 30 years, the prevailing view has been that the hydrophobic effect contributes considerably more than hydrogen bonding to the conformational stability of globular proteins . The results and reasoning presented here suggest that hydrogen bonding and the hydrophobic effect make comparable contributions to the conformational stability of ribonuclease T1 (RNase T1) . When RNase T1 folds, 86 intramolecular hydrogen bonds with an average length of 2.95 A are formed . Twelve mutants of RNase T1 {Tyr----Phe (5), Ser----Ala (3), and Asn----Ala (4)} have been prepared that remove 17 of the hydrogen bonds with an average length of 2.93 A . On the basis of urea and thermal unfolding studies of these mutants, the average decrease in conformational stability due to hydrogen bonding is 1.3 kcal/mol per hydrogen bond . This estimate is in good agreement with results from several related systems . Thus, we estimate that hydrogen bonding contributes about 110 kcal/mol to the conformational stability of RNase T1 and that this is comparable to the contribution of the hydrophobic effect . Accepting the idea that intramolecular hydrogen bonds contribute 1.3 +/- 0.6 kcal/mol to the stability of systems in an aqueous environment makes it easier to understand the stability of the "molten globule" states of proteins, and the alpha-helical conformations of small peptides.

Biochemistry, 1992 Jan 28, 31(3), 886 - 91
Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase; al-Shawi MK et al.; (1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v) . Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax . The inhibition was rapidly reversed on dilution into aqueous buffer . (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM . This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic . (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed . The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site) . (4) Significant Pi binding to E . coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure . (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.

Biochemistry, 1992 Jan 28, 31(3), 878 - 85
Catalytic sites of Escherichia coli F1-ATPase . Characterization of unisite catalysis at varied pH; al-Shawi MK et al.; Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and {gamma-32P}ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound {32P}Pi equal to 3.5 x 10(-3) s-1, similar to previously published values . Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase {Noumi, T., Maeda, M., & Futai, M . (1987) FEBS Lett . 213, 381-384} . In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57 . These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly . The data justify the validity of previously published rate constants for unisite catalysis . Unisite catalysis in E . coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate . Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis . ATP binding was pH-independent; ATP release accelerated at higher pH . The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jan 28, 31(3), 780 - 6
Reduction of the small subunit of Escherichia coli ribonucleotide reductase by hydrazines and hydroxylamines; Gerez C et al.; Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and an antiferromagnetically coupled diferric center . Recent crystallographic studies {Nordlund, P., Eklund, H., & Sjoberg, B.-M . (1990) Nature 345, 593-598} have shown that both the radical and the diiron site are deeply buried inside the protein and thus strongly support the hypothesis of long-range electron-transfer processes within protein R2 . This study shows that monosubstituted hydrazines and hydroxylamines are able to reduce the tyrosyl radical and the ferric ions, under anaerobic conditions . It allows characterization of the site from which those compounds transfer their electrons to the iron/radical center . The efficiency of any given reducing agent is not solely governed by its redox potential but also by its size, its charge, and its hydrophobicity . We suggest, as a possible alternative to the long-range electron-transfer hypothesis, that conformational flexibility of the polypeptide chain might exist in solution and allow small molecules to penetrate the protein and react with the iron/radical center . This study also shows that two reduction mechanisms are possible, depending on which center, the radical or the metal, is reduced first . Full reduction of protein R2 yields reduced R2, characterized by a normal tyrosine residue and a diferrous center . Both the radical and the diferric center are regenerated from reduced R2 by reaction with oxygen, while only the diferric center is formed by reaction with hydrogen peroxide.

FEBS Lett, 1992 Jan 27, 296(3), 283 - 6
Function of the lipopolysaccharide-binding protein of Periplaneta americana as an opsonin; Jomori T et al.; Previously, we reported the purification of an LPS-binding protein from the hemolymph of the American cockroach that was specific for E . coli LPS . In this study we found that this protein participated in the clearance of E . coli cells injected into the abdominal cavity of the cockroach, and that hemocytes ingested E . coli cells treated with this LPS-binding protein in vitro . These findings suggest that this LPS-binding protein acts as an opsonin.

J Biol Chem, 1992 Jan 15, 267(2), 1231 - 8
Synthesis of precursor maltose-binding protein with proline in the +1 position of the cleavage site interferes with the activity of Escherichia coli signal peptidase I in vivo; Barkocy-Gallagher GA et al.; The residues occupying the -3 and -1 positions relative to the cleavage site of secretory precursor proteins are usually amino acids with small, neutral side chains that are thought to constitute the recognition site for the processing enzyme, signal peptidase . No restrictions have been established for residues positioned +1 to the cleavage site, although there have been several indications that mutant precursor proteins with a proline at +1 cannot be processed by Escherichia coli signal peptidase I (also called leader peptidase) . A maltose-binding protein (MBP) species with proline at +1, designated MBP27-P, was translocated efficiently but not processed when expressed in E . coli cells . Unexpectedly, induced expression of MBP27-P was found to have an adverse effect on the processing kinetics of five different nonlipoprotein precursors analyzed, but not precursor Lpp (the major outer membrane lipoprotein) processed by a different enzyme, signal peptidase II . Cell growth also was inhibited following induction of MBP27-P synthesis . Substitutions in the MBP27-P signal peptide that blocked MBP translocation across the cytoplasmic membrane and, hence, access to the processing enzyme or that altered the signal peptidase I recognition site at position -1 restored both normal growth and processing of other precursors . Since overproduction of signal peptidase I also restored normal growth and processing to cells expressing unaltered MBP27-P, it was concluded that precursor MBP27-P interferes with the activity of the processing enzyme, probably by competing as a noncleavable substrate for the enzyme's active site . Thus, although signal peptidase I, like many other proteases, is unable to cleave an X-Pro bond, a proline at +1 does not prevent the enzyme from recognizing the normal processing site . When the RBP signal peptide was substituted for the MBP signal peptide of MBP27-P, the resultant hybrid protein was processed somewhat inefficiently at an alternate cleavage site and elicited a much reduced effect on cell growth and signal peptidase I activity . Although the MBP signal peptide also has an alternate cleavage site, the different properties of the RBP and MBP signal peptides with regard to the substitution of proline at +1 may be related to their respective secondary structures in the processing site region.

J Biol Chem, 1992 Jan 15, 267(2), 855 - 63
The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase; Li SJ et al.; We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase . The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase . The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role . The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed . As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass . However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein . Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties . Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene . We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin . Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.

Nucleic Acids Res, 1992 Jan 25, 20(2), 337 - 41
The NH2-terminal arms of trp repressor participate in repressor/operator association; Hurlburt BK et al.; The 3-dimensional structure of the trp repressor, aporepressor, and repressor/operator complex have been described . The NH2-terminal arms of the protein, comprising approximately 12-14 residues, were not well resolved in any of these structures . Previous studies by Carey showed that the arms are required for full in vitro repressor activity . To examine the roles of the arms more fully we have removed codons 2-5 and 2-8 of the trpR gene and analyzed the resulting truncated repressors in vivo and in vitro . The delta 2-5 trp repressor was found to be approximately 25% as active as the wild type repressor in vivo . In in vitro equilibrium binding experiments, the delta 2-5 trp repressor was shown to be five-fold less active in operator binding . The rate of dissociation of the complex formed between the delta 2-5 trp repressor and operator was essentially the same as the rate of dissociation of the wild type trp repressor/operator complex . However association of the delta 2-5 trp repressor with operator was clearly defective . Since the NH2-terminal arms of the trp repressor appear to affect association predominantly they may play a role in facilitating non-specific association of repressor with DNA as repressor seeks its cognate operators . The delta 2-8 trp repressor was unstable in vivo and in vitro, suggesting that some portion of the NH2-terminal arm is required for proper folding of the remainder of the molecule.

Nucleic Acids Res, 1992 Jan 25, 20(2), 257 - 61
Promoter selectivity of the stationary-phase forms of Escherichia coli RNA polymerase and conversion in vitro of the S1 form enzyme into a log-phase enzyme-like form; Ozaki M et al.; Upon growth transition of Escherichia coli cells from exponential to stationary phase, RNA polymerase is converted into at least three different forms (S1, S2 and S3), which could be separately isolated by phosphocellulose column chromatography (Ozaki et al., 1991 (2)) . Here, the promoter selectivity of these three stationary-phase enzymes was examined using an in vitro mixed transcription system and an E . coli promoter collection . These altered forms of RNA polymerase showed different recognition properties of promoters from that by the log-phase holoenzyme (L1) . One of the stationary-phase RNA polymerases, S1, was found to be converted in vitro into an enzyme like the log-phase form following incubation with nucleotides or pyrophosphate . The conversion was indicated by not only the shift of elution position from a phosphocellulose column but also the change in the promoter selectivity . These results may suggest that RNA polymerase is interconvertible between different forms with different promoter selectivity by interaction with a phosphorylated compound(s).

