J Cell Biol, 1991 Jan, 112(2), 237 - 43 42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor; Coppard NJ et al.; We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe . The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established . The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci . 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA . 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus . Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E . coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis. Carcinogenesis, 1991 Jan, 12(1), 29 - 34 The mutational specificity of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF2) in the lacI gene of Escherichia coli; Lambert IB et al.; We have determined the mutational specificity of the 5-nitrofuran derivative furylfuramide (AF2) in the lacI gene of Escherichia coli . Treatment of a delta uvrB, pKM101 strain with 1 M AF2 yielded a mutation frequency approximately 300 times greater than that of untreated controls . Of the 165 AF2-induced mutants analysed by DNA sequencing, 145 were base substitution mutations, 11 were frameshifts, and the remainder small deletions, tandem base substitutions and complex mutations . Base substitution occurred primarily (greater than 93%) at G:C base pairs . The proportions of the various mutations are very similar to those that have been reported for AP sites . We suggest that the principal mechanism for AF2 mutagenesis is the formation of an adduct which depurinates to yield AP sites that serve as a substrate for error-prone repair . Seventy-two of the mutations occurred at four 5'-TGC-3' sites . The majority (10/11) of the frameshift mutations occurred at one such hotspot and could have been templated by an inverted repeat less than 100 bp removed from the site of the mutation. Am J Pathol, 1991 Jan, 138(1), 183 - 93 Anatomic pathway of pulmonary fluid leakage in endotoxemia induced in rats . The red blood cell packing method and its application; Nagata N et al.; The anatomic site of pulmonary fluid leakage in endotoxemia in rats was investigated using the red blood cell packing method and low-dose horseradish peroxidase as a tracer . To differentiate between arterioles and venules in a given section by light and electron microscopy, human red blood cells fixed with 4% paraformaldehyde were administered to the rat pulmonary arterial trunk at a pressure of 40 cm water . Fixed red blood cells were packed in the lumina of arteries, arterioles, and a few capillaries surrounding arterioles, while veins, venules, and almost all capillaries were void of red blood cells in the lumina . Fifteen minutes after the intravenous administration of 3 and 30 mg/kg of Escherichia coli endotoxin, extravascular leakage of horseradish peroxidase from venules (nonmuscular veins) was evident . Two hours after the intravenous injection of the same doses of endotoxin, some arterioles (nonmuscular arteries) and venules (non-muscular veins) showed extravascular leakage of horseradish peroxidase, while few capillaries showed this leakage . These results suggest that pulmonary fluid leakage occurs predominantly through venules in the early phase of endotoxemia (at 15 minutes), while the arterioles contribute to fluid leakage in addition to venules in the late phase of endotoxemia (at 2 hours). J Bacteriol, 1991 Jan, 173(2), 921 - 5 Transformation of freshwater and marine caulobacters by electroporation; Gilchrist A et al.; We performed plasmid electrotransformation of Caulobacter crescentus strains and obtained up to 3 x 10(8) transformants per micrograms of pKT230 . The presence and integrity of the paracrystalline protein surface (S) layer influenced electroporation; caulobacters lacking the S layer were electrotransformed 10 times more efficiently than caulobacters possessing the S layers . A procedure yielding 1,500 transformants per micrograms of pKT230 was developed for a marine caulobacter . Electroporation was used in combination with several genetic techniques, including introduction of ligation mixtures, suicide transposon mutagenesis, gene replacement, and plasmid electrotransfer from Escherichia coli to caulobacters. J Bacteriol, 1991 Jan, 173(2), 826 - 33 The korF region of broad-host-range plasmid RK2 encodes two polypeptides with transcriptional repressor activity; Jagura-Burdzy G et al.; Broad-host-range IncP plasmid RK2 possesses a series of operons involved in plasmid maintenance, whose expression is coordinated by a number of regulators, most of which are encoded in the central regulatory korA-korB operon . The nucleotide sequence of two new cistrons in this operon, comprising what we have previously designated the korF locus located between coordinates 57.0 and 56.0 kb on the genome of the IncP alpha plasmid RK2, is presented . The cistrons encode polypeptides of 173 and 175 amino acids . Each can repress transcription from the promoters for the kfrA (a monocistronic operon which follows the korA-korB operon) and trfA (a polycistronic operon encoding a putative single-stranded-DNA-binding protein as well as the essential plasmid replication protein TrfA) operons . In addition, the korF loci allow korB to repress kfrA transcription . Both polypeptides contain hydrophobic segments, suggesting that they may be membrane associated . KorFI is highly basic protein whose predicted properties