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