Nucleic Acids Res, 1992 Jan 25, 20(2), 179 - 86
Purification and biochemical characterisation of the EcoR124 type I modification methylase; Taylor I et al.; Large scale purification of the type I modification methylase EcoR124 has been achieved from an over-expressing strain by a two step procedure using ion-exchange and heparin chromatography . Pure methylase is obtained at a yield of 30 mg per gm of cell paste . Measurements of the molecular weight and subunit stoichiometry show that the enzyme is a trimeric complex of 162 kDa consisting of two subunits of HsdM (58 kDa) and one subunit of HsdS (46 kDa) . The purified enzyme can methylate a DNA fragment bearing its cognate recognition sequence . Binding of the methylase to synthetic DNA fragments containing either the EcoR124 recognition sequence GAAN6RTCG, or the recognition sequence GAAN7RTCG of the related enzyme EcoR124/3, was followed by fluorescence competition assays and by gel retardation analysis . The results show that the methylase binds to its correct sequence with an affinity of the order 10(8) M-1 forming a 1:1 complex with the DNA . The affinity for the incorrect sequence, differing by an additional base pair in the non-specific spacer, is almost two orders of magnitude lower.

J Biol Chem, 1992 Jan 25, 267(3), 2080 - 6
The site of hydrolysis by rabbit reticulocyte peptidyl-tRNA hydrolase is the 3'-AMP terminus of susceptible tRNA substrates; Gross M et al.; The preceding paper (Gross, M., Starn, T.K., Rundquist, C., Crow, P., White, J., Olin, A., and Wagner, T . (1992) J . Biol . Chem . 267, 2073-2079) reported the purification and partial characterization of rabbit reticulocyte peptidyl-tRNA hydrolase . In this article we demonstrate that, unlike bacterial and yeast peptidyl-tRNA hydrolase which act by deacylation, the reticulocyte enzyme hydrolyzes N-acylaminoacyl-tRNA to N-acylaminoacyl-AMP . Reticulocyte lysate has a separate enzyme, that we have isolated and termed aminoacyl-AMP deacylase, which hydrolyzes N-acylaminoacyl-AMP and aminoacyl-AMP, recycling the amino acid and nucleotide components . The action of this enzyme is relatively specific for the N-acylaminoacyl-AMP generated by peptidyl-tRNA hydrolase, since it is much less active with N-acylaminoacyl-adenosine and inactive with N-acylaminoacyl-ACCAC, N-acylaminoacyl-tRNA, or aminoacyl-tRNA . The tRNA product of peptidyl-tRNA hydrolase action is tRNA missing only its 3'-AMP terminus (tRNA(c-c)), since reaminoacylation requires tRNA nucleotidyltransferase but not CTP . The 3' exonucleolytic action of reticulocyte peptidyl-tRNA hydrolase is specific to susceptible tRNA substrates, since it does not hydrolyze CACCA, CACCA-N-acylamino acid, polyuridylic acid, or the 3' polyadenylate tail of globin mRNA, and, since its ability to hydrolyze Escherichia coli f{3H}Met-tRNA(fMet) is not reduced by excess 5 S or 28 S ribosomal RNA and is reduced only slightly by excess tRNA(c-c) . Reticulocyte peptidyl-tRNA hydrolase also hydrolyzes th 3'-AMP terminus of deacylated tRNA . This property may explain why the 3'-terminal AMP of tRNA undergoes turnover in reticulocytes and reticulocyte lysate, since we find that such turnover in gel-filtered reticulocyte lysate is increased under conditions where aminoacylation is reduced.

J Biol Chem, 1992 Jan 25, 267(3), 2038 - 45
Multidegenerate DNA recognition by the OxyR transcriptional regulator; Tartaglia LA et al.; The Escherichia coli OxyR protein, a regulator of hydrogen peroxide-inducible genes, is a potent stimulator of transcription in its oxidized form but not in its reduced form . OxyR protein purified in its oxidized form was found to bind four of its non-homologous, functional DNA-binding sites with over 10(6)-fold higher affinity than random DNA sequences . A similarly high DNA binding specificity was observed for the reduced (transcriptionally inactive) form of OxyR, consistent with a model in which the OxyR protein is bound to its recognition sequences even in the absence of an oxidative stress . Alignment of five functional OxyR-binding sites revealed a marked lack of perfectly conserved positions, yet an unusually high number of degenerate homologies (positions at which only two of the four possible base pairs are represented) . Methylation interference assays on two OxyR-binding sites showed that OxyR contacts its recognition sequences predominantly at positions of degenerate homology . These results suggest that the OxyR protein specifically recognizes seemingly dissimilar sequences through the use of a multidegenerate recognition code . The chemical basis for a plausible degenerate recognition system is discussed.

J Biol Chem, 1992 Jan 25, 267(3), 1881 - 7
Cloning and expression in Escherichia coli of mature E1 beta subunit of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex . Mapping of the E1 beta-binding region on E2; Wynn RM et al.; A cDNA encoding the mature E1 beta subunit of the bovine branched-chain alpha-keto acid dehydrogenase complex was isolated from a lambda ZAP expression library . The bovine E1 beta cDNA is 1,393 base pairs in length . It encodes the entire mature E1 beta subunit consisting of 342 amino acid residues and a partial mitochondrial targeting presequence of 26 residues . The calculated molecular mass of the mature bovine E1 beta subunit is 37,776 daltons, and the calculated isoelectric point is pI 5.04 . The mature bovine E1 beta subunit was expressed in Escherichia coli via the pKK233-2 vector in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG) . When expression was induced by IPTG at 37 degrees C, the soluble recombinant E1 beta subunit existed as a single high molecular weight form (Mr congruent to 3.5 x 10(5)), which sedimented during sucrose gradient ultracentrifugation at 2 x 10(5) x g . However, lowering the induction temperature to 25 degrees C resulted in the occurrence of both high and low molecular weight forms of the recombinant E1 beta protein . The low molecular weight form (Mr congruent to 9.1 x 10(4)) remained soluble after sucrose gradient centrifugation and was utilized in binding studies with a series of truncated recombinant E2 proteins . The results showed that the E1 beta subunit bound to the region between Ala-115 and Lys-150 of the E2 chain, which lay within the putative E3-binding domain . In contrast, the recombinant E1 alpha subunit did not bind the E2 component . The data suggest an apparent binding order of E2-E1 beta-E1 alpha, which supports and extends the model of E2 inner core deduced previously from the data of scanning transmission electron microscopy (Hackert, M.L., Xu, W.-X., Oliver, R.M., Wall, J.S., Hainfeld, J.F., Mullinax, T.R., and Reed, L.J . (1989) Biochemistry 28, 6816-6821) . The relatively inaccessible topology of E1 beta may explain the lack of antigenicity and resistance to limited proteolysis of this subunit as it exists in the complex.

J Biol Chem, 1992 Jan 25, 267(3), 1776 - 85
Competing B-Z and helix-coil conformational transitions in supercoiled plasmid DNA; Aboul-ela F et al.; The formation of melted regions from A + T-rich sequences and left-handed Z-DNA by alternating purine-pyrimidine sequences will both be facilitated by negative supercoiling, and thus if the sequences are present within the same plasmid molecule they will compete for the free energy of supercoiling . We have studied a series of plasmids that contain either (CG)8 or (TG)12 sequences in either G + C or A + T-rich contexts, by means of two-dimensional gel electrophoresis and chemical modification . We observe both B-Z and helix-coil transitions in all plasmids at elevated temperatures and low ionic strength . The plasmids fall into a number of different classes, in terms of the conformational behavior . As the superhelix density is increased, pCG8/vec ((CG)8 in G + C-rich context) undergoes an initial B-Z transition, followed by melting transitions in sequences remote from the (CG)8 sequence . The two transitions are coupled through the topology of the molecule but are otherwise independent . When the (CG)8 sequence was placed in an A + T-rich context (pCG8/col), the helix-coil transition was perturbed by the presence of the Z-DNA segment . Replacement of the (CG)8 tracts by (TG)12 sequences resulted in a further level of interaction between the transitions . Statistical mechanical modeling of the transitions suggested that at intermediate levels of negative supercoiling the Z-DNA formed by the (TG)12 sequence has a lowered probability due to the helix-coil transition in the A + T-rich sequences . These studies illustrate the complexities of competing conformational equilibria in supercoiled DNA molecules.

J Biol Chem, 1992 Jan 25, 267(3), 1727 - 32
Alteration by site-directed mutagenesis of the conserved lysine residue in the ATP-binding consensus sequence of the RecD subunit of the Escherichia coli RecBCD enzyme; Korangy F et al.; The RecD subunit of the RecBCD enzyme from Escherichia coli contains an amino acid sequence common to many enzymes which bind ATP or GTP (Gly-X-X-Gly-X-Gly-Lys-Thr) . We have changed the conserved lysine residue (amino acid number 177) in the RecD protein to glutamine to investigate the role of RecD, and ATP-binding to RecD, in the enzymatic activities of RecBCD . The mutant RecD protein assembles with the RecB and RecC subunits and the mutant enzyme, designated RecBCD-K177Q, can be purified in the same way as the wild-type RecBCD enzyme . The mutant RecD subunit in RecBCD-K177Q is photolabeled to a lesser extent by the ATP analogue 8-azido-adenosine-5'-triphosphate than is the wild-type RecD subunit in RecBCD, suggesting that the mutation has reduced the affinity of RecD for ATP.