are similar to those of histone like proteins. J Bacteriol, 1991 Jan, 173(2), 776 - 82 A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium; Randall T et al.; A stable extrachromosomally maintained transformation vector (pG12-1) for the lignin-degrading filamentous fungus Phanerochaete chrysosporium is described . The vector is 6.3 kb and contains a Kanr marker, pBR322 ori, and a 2.2-kb fragment (ME-1) derived from an endogenous extrachromosomal DNA element of P . chrysosporium . Vector pG12-1 was able to transform P . chrysosporium to G418 resistance and was readily and consistently recoverable from the total DNA of transformants via Escherichia coli transformation . Southern blot analyses indicated that pG12-1 is maintained at a low copy number in the fungal transformants . The vector is demonstrable in the total DNA of individual G418-resistant basidiospore progeny of the transformants only after amplification by polymerase chain reaction . Exonuclease III and dam methylation analyses, respectively, indicated that pG12-I undergoes replication in P . chrysosporium and that it is maintained extrachromosomally in a circular form . The vector is stably maintained in the transformants even after long-term nonselective growth . There is no evidence for integration of the vector into the chromosome at any stage. J Bacteriol, 1991 Jan, 173(2), 751 - 6 Direct proof of a "more-than-single-layered" peptidoglycan architecture of Escherichia coli W7: a neutron small-angle scattering study; Labischinski H et al.; A neutron small-angle scattering study was performed to determine the thickness and the scattering density profile of isolated peptidoglycan sacculi of Escherichia coli W7 in aqueous suspension (D2O) . The maximum thickness (7 +/- 0.5 nm) of the sacculus from the exponential-phase cells was large enough to suggest the existence of a more-than-single-layered architecture . The experimental density profile across the thickness of the sacculus did not allow an unambiguous differentiation between a single-layered architecture characterized by completely extended peptide side chains projecting from the sugar strands or, alternatively, a partially triple layered structure . To resolve this ambiguity, sacculi were labeled with deuterated wall peptides . Comparison of the two experimental profiles indicated that the sacculus is more than single layered across its surface, with about 75 to 80% of its surface single layered and 20 to 25% triple layered. J Bacteriol, 1991 Jan, 173(2), 720 - 6 Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation; Barry EM et al.; In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E . coli hlyC gene, which is required for the production of a functional hemolysin molecule in E . coli . Mutations produced in the chromosome of B . pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule . These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis . The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays . The upstream region restored hemolytic activity when returned in trans to the mutant strains . This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally . Sequence analysis of the upstream region defined an open reading frame with homology to the E . coli hlyC gene . In contrast to E . coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene. J Bacteriol, 1991 Jan, 173(2), 697 - 703 16S ribosomal DNA amplification for phylogenetic study; Weisburg WG et al.; A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples . One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences . Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined . An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed . Anaplasmas are related to the genera Rickettsia and Ehrlichia . In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection . By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them . In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture. J Bacteriol, 1991 Jan, 173(2), 687 - 96 Genetic analysis of potassium transport loci in Escherichia coli: evidence for three constitutive systems mediating uptake potassium; Dosch DC et al.; The analysis of mutants of Escherichia coli that require elevated concentrations of K+ for growth has revealed two new genes, trkG, near minute 30 within the cryptic rac prophage, and trkH, near minute 87, the products of which affect constitutive K+ transport . The analysis of these and other trk mutations suggests that high rates of transport, previously considered to represent the activity of a single system, named TrkA, appear to be the sum of two systems, here named TrkG and TrkH . Each of these two is absolutely dependent on the product of the trkA gene, a cytoplasmic protein associated with the inner membrane (D . Bossemeyer, A . Borchard, D . C . Dosch, G . C . Helmer, W . Epstein, I . R . Booth, and E . P . Bakker, J . Biol . Chem . 264:16403-16410, 1989) . The TrkH system is also dependent on the products of the trkH and trkE genes, while the TrkG system is also dependent on the product of the trkG gene and partially dependent on the product of the trkE gene . It is suggested that the trkH and trkG products are membrane proteins that form the transmembrane path for the K+ movement of the respective systems . Two mutations altering the trkA product reduce the affinity for K+ of both TrkG and TrkH, indicating