J Biol Chem, 1992 Jan 25, 267(3), 1615 - 22
Vibrational spectroscopy of bacteriorhodopsin mutants . Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds; Rothschild KJ et al.; The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time-resolved visible absorption spectroscopy of site-directed mutants . Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin . The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type . During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature . In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89 . Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212 . In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha . These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212 . In addition, a transient interaction between Thr-46 and Asp-96 occurs early in the photocycle . In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues . This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin.

J Biol Chem, 1992 Jan 25, 267(3), 1484 - 90
Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli; Zhang AJ et al.; The catalytic subunit of rabbit skeletal muscle protein phosphatase-1 was expressed in Escherichia coli . Expression of phosphatase-1 in the pET3a vector, which is based on the use of the T7 promoter, resulted in the expression of the enzyme as an insoluble aggregate . The insoluble enzyme could be renatured by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn2+, and high NaCl concentrations . However, under all conditions tested, only partial (less than 5%) renaturation was achieved . A second attempt was made using a vector with the trp-lac hybrid promoter . In this case it was possible to express the enzyme as a soluble protein at levels of 3-4% of the soluble E . coli protein . The recombinant enzyme was purified by DEAE-Sepharose and heparin-Sepharose chromatography . Approximately 20 mg of purified enzyme was reproducibly obtained from the cells derived from 2 liters of culture . The purified enzyme had a specific activity toward phosphorylase alpha comparable to that reported for the authentic protein and had an Mr of 37,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid, and microcystin-LR as for the protein isolated from rabbit muscle . At all stages of purification the recombinant phosphatase behaved as an essentially inactive enzyme that required the presence of microM Mn2+ for full expression of its activity.

J Biol Chem, 1992 Jan 25, 267(3), 1415 - 8
Asparagine 26, glutamic acid 31, valine 45, and tyrosine 64 of Ras proteins are required for their oncogenicity; Nur-E-Kamal MS et al.; Ras and Rap1 proteins are related GTP-dependent signal transducers which require Gly-12, the effector domain (residues 32-40), and Ala-59 for stimulation of their GTPase activities by GAP1 and GAP3, respectively . The replacement of Gly-12 by Val or Ala-59 by Thr potentiates the Ras oncogenicity and Rap1A antioncogenicity . However, the mutations in the effector domain, in particular the replacement of Thr-35 by Ala, abolish both Ras oncogenicity and Rap1A antioncogenicity, indicating that the effector domain is involved in interactions of these signal transducers with their targets as well as the GAPs . In this paper, we demonstrate that (i) replacement of Tyr-64 of the Ha-Ras protein or Phe-64 of the Rap1A protein by Glu or other non-hydrophobic amino acids reduces their intrinsic GTPase activities and abolishes their stimulation by GAP1 or GAP3, respectively, (ii) replacement of Tyr-64 by Gly and other non-hydrophobic amino acids results in complete loss of the oncogenicity of the v-Ha-Ras protein, indicating that the hydrophobic residue 64, in addition to the known effector domain, is essential for the Ras protein to interact with its target as well as GAP1 . In addition we have found that Asn-26, Glu-31, and Val-45 of the v-Ha-Ras protein are required for its oncogenicity . Replacement of the Ras residues at either positions 26, 31, or 45 by the corresponding Rap1A residues abolishes the Ras oncogenicity.

J Biol Chem, 1992 Jan 25, 267(3), 1407 - 10
Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli; Sweasy JB et al.; Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA . Since some catalytic functions of DNA polymerase beta and E . coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria . We found that the expression of mammalian DNA polymerase beta in E . coli restored growth in a DNA polymerase I-defective bacterial mutant . Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments . These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication . Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.

J Biol Chem, 1992 Jan 25, 267(3), 1712 - 8
On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase; Weber J et al.; 1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F1-ATPase . The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F1, with Kd1 = 0.20 microM and Kd2,3 = 5.5 microM . lin-Benzo-ATP was a good substrate for hydrolysis . 2) The mutants investigated were beta Y331F, L, A and E . kcat/KM for ATP hydrolysis in purified F1 was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen . 3) Fluorescence properties of lin-benzo-ADP bound to F1 differed widely, depending on the residue present at position beta 331 . Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E . There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E . 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same . 5) F1 from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal . Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.

J Biol Chem, 1992 Jan 25, 267(3), 1455 - 63
Molecular cloning and enzymatic analysis of the rat homolog of "PhK-gamma T," an isoform of phosphorylase kinase catalytic subunit; Calalb MB et al.; Messenger RNA encoding a protein kinase closely related to the catalytic subunit of skeletal muscle phosphorylase kinase has previously been isolated from a human HeLa cell cDNA library, and cross-species Northern hybridization analysis has shown that the rat homolog of this transcript is abundant in the adult testis (Hanks, S.K . (1989) Mol . Endocrinol . 3, 110-116) . We now propose that the protein encoded by this transcript be designated as "PhK-gamma T." In this article, the primary structure of the rat homolog of PhK-gamma T is described, as deduced from nucleotide sequences of cDNA and genomic clones . RNase protection analysis reveals that PhK-gamma T transcripts are actually present in a wide variety of adult rat tissues, but at levels 20-100-fold less than what is observed in the testis . In the testis, transcription of the PhK-gamma T gene is initiated at multiple sites as shown by RNase protection and primer extension . Enzymatic activity of PhK-gamma T was demonstrated using renatured bacterially expressed protein . In the presence of Ca2+/calmodulin, PhK-gamma T is able to efficiently phosphorylate glycogen phosphorylase and convert it from an inactive to an active form . We conclude that PhK-gamma T represents a true isoform of phosphorylase kinase catalytic subunit.

J Biol Chem, 1992 Jan 25, 267(3), 1449 - 54
The NusA and NusG proteins of Escherichia coli increase the in vitro readthrough frequency of a transcriptional attenuator preceding the gene for the beta subunit of RNA polymerase; Linn T et al.; The genes for the beta (rpoB) and beta' (rpoC) subunits of Escherichia coli RNA polymerase are the distal members of a complex transcriptional unit that contains four upstream ribosomal protein genes . The RNA polymerase subunit genes are transcribed at a lower frequency than the ribosomal protein genes as a result of termination at an attenuator preceding rpoB . A purified in vitro transcription system was developed using linear DNA templates that carry the attenuator . The ability of known termination and antitermination proteins to modulate termination at the attenuator was tested . Both NusA and NusG increase the frequency of transcriptional readthrough at the attenuator whereas NusB, S10, and Rho had no significant effect in this system.

J Biol Chem, 1992 Jan 25, 267(3), 1872 - 80
Oxidative modification of Escherichia coli glutamine synthetase . Decreases in the thermodynamic stability of protein structure and specific changes in the active site conformation; Fisher MT et al.; Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation . After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis . The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer . This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer . The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding . Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements . Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer . Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined . This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively . Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.

J Biol Chem, 1992 Jan 25, 267(3), 1491 - 5
Different positively charged amino acids have similar effects on the topology of a polytopic transmembrane protein in Escherichia coli; Andersson H et al.; Integral membrane proteins from a wide variety of sources conform to a "positive-inside rule," with many more positively charged amino acids in their cytoplasmic as compared to extracytoplasmic domains . A growing body of experimental work also points to positively charged residues in regions flanking the apolar transmembrane segments as being the main topological determinants . In this paper, we report a systematic comparison of the effects of positively (Arg, Lys, His) as well as negatively (Asp, Glu) charged residues on the membrane topology of a model Escherichia coli inner membrane protein . Our results show that positive charge is indeed the major factor determining the transmembrane topology, with Arg and Lys being of nearly equal efficiency . His, although normally a very weak topological determinant, can be potentiated by a lowering of the cytoplasmic pH . Asp and Glu affect the topology to similar extents and only when present in very high numbers.

J Biol Chem, 1992 Jan 25, 267(3), 2105 - 13
Determination of the ligands of the low spin heme of the cytochrome o ubiquinol oxidase complex using site-directed mutagenesis; Lemieux LJ et al.; The cytochrome o complex of Escherichia coli is a ubiquinol oxidase which is the predominant respiratory terminal oxidase when the bacteria are grown under high oxygen tension . The amino acid sequences of three of the subunits of this quinol oxidase reveal a substantial relationship to the aa3-type cytochrome c oxidases . The two cytochrome components (b563.5 and o) and the single copper (CuB) present in the E . coli quinol oxidase appear to be equivalent to cytochrome a, cytochrome a3, and CuB of the aa3-type cytochrome c oxidases, respectively . These three prosthetic groups are all located within subunit I of the oxidase . Sequence alignments indicate only six totally conserved histidine residues among all known sequences of subunit I of the cytochrome c oxidases of various species plus the E . coli quinol oxidase . Site-directed mutagenesis has been used to change each of these totally conserved histidines with the presumption that two of these six must ligate to the low spin cytochrome center of the E . coli oxidase . The presence of the low spin cytochrome b563.5 component of the oxidase can be evaluated both by visible absorbance properties and by its EPR spectrum . The results unambiguously indicate that His-106 and His-421 are the ligands of the six-coordinate low spin cytochrome b563.5 . Although the data are not definitive in making additional metal ligation assignments of the remaining four totally conserved histidines, a reasonable model is suggested for the structure of the catalytic core of the cytochrome o complex and, by extrapolation, of cytochrome c oxidase.

J Biol Chem, 1992 Jan 25, 267(3), 2096 - 104
Identification of heme and copper ligands in subunit I of the cytochrome bo complex in Escherichia coli; Minagawa J et al.; The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli (Kita, K., Konishi, K., and Anraku, Y . (1984) J . Biol . Chem . 259, 3368-3374) and functions as a proton pump . It belongs to the heme-copper oxidase superfamily with the aa3-type cytochrome c oxidases in mitochondria and aerobic bacteria . In order to identify ligands of hemes and copper, we have substituted eight conserved histidines in subunit I by alanine and, in addition, His-106, -284, and -421 by glutamine and methionine . Western immunoblotting analysis showed that all the mutations do not affect the expression level of subunit I in the cytoplasmic membrane, indicating that these histidines are not crucial for its stability . A single copy expression vector carrying a single mutation at the invariant histidines, His-106, His-284, His-333, His-334, His-419, and His-421, of subunit I was unable to support the aerobic growth of a strain in which the chromosomal terminal oxidase genes (the cyo and cyd operons) have been deleted . The same mutations caused a complete loss of ubiquinol oxidase activity of the partially purified enzymes . Spectroscopic analysis of mutant oxidases in the cytoplasmic membrane revealed that substitutions of His-106 and -421 specifically eliminated a 563.5 nm peak of the low spin heme and that replacements of His-106, -284, and -419 reduced the extent of the CO-binding high spin heme . These spectroscopic properties of mutant oxidases were further confirmed with partially purified preparations . Atomic absorption analysis showed that substitutions of His-106, -333, -334, and -419 eliminated CuB almost completely . Based on these findings, we conclude that His-106 and -421 function as the axial ligands of the low spin heme and His-284 is a possible ligand of the high spin heme . His-333, -334, and -419 residues are attributed to the ligands of CuB . We present a helical wheel model of the redox center in subunit I, which consists of the membrane-spanning regions II, VI, VII, and X, and discuss the implications of the model.

J Biol Chem, 1992 Jan 25, 267(3), 1945 - 52
The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis; Berens C et al.; The N-terminal residues preceding the alpha-helix-turn-alpha-helix motif on the Tn10 Tet repressor protein were probed by oligonucleotide-directed deletion mutagenesis for their role in protein activity . All deletion mutants showed decreased repression in vivo, emphasizing the importance of the N terminus for tet operator binding . Only two of the mutants, TetR delta 2-23 and TetR delta 3-8 displayed a reduced intracellular protein level . The remaining deletion mutants showed either reduced binding to tet operator and inducibility by tetracycline or transdominance . We conclude that these deletions do not affect stability and overall protein structure . DNA binding activities of residue-wise increasing deletions, TetR delta 9 through TetR delta 9-13, reveal a pattern consistent with an alpha-helical structure of the affected residues . This conclusion is supported by the helical wheel projection and the hydrophobic moment profile calculated for the protein segment ranging from residues S7-V20 . We propose that these residues form an amphipathic alpha-helix which packs closely against the alpha-helix-turn-alpha-helix motif and is essential for Tet repressor activity . The residues preceding this putative alpha-helix contribute to DNA binding, but no direct interactions with base pairs of tet operator were revealed in a loss of contact analysis . Individual mutation of the 4 charged residues to alanine at the N terminus shows that no single residue can account for the reduction in repression observed for the deletion mutants.

J Biol Chem, 1992 Jan 25, 267(3), 1733 - 40
Enzymatic effects of a lysine-to-glutamine mutation in the ATP-binding consensus sequence in the RecD subunit of the RecBCD enzyme from Escherichia coli; Korangy F et al.; The RecBCD-K177Q enzyme has a lysine-to-glutamine mutation in the putative ATP-binding sequence of the RecD protein (Korangy, F., and Julin, D.A . (1992) J . Biol . Chem . 267, 1727-1732) . We have compared the enzymatic properties of the RecBCD-K177Q enzyme with those of the wild-type RecBCD enzyme from Escherichia coli . The purified RecBCD-K177Q enzyme has ATP-dependent nuclease activity on double-stranded or denatured DNA which is reduced (4-14-fold less) compared with the wild type . The kcat and Km(ATP) for ATP hydrolysis stimulated by double-stranded DNA are both reduced in RecBCD-K177Q, so that kcat/Km(ATP) is relatively unaffected . The mutant enzyme is impaired in its ability to unwind DNA in an assay where single-stranded DNA is trapped by the single-stranded DNA binding protein and subsequently degraded by S1 nuclease . The mutant enzyme also produces fewer acid-soluble DNA nucleotides per ATP hydrolyzed than does the wild type, at low ATP concentrations (less than 20 microM).

J Biol Chem, 1992 Jan 25, 267(3), 1705 - 11
Cloning of the vaccinia virus ribonucleotide reductase small subunit gene . Characterization of the gene product expressed in Escherichia coli; Howell ML et al.; During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K . (1984) J . Virol . 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K . (1984) J . Virol . 52, 507-514) . We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system . After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein . The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution . Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract . A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure . Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy . The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells . Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E . coli R2 . By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer . Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.

J Biol Chem, 1992 Jan 25, 267(3), 1411 - 4
Purification and characterization of the carboxyl-terminal transactivation domain of Vmw65 from herpes simplex virus type 1; Donaldson L et al.; A glutathione S-transferase fusion to the COOH-terminal acidic transactivation domain of Vmw65 from herpes simplex virus type 1 was overexpressed in Escherichia coli and isolated by affinity chromatography on glutathione-Sepharose . Following cleavage of the fusion protein with thrombin, the transactivation domain was purified to homogeneity by ion exchange chromatography yielding approximately 0.6 mg of protein/liter of bacterial culture . Equilibrium sedimentation analysis showed the purified polypeptide to be monomeric; however, it displayed aberrant electrophoretic and chromatographic properties . Contrary to secondary structure predictions, circular dichroism spectroscopy demonstrated that this transactivation domain was devoid of significant alpha-helical structure at physiological conditions . The polypeptide, however, became notably more structured under hydrophobic conditions or at low pH, suggesting that it was sensitive to its environment . Near-UV circular dichroism suggested that phenylalanyl and tyrosyl residues were under influence from tertiary structure.

J Med Chem, 1992 Jan 24, 35(2), 397 - 401
Synthesis and biologic activity of 2'-fluoro-2-halo derivatives of 9-beta-D-arabinofuranosyladenine; Montgomery JA et al.; The synthesis of 2-halo-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenines (4b and 4d) by coupling the 2,6-dihalopurine with 3-acetyl-5-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl bromide (2) followed by replacement of the 6-halogen with concomitant removal of the acyl blocking groups is described . 2-Fluoroadenine derivative 4g had to be prepared by the diazotization-fluorination of 2-aminoadenine nucleoside 4e . All three nucleosides provided good increases in life span of mice inoculated with P388 leukemia . The best results were obtained when the compounds were administered q3h x 8 on days 1, 5, and 9 after implantation of the leukemia cells . The 2',3'-dideoxynucleoside 5b, prepared by deacetylation of 4f and deoxygenation of the resultant 4h followed by removal of the benzoyl group of 5a, was slightly active against HIV in cell culture.

Biochim Biophys Acta, 1992 Jan 24, 1123(2), 191 - 7
Photoaffinity cross-linking of acyl carrier protein to Escherichia coli membranes; Bayan N et al.; Acyl Carrier Protein (ACP) is a small acidic protein which interacts with the various enzymes implicated in the biosynthesis of fatty acids in E . coli . It also interacts with the inner membrane proteins implicated in the biosynthesis of phospholipids . Samples of radioactive ACP were prepared with high specific activities and bearing photoactivable aryl azide derivatives . Two photoactivable reagents were used: para azido phenacyl bromide (pAPA) which reacts with the SH of the ACP prosthetic group and the N-hydroxysuccinimide ester of 4-azido salicylic acid (NHS-ASA) which reacts with the amino groups of the protein . Various methods were used to demonstrate that ACP could be cross-linked specifically to an inner membrane protein of E . coli, most probably to the glycerol-3-phosphate acyl transferase (GPAT) . This covalent link should provide a powerful tool for further analysis of the structure of GPAT and its role in phospholipid biosynthesis . These photoactivable aryl azide derivatives of ACP could also be very useful for studying the interaction of ACP with the soluble enzymes implicated in fatty acid biosynthesis.

J Immunol Methods, 1992 Jan 21, 146(1), 25 - 32
Escherichia coli heat-stable enterotoxin (STa)-biotin conjugates for the titration of STa antisera by an enzyme-linked immunosorbent assay; Germani Y et al.; The development of a new approach to the diagnosis of infectious diarrhoea, caused by Escherichia coli heat-stable enterotoxin (ST), was preceded by a preliminary study . The purpose of the latter was to establish whether three preparations of ST produced by a human isolate of enterotoxigenic E . coli (STa), obtained at different steps of the purification procedure (involving Amberlite XAD2 resin chromatography (P3), a gel filtration chromatography on a Biogel P4 (P2) or a disc-gel electrophoresis (P1)), could be employed to titrate antisera to STa using an ST-biotin enzyme-linked immunosorbent assay (ELISA) . The solid-phase STa was obtained by first coupling the toxin to biotinyl-N-hydroxysuccinimide and then binding this conjugate to avidin adsorbed to flat-bottomed polystyrene microtitre plates . Using these reagents, the assay conditions were examined . Checkerboard tests determined optimal biotin-P3, P2 or P1 toxin conjugate concentrations to be used as the immunosorbent for P3, P2 and P1 antiserum titration . The immunosorbent prepared with STa purified only on Amberlite XAD2 resin was unable to differentiate significantly between P3, P2 or P1 antisera . Immunosorbent prepared with P2 or P1 detected widely differing titres between the three antisera and gave more sensitive results . Only small but questionable differences were observed between P2 and P1 toxin preparations.

Biochemistry, 1992 Jan 21, 31(2), 603 - 9
Enzymatic synthesis and structure of precorrin-3, a trimethyldipyrrocorphin intermediate in vitamin B12 biosynthesis; Warren MJ et al.; The trimethylated intermediate of vitamin B12 (corrin) biosynthesis, precorrin-3, was produced from various 13C-enriched isotopomers of 5-aminolevulinic acid (ALA), using a multiple-enzyme system containing ALA dehydratase, porphobilinogen deaminase, uro'gen III synthetase, and the S-adenosyl-L-methionine-(SAM)-dependent uro'gen III methyltransferase (M-1) and precorrin-2 methyltransferase (M-2) in the presence of {13C}SAM . Structural analysis of the resulting product, precorrin-3, reveals a close similarity to precorrin-2 but with several subtle differences in the conjugated array of C = C and C = N bonds which reflect the presence of the new C-methyl group at C20 and its influence on the electronic distribution in the dipyrrocorphin chromophore . The implications of this structure for corrin biosynthesis are discussed.

Biochemistry, 1992 Jan 21, 31(2), 519 - 25
Amino acid substitutions in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that influence catalytic activity of the holoenzyme; Read BA et al.; Four unique amino acid substitutions were introduced by site-directed mutagenesis into the third conserved region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans (Synechococcus sp., PCC6301), resulting in the formation of four mutant enzymes, I87V, R88K, G91V, and F92L . Wild-type and mutant proteins were purified after synthesis in Escherichia coli . These single amino acid substitutions do not appear to perturb intersubunit interactions or induce any gross conformational changes; purified mutant proteins are stable, for the most part like the wild-type holoenzyme, and exhibit nearly identical CD spectra . Three of the four mutants, however, are severely deficient in carboxylase activity, with kcat less than or equal to 35% of the wild-type enzyme . While the substrate specificity factors were the same for the mutant and wild-type enzymes, significant alterations in some kinetic parameters were observed, particularly in the Michaelis constants for CO2, O2, and RuBP . All four mutant proteins exhibited lower KCO2 values, ranging from 37 to 88% of the wild-type enzyme . Two of the mutants, in addition, exhibited significantly lower KRuBP values, and one mutant showed a substantial decrease in KO2 . The effects of the single-site mutations in rbcS of this study strengthen the hypothesis that small subunits may not contribute directly to substrate specificity; however, individual residues of the small subunit substantially influence catalysis by large subunits.

Biochemistry, 1992 Jan 21, 31(2), 475 - 81
Purification, characterization, and partial sequence of the glutathione-dependent formaldehyde dehydrogenase from Escherichia coli: a class III alcohol dehydrogenase; Gutheil WG et al.; The glutathione-dependent formaldehyde dehydrogenase from Escherichia coli has been purified to homogeneity and characterized . It is a 83,000-kDa homodimer containing 4 g-atom of zinc per dimer with a specific activity of 60 units/mg toward S-(hydroxymethyl)glutathione and NAD+ as substrates . Its isoelectric point, 4.4, is consistent with both its amino acid composition and chromatographic behavior on DEAE HPLC . The N-terminus is unblocked, and 47 residues from the N-terminus were sequenced . A computer search of the Swiss-Prot protein sequence data bank shows that the N-terminal sequence, {sequence; see text}, is homologous with the mammalian class III alcohol dehydrogenases with 27 identities when compared to the human enzyme . Like the human, rat, and rabbit enzymes, it has high formaldehyde dehydrogenase activity in the presence of glutathione and catalyzes the oxidation of normal alcohols (ethanol, octanol, 12-hydroxydodecanoate) in a reaction that is not GSH-dependent . In addition, hemithiolacetals other than those formed from GSH, including omega-thiol fatty acids, also are substrates . The wide distribution and high degree of similarity of this enzyme to the plant and animal alcohol dehydrogenases suggest that the E . coli enzyme is closely related to the ancestor of the plant and animal dimeric zinc alcohol dehydrogenases.

Biochemistry, 1992 Jan 21, 31(2), 350 - 9
Metal activation of synthetic and degradative activities of phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication; Esteban JA et al.; Analysis of metal activation on the synthetic and degradative activities of phi 29 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment) . In the three DNA polymerases studied, both the polymerization and the 3'----5' exonuclease activity had clear differences in their metal ion requirements . The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi 29 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3'----5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and phi 29 DNA polymerase . Furthermore, DNA competition experiments using phi 29 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzyme-DNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases . Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi 29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator . The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP.

J Mol Biol, 1992 Jan 20, 223(2), 477 - 507
Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution; Wilmanns M et al.; The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation . The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules . The final R-factor is 17.3% for all observed data between 15 and 2 A resolution . The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively . The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E . coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs . The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis . The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size . The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate . These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein . The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates . These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E . coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.

FEBS Lett, 1992 Jan 20, 296(2), 179 - 83
The precursor form of the rat liver non-specific lipid-transfer protein, expressed in Escherichia coli, has lipid transfer activity; Ossendorp BC et al.; The cDNA encoding the precursor form of non-specific lipid-transfer protein (pre-nsL-TP) from rat liver was cloned into the expression vector pET3d . The resulting plasmid was transformed to the Escherichia coli strain BL21(DE3) . After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside (IPTG) pre-nsL-TP was purified from the bacterial lysate by anion exchange chromatography followed by gelfiltration . From 11 of culture, 6-7 mg of pre-nsL-TP was obtained, equal to approximately 7% of the cytoplasmic protein . By use of a fluorescence lipid transfer assay, pre-nsL-TP was found to have lipid transfer activity identical to mature nsL-TP.

Nature, 1992 Jan 16, 355(6357), 273 - 5
MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis; Maki H et al.; Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low . We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis . This error-avoiding process is catalysed by a protein encoded by the mutT gene of Escherichia coli, mutations of which increase the occurrence of A.T----C.G transversions 100 to 10,000 times the level of the wild type . Spontaneous oxidation of dGTP forms 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP), which is inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein specifically degrades 8-oxodGTP to the monophosphate . This indicates that elimination from the nucleotide pool of the oxidized form of guanine nucleotide is important for the high fidelity of DNA synthesis.

Biochem Biophys Res Commun, 1992 Jan 15, 182(1), 412 - 9
Cloning and characterization of a highly conserved HMG-like protein (PF16) gene from Plasmodium falciparum; Guntaka RV et al.; A novel gene encoding a protein of 147 amino acids (Pf16) has been cloned from Plasmodium falciparum and expressed in E . coli . The protein contains 19 methionines, all of which are localized in the NH2-terminal 35 amino acid residues, and it is also rich in lysine . Pf16 is highly basic, contains a polyacidic domain consisting of aspartic acid and is related to the non-histone high mobility group proteins of higher eukaryotes . The gene is conserved among eight different species of Plasmodium so far examined, suggesting an important function for this gene product in the parasite's life cycle.

Biochem Biophys Res Commun, 1992 Jan 15, 182(1), 355 - 60
Drosophila glutathione S-transferases have sequence homology to the stringent starvation protein of Escherichia coli; Toung YP et al.; The Drosophila glutathione S-transferase D genes encode a family of isozymes . We have determined the amino acid sequence of a new member of this family by nucleotide sequence analysis of a genomic DNA clone . The open reading frame of this intronless gene should encode an isozyme subunit of 211 amino acids . This sequence has significant homology to the E . coli stringent starvation protein, SSP, which is also a protein of two identical 211 amino acid subunits . The two proteins have very similar overall amino acid composition as well . It is possible that SSP may be a glutathione S-transferase(s) in E . coli or is evolutionarily related to glutathione S-transferases . Because SSP is known to be tightly associated with the RNA polymerase holoenzyme during purification, it is conceivable that Drosophila glutathione S-transferase(s) may potentially interact with the transcription machinery in a fashion similar to SSP's interaction with E . coli RNA polymerase holoenzyme.

Proc Natl Acad Sci U S A, 1992 Jan 15, 89(2), 683 - 7
Cloning of Drosophila transcription factor Adf-1 reveals homology to Myb oncoproteins; England BP et al.; The Drosophila sequence-specific DNA binding protein, Adf-1, is capable of activating transcription of the alcohol dehydrogenase gene, Adh, and is implicated in the transcriptional control of other developmentally regulated genes . We have cloned the cDNA encoding Adf-1 by generating specific DNA probes deduced from partial amino acid sequence of the protein . Several cDNA clones encoding an extended open reading frame were isolated from a phage lambda library . The complete amino acid sequence of Adf-1 deduced from the longest cDNA reveals structural similarities to the putative helix-turn-helix DNA binding motif of Myb and Myb-related proteins . DNA sequence analysis of genomic clones and Northern blot analysis of mRNA suggest that Adf-1 is a single-copy gene encoding a 1.9-kb transcript . Purified recombinant Adf-1 expressed in Escherichia coli binds specifically to Adf-1 recognition sites and activates transcription of a synthetic Adh promoter in vitro in a manner indistinguishable from the protein purified from Drosophila . Temporally staged Drosophila embryos immunochemically stained with affinity-purified anti-Adf-1 antibodies indicate that Adf-1 protein is not detectable in very early embryos and does not appear to be maternally inherited . During later stages of embryogenesis, Adf-1 appears to be expressed in the nucleus of most somatic cells in the embryo with possibly higher concentrations found in some tissues.

Proc Natl Acad Sci U S A, 1992 Jan 15, 89(2), 504 - 8
Specific inhibition of transcription by triple helix-forming oligonucleotides; Duval-Valentin G et al.; Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation . The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site . A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro . We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature . The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix . Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition . Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action . The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm.

Proc Natl Acad Sci U S A, 1992 Jan 15, 89(2), 485 - 9
Mutagenesis of some conserved residues in human 5-lipoxygenase: effects on enzyme activity; Zhang YY et al.; Recombinant human 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) was expressed in Escherichia coli . In incubations of E . coli supernatants with arachidonic acid, 5-hydroxy-7,9,11,14-eicosatetraenoic acid and leukotriene A4 were formed, while incubation with 8,11,14-eicosatrienoic acid gave 8-hydroxy-9,11,14-eicosatrienoic acid . Six conserved histidine residues in 5-lipoxygenase were subjected to site-directed mutagenesis . Exchanges of His-367, -372, or -551 gave mutants for which no enzyme activities were detectable . On the other hand, exchanges of His-362, -390, or -399 gave mutants that were enzymatically active, but less so than the nonmutated control . For two of these (exchanges of His-390 or -399), the activities of the mutants were dependent on the expression temperature . Thus, the histidines in the first group (His-367, -372, -551) were crucial for 5-lipoxygenase activity, possibly because of a function of these residues as metal ligands . Mutagenesis aimed at two other conserved elements in 5-lipoxygenase, Gln-558 and the C terminus, gave mutated proteins with only a small residual activity (substitution of Gln-558), or with no detectable activity (deletion of six C-terminal amino acids), indicating that these regions are important for the function of 5-lipoxygenase.

J Biol Chem, 1992 Jan 15, 267(2), 997 - 1000
A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus; Sung CY et al.; The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins . Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M . (1982) Proc . Natl . Acad . Sci . U.S.A . 79, 3438-3441; Vlasuk, G . P., Inouye, S., Ito, H., Itakura, K., and Inouye, M . (1983) J . Biol . Chem . 258, 7141-7148) . We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus . Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein . This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so . Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis . This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus.

J Biol Chem, 1992 Jan 15, 267(2), 975 - 8
Sequence of a cDNA clone encoding pig heart mitochondrial CoA transferase; Lin TW et al.; We have isolated a full-length cDNA clone encoding the cytoplasmic precursor to pig heart mitochondrial CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase (3-oxoacid CoA transferase, EC 2.8.3.5}, a key enzyme for ketone body catabolism . The deduced amino acid sequence indicates the presence of a 39-residue mitochondrial signal sequence and a 481-residue mature protein of molecular weight 52,197 . CoA transferase is known to be susceptible to proteolytic cleavage to produce a nicked but active enzyme . We have identified the site of proteolysis, and analysis of the sequence in its vicinity suggests that the polypeptide may fold into two domains connected by a highly hydrophilic bridge.

J Biol Chem, 1992 Jan 15, 267(2), 1382 - 90
Structure function relationships in the ribosomal stalk proteins of archaebacteria; Kopke AK et al.; The ribosomal L12 protein gene of Sulfolobus solfataricus (SsoL12) has been subcloned and overexpressed in Escherichia coli . Five protein L12 mutants were designed: two NH2-terminal and two COOH-terminal truncated mutants and one mutant lacking the highly charged part of the COOH-terminal region . The mutant protein genes were overexpressed in E . coli and the products purified . The amino acid composition was verified and the NH2 terminally truncated mutants were subjected to Edman degradation . The SsoL12 protein was selectively removed from entire S . solfataricus ribosomes by an ethanol wash . The remaining ribosomal core particles showed a substantial decrease in the in vitro translational activity . S . solfataricus L12 protein overexpressed in E . coli (SsoL12e) was incorporated into these ribosomal cores and restored their translational activity . Mutants lacking any part of the COOH-terminal region could be incorporated into these cores, as proven by two-dimensional polyacrylamide gels of the reconstituted particles . Mutant SsoL12 MC2 (residue 1-70) was sufficient for dimerization and incorporation into ribosomes . In contrast to the COOH terminally truncated mutants, L12 proteins lacking the 12 highly conserved NH2-terminal residues or the entire NH2-terminal region (44 amino acids) are unable to bind to ribosomes, suggesting that the SsoL12 protein binds with its NH2-terminal portion to the ribosome . None of the mutants could significantly increase the translational activity of the core particles suggesting that every deleted part of the protein was needed directly or indirectly for translational activity . Our results suggest that the COOH terminally truncated mutants were bound to ribosomes but not functional for translation . Cores preincubated with these COOH terminally truncated mutants regained activity when a second incubation with the entire overexpressed SsoL12e protein followed . This finding suggests that archaebacterial L12 proteins are freely exchanged on the ribosome.

J Biol Chem, 1992 Jan 15, 267(2), 1303 - 9
Functional analysis of a recombinant glycoprotein Ib alpha polypeptide which inhibits von Willebrand factor binding to the platelet glycoprotein Ib-IX complex and to collagen; Cruz MA et al.; By deletion mutagenesis and transient expression in COS cells, a 96-amino acid hydrophilic sequence in the glycoprotein Ib alpha polypeptide located between L220 and L318 was identified which appeared to contain its von Willebrand factor- (vWF) binding site . The cDNA encoding this fragment was then expressed in Escherichia coli and purified from the bacterial cell lysate . The recombinant polypeptide, rGpIb alpha Q221-L318, was monomeric and had an apparent molecular weight of 14,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . It inhibited both ristocetin-induced binding of 125I-vWF to fixed washed platelets and ristocetin-induced platelet agglutination . The recombinant polypeptide also inhibited the binding of 125I-vWF to immobilized type I and III collagen . Inhibition of 125I-vWF binding to platelets and collagen was dose-dependent, with IC50 values of 500 and 200 nM rGpIb alpha Q221-L318, respectively . Fifty % inhibition of ristocetin-induced platelet agglutination required 500 nM rGpIb alpha Q221-L318 . Although rGpIb alpha Q221-L318 inhibited vWF binding to collagen it did not, itself, bind to collagen-coated surfaces . Reduction of the disulfide bond between C248 and C264 abolished activity . 125I-rGpIb alpha Q221-L318 bound directly to GpIb/IX sites on multimeric vWF . These studies document that a portion of the sequence between Q221 and L318 is needed for recognition and binding to vWF and that binding requires an intact disulfide bond between C248 and C264 . The binding of this recombinant polypeptide to vWF multimers inhibits vWF interaction with two important substrates, platelet GpIb/IX and collagen.

J Biol Chem, 1992 Jan 15, 267(2), 1212 - 8
The G226A mutant of Gs alpha highlights the requirement for dissociation of G protein subunits; Lee E et al.; Adenylylcyclase cannot be activated by hormones or guanine nucleotide analogs in membranes from cells that express the G226A mutant form Gs alpha instead of the wild-type protein . The mutant Gs alpha protein appears incapable of undergoing the conformational change necessary for guanine nucleotide-induced dissociation of the G protein alpha subunit from the beta gamma subunit complex (Miller, R.T., Masters, S.B., Sullivan, K.A., Beiderman, B., and Bourne, H.R . (1988) Nature 334, 712-715) . G226A Gs alpha was synthesized in Escherichia coli, purified, and characterized . Examination of the kinetics of dissociation of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) suggests that G226A Gs alpha is incapable of assuming the conformation necessary for high affinity binding of Mg2+ to the alpha subunit-GTP gamma S complex . Associated changes include the failure of Mg2+ and GTP gamma S to confer resistance to tryptic proteolysis upon the protein, to enhance intrinsic tryptophan fluorescence, or to cause dissociation of alpha from beta gamma . However, the GTPase activity of the mutant protein is near normal (at high Mg2+ concentrations), and the protein is capable of activating adenylylcyclase . A similar defect is present in G49V Gs alpha . Failure of G protein subunit dissociation appears to be the explanation for the phenotypic properties of cells that express G226A Gs alpha, and this mutation thus highlights the crucial nature of this reaction as a component of G protein action.

J Biol Chem, 1992 Jan 15, 267(2), 1062 - 6
Maxadilan . Cloning and functional expression of the gene encoding this potent vasodilator peptide; Lerner EA et al.; Maxadilan is a potent vasodilator peptide released into the skin when the sand fly Lutzomyia longipalpis, an important vector of leishmania, probes for a blood meal . As several lines of evidence suggest that this peptide may play a critical role in the enhancement of leishmania infectivity attributed to sand fly saliva, the peptide has been proposed as a candidate antigen for a leishmanial vaccine . Although maxadilan is the most potent vasodilator peptide known and shares several properties with calcitonin gene-related peptide (CGRP), studies of its structure, physiological effects, and biological roles have been limited by the miniscule quantities available . Here we report the isolation of cDNA and genomic DNA clones that encode maxadilan . The predicted translation product shows no significant homology with any previously isolated proteins . The coding DNA has been expressed in Escherichia coli and the purified recombinant peptide is biologically active with a specific activity comparable to the natural peptide . Recombinant maxadilan will be useful in studies of vascular biology and could lead to novel therapeutic and prophylactic agents.

J Biol Chem, 1992 Jan 15, 267(2), 1001 - 7
Ca(2+)-binding properties of the platelet glycoprotein IIb ligand-interacting domain; Gulino D et al.; Glycoprotein (GP) IIb is the alpha subunit of platelet integrin GPIIb-IIIa . Analysis of the primary structure of this subunit has indicated the presence of four stretches of amino acid residues that are highly conserved among various integrin alpha subunits and that have been suggested to be putative calcium-binding sites . To verify the Ca(2+)-binding capacity of these conserved domains and their implication in integrin adhesive functions, a fragment corresponding to the amino acid sequence of GPIIb from positions 171 to 464 was expressed . The nucleotide sequence coding for this GPIIb domain was generated by polymerase chain reaction, cloned into the pTG1924 expression vector, and expressed in Escherichia coli strain TGE901 . The recombinant protein was purified by gel exclusion chromatography and used in equilibrium dialysis experiments . The results demonstrate that the four binding sites can be occupied by Ca2+ . Two classes of binding sites can be detected, including two sites with a Kd of 30 microns and two sites of lower affinity with a Kd of 120 microns . Interaction of Ca2+ with these two classes of sites was inhibited by a large excess of Mg2+ or Mn2+, suggesting that these cations are competitive for the same sites on GPIIb . Thus, the four Ca(2+)-binding sites of GPIIb are not similar and exhibit different affinities for divalent ions . To verify the functional implication of these Ca(2+)-binding sites, the effect of Ca2+ on the binding of fibrinogen to the recombinant protein was analyzed using a solid-phase assay . The results indicate that optimal fibrinogen binding occurs when the four calcium-binding sites are occupied and establish the functional importance of this Ca(2+)-binding domain in the ligand-binding activity of GPIIb.

Eur J Biochem, 1992 Jan 15, 203(1-2), 89 - 98
Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli . Cleavage of a fusion protein with cyanogen bromide; Franken PA et al.; Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity . The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr . The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence . This protein was expressed in E . coli as a 68-kDa Cro-LacZ fusion protein . CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro . The {Met8----Leu}HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8 . p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect . The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively . In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine . The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration . The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine . The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2 . In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested . The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors . The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol . The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.

Blood, 1992 Jan 15, 79(2), 406 - 16
Endotoxin enhances the expression of monocyte prothrombinase activity; Robinson RA et al.; Thrombin is generated on the surface of mononuclear cells (MNCs) through the assembly and function of the prothrombinase complex consisting of the enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate membrane surface for proper assembly of the protein constituents . Assays performed in the presence of factors Va and Xa indicated that endotoxin significantly enhanced the prothrombinase activity (1.5- to 2.5-fold; P less than .001) expressed by MNCs in a dose- and time-dependent manner . Monocytes present in the MNC suspensions were responsible for this increased activity through processes resulting in both enhanced cellular activity and the enhanced release of membranous vesicles . Endotoxin was without effect on the expression of lymphocyte prothrombinase activity . Scanning electron microscopy techniques indicated that endotoxin resulted in extensive membrane blebbing of the monocytes present in the MNC suspensions with no effect on the morphology of the lymphocytes . Within 5 hours, endotoxin maximally enhanced the prothrombinase activity expressed by the monocyte membrane surface 2.8-fold, whereas 8 hours was required to maximally enhance the activity associated with the released vesicles by twofold . The observed increase in activity expressed by the monocyte membrane surface was due solely to endotoxin, since the activity expressed by the unstimulated monocyte membrane surface remained unaltered over time . In contrast, cell vesiculation, which occurred in the absence of any stimulus, was further enhanced by endotoxin . The increase in activity associated with the released vesicles from both stimulated and unstimulated cells paralleled an increase in the vesicle number as determined by flow cytometric analyses . The vesicle released from both unstimulated and stimulated monocytes were indistinguishable in size as determined by image analysis and ranged between 0.05 and 0.3 microns in diameter . 2-Deoxy-D-glucose (2DG) significantly enhanced the prothrombinase activity expressed by the monocyte membrane surface, as well as the released vesicle fraction, when used alone or in addition to endotoxin . The enhanced activity associated with the vesicle fraction again was attributed to the release of more vesicles . In contrast, cycloheximide decreased the prothrombinase activity expressed by the monocyte membrane surface, as well as the activity associated with vesicles released from both stimulated and unstimulated cells . These data suggest that the expression of monocyte prothrombinase activity can be significantly enhanced by endotoxin through processes that alter the monocyte membrane surface and augment the vesiculation process . Both processes appear to be regulated by protein synthesis and adenosine triphosphate (ATP)-dependent mechanisms.

FEMS Microbiol Lett, 1992 Jan 15, 69(3), 249 - 52
Action of Escherichia coli heat-stable enterotoxin II on isolated sections of mouse ileum; Hitotsubashi S et al.; When Escherichia coli STII was applied to the serosa of the ileum at a concentration of 40 micrograms/ml, an acceleration of the spontaneous motility and a weak contraction were induced 2-3 min later . The induction was not affected by the addition of atropine (10(-6) M), but was abolished by the addition of papaverine (10(-4) M) . When STII was applied to the mucosa, the acceleration of the spontaneous motility appeared 7-8 min later, but a contraction was not induced . These results suggest that STII acts directly on muscle cell of the ileum and enhances the spontaneous motility of the intestine.

FEMS Microbiol Lett, 1992 Jan 15, 69(3), 229 - 34
Bordetella bronchiseptica dermonecrotizing toxin suppresses in vivo antibody responses in mice; Horiguchi Y et al.; The effects of Bordetella bronchiseptica dermonecrotic toxin (DNT) on the in vivo antibody response of mice were investigated . Intravenous injection of DNT at doses of 0.5 and 2.0 ng resulted in a significant suppression of the antibody response both to sheep red blood cells and to Escherichia coli lipopolysaccharide as measured by plaque-forming cell and hemagglutination assays . Spleen weights of mice given the same doses of DNT were significantly reduced, while the weights of thymuses and mesenteric lymph nodes were not . Numbers of Thy-1,2+ T lymphocytes, L3T4+ T lymphocytes, Lyt-2+ T lymphocytes and surface-immunoglobulin-positive lymphocytes decreased in spleens of the DNT-treated mice . Since the ratio of each lymphocyte population to the total number of splenic lymphocytes was not significantly different between the DNT-treated and non-treated mice, it is unlikely that DNT has a cytotoxic activity or a mitogen activity to some specific population of lymphocytes . Thus, we considered that the immunosuppression was attributable to a dysfunction of the spleen atrophied by the DNT.

J Biol Chem, 1992 Jan 15, 267(2), 780 - 8
Post-incision steps of nucleotide excision repair in Escherichia coli . Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I; Orren DK et al.; UvrA, UvrB, and UvrC initiate nucleotide excision repair by incising a damaged DNA strand on each side of the damaged nucleotide . This incision reaction is substoichiometric with regard to UvrB and UvrC, suggesting that both proteins remain bound following incision and do not "turn over." The addition of only helicase II to such reaction mixtures turns over UvrC; UvrB turnover requires the addition of helicase II, DNA polymerase I, and deoxynucleoside triphosphates . Column chromatography and psoralen photocross-linking experiments show that following incision, the damaged oligomer remains associated with the undamaged strand, UvrB, and UvrC in a post-incision complex . Helicase II releases the damaged oligomer and UvrC from this complex, making repair synthesis possible; DNase I footprinting experiments show that UvrB remains bound to the resulting gapped DNA until displaced by DNA polymerase I . The specific binding of UvrB to a psoralen adduct in DNA inhibits psoralen-mediated DNA-DNA cross-linking, yet promotes the formation of UrvB-psoralen-DNA cross-links . The discovery of psoralen-UvrB photocross-linking offers the potential of active-site labeling.

Gene, 1992 Jan 15, 110(2), 137 - 44
Hepatitis B virus envelope epitopes: gene assembly and expression in Escherichia coli of an immunologically reactive novel multiple-epitope polypeptide 1 (MEP-1); Kumar V et al.; A novel synthetic 323-bp gene with the open reading frame of a multiple-epitope polypeptide has been assembled and cloned . The gene is engineered by contiguous alignment of selected epitopes and functional domains of the hepatitis B virus envelope proteins separated by pairs of glycine residues . High-level bacterial production of this 100-amino acid (approx . 10 kDa) protein has been achieved and the gene product is stable . ELISA and Western blot experiments using epitope-specific antisera confirm that the corresponding epitopes are present in the engineered protein.

J Biol Chem, 1992 Jan 15, 267(2), 1293 - 7
The effects of cysteine mutations on the reverse transcriptases of human immunodeficiency virus types 1 and 2; Hizi A et al.; Chemical modification of HIV-1 and HIV-2 (human immunodeficiency virus, types 1 and 2) reverse transcriptases (RT) with three thiol reactive compounds selectively inhibits the RNase H function of the enzyme . HIV-1 RT has 2 cysteines (at positions 38 and 280); HIV-2 RT has 3 (38, 280, 445) . Both of the cysteines in HIV-1 RT are in the polymerase domain . To investigate the role of the cysteines in the structure and function of the HIV RTs, we have converted each cysteine to serine and made combinations of the mutations . Since HIV-1 RT has alanine at position 445, we have also substituted alanine for serine at this position in HIV-2 RT . Neither of the single mutations in HIV-1 RT nor the double mutation mimics the effects of the chemical modification . The serine 280 mutation has little effect on either polymerase or RNase H; the serine 38 mutation affects both activities, as does the 38/280 double mutant . The 38 and 280 serine mutations in HIV-2 RT resemble the equivalent mutations in HIV-1 RT . Substitution of serine or alanine at position 445 (which lies in the RNase H domain) diminishes, but does not abolish, the RNase H activity of HIV-2 without affecting polymerase activity . The RNase H activity of a mutant HIV-1 RT with serine at position 280 is completely resistant to inactivation by the three thiol reactive compounds we tested, which demonstrates that cysteine 280 is the critical residue . We suggest that the reason the mutation (cysteine 280 to serine) does not mimic the chemical modification is because the chemical modification produces a greater change in the structure of the protein . We also suggest that position 280 lies at or near the important points of contact between the RNase H and polymerase domains, so that chemical modification of this position, which lies within the polymerase domain, distorts the RNase H domain.

J Biol Chem, 1992 Jan 15, 267(2), 1054 - 61
Secondary structure of the mRNA for ribosomal protein S20 . Implications for cleavage by ribonuclease E; Mackie GA; A synthetic RNA transcript containing the entire sequence of one of the two natural mRNAs for Escherichia coli ribosomal protein S20 is a substrate for specific cleavage by an endonuclease which is or depends on ribonuclease E (Mackie, G . A . (1991) J . Bacteriol . 173, 2488-2497) . Partial cleavage with ribonucleases T1 or CL3 and limited modification with dimethyl sulfate have been employed to identify residues that are likely to be single stranded in the S20 mRNA's native state . The data show that the 5' one-third of the mRNA is relatively unstructured whereas the 3' one-third is extensively folded . The latter property can account for the previously observed accumulation of a 147-residue product co-terminal with the 3' end of the S20 mRNA (Mackie, G . A . (1989) J . Bacteriol . 171, 4112-4120) . Sites of cleavage by the ribonuclease E-dependent activity map to single-stranded regions of the RNA . In addition, denaturation of the RNA substrate results in loss of susceptibility to the ribonuclease E-dependent activity and simultaneous loss of the single-stranded character of the two most prominent cleavage sites . It is proposed that ribonuclease E is a single-strand-specific enzyme with few primary structural constraints but a preference for an AU dinucleotide 3' to the site of cleavage.

J Biol Chem, 1992 Jan 15, 267(2), 695 - 8
Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring; Viitanen PV et al.; Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts . While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown . We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity . Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each . The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP . Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded {35S}ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP . We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.

Mol Cell Biochem, 1992 Jan 15, 109(1), 61 - 9
NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase; Martinez-Galisteo E et al.; The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP+, NAD+ and oxidized E . coli thioredoxin activated both enzymes significantly, particularly the bacterial one . The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E . coli and calf-liver enzymes were 13.5 and 2 min, respectively . Oxidized E . coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30 degrees C . Lower but significant protection and reactivation was also observed with NADP+ and NAD+ . EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation . Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O2 or oxygen active species in redox inactivation . The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD+ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide . Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase . This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.

J Biol Chem, 1992 Jan 15, 267(2), 1093 - 8
Purification and molecular cloning of a butyrolactone autoregulator receptor from Streptomyces virginiae; Okamoto S et al.; In streptomyces, low molecular weight compounds termed "autoregulators" have been isolated as primary signal molecules for triggering secondary metabolism and/or cytodifferentiation . Streptomyces virginiae produces a set of autoregulators termed virginiae butanolide A-E which trigger virginiamycin production, and possesses a high-affinity virginiae butanolide receptor (Kim, H.S., Nihira, T., Tada, H., Yanagimoto, M., and Yamada, Y . (1989) J . Antibiot . (Tokyo) 42, 769-778) . The virginiae butanolide receptor has now been purified to apparent homogeneity with 14,000-fold purification and an 8.6% activity yield . The purified receptor showed a Mr of 36,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a maximum ligand binding of 33.0 nmol/mg protein, indicating a 1:1 binding stoichiometry (1.18 mol of virginiae butanolide/36 kDa of protein) between virginiae butanolides and the receptor . Due to a blockage at the amino terminus, fragment peptides were generated by lysyl endopeptidase and five partial amino acid sequences were determined . The gene (vbrA) encoding the virginiae butanolide receptor was identified on a 5.0-kbp BamHI fragment by hybridization to synthetic oligonucleotide probes, cloned, and sequenced . Nucleotide-sequence analysis predicted a 319-amino acid open reading frame (vbrA) in which all the partial amino acid sequences of the receptor appeared, and 166 bp downstream from it another open reading frame for a 144-amino acid protein which was designated as a ribosomal protein L11 from its high homology (62-64%) to the amino acid sequences of ribosomal protein L11 of several origins, and thus denoted as rplK . The C-terminal half of VbrA showed 36% overall homology to the amino acid sequence of an essential protein (NusG) of Escherichia coli . Furthermore, the gene assembly of vbrA-rplk of S . virginiae closely resembled that of nusG-rplK of E . coli, suggesting that vbrA may constitute a part of an essential gene cluster encoding components of transcriptional and translational apparatuses.

J Immunol, 1992 Jan 15, 148(2), 457 - 65
Cytokine-stimulated human mesothelial cells produce chemotactic activity for neutrophils including NAP-1/IL-8; Goodman RB et al.; To test the hypothesis that mesothelial cells play a role in regulating inflammatory responses within the pleural space, we examined neutrophil chemotactic activity released by cytokine-stimulated mesothelial cells . Human mesothelial cells were isolated from patients with transudative pleural effusions and cultured . The purity of the cell population was assessed by morphologic, immunocytochemical, and biochemical characteristics . Confluent fourth passage mesothelial cell plates were exposed to varying concentrations of the recombinant human cytokines IL-1 alpha, TNF-alpha, or IFN-gamma, or Escherichia coli endotoxin (LPS) . Polymorphonuclear neutrophil (PMN) chemotactic activity in the conditioned media was measured in microchemotaxis chambers . Although none of the cytokines demonstrated inherent chemotactic activity, each stimulated mesothelial cells to produce PMN chemotactic activity in a dose-dependent manner . TNF-alpha stimulated the release of the greatest quantity, whereas stimulation with IFN-gamma and IL-1 alpha resulted in the release of lesser but still significant quantities of PMN chemotactic activity . By contrast, LPS did not increase the basal level of chemotactic activity produced by the cells . The cytokine-induced chemotactic activity was proteinaceous, required de novo synthesis, and had a predominant m.w . of 10,000 . Significant quantities of immunoreactive neutrophil-activating peptide-1 (NAP-1)/IL-8 were detected in mesothelial cell supernatants after stimulation with each of the cytokines . The neutrophil chemotactic activity of supernatants from mesothelial cells stimulated with either IL-1 alpha or IFN-gamma was completely neutralized with rabbit anti-human NAP-1/IL-8 polyclonal antiserum . The same antiserum neutralized the majority, but not all, of the neutrophil chemotactic activity in supernatants from TNF-stimulated mesothelial cells . Stimulated mesothelial cells also expressed an inducible mRNA transcript that hybridized with a specific oligonucleotide probe for human NAP-1/IL-8 . These observations provide a mechanism whereby mesothelial cells could respond to inflammatory stimuli in the underlying lung and regulate inflammatory responses within the pleural space.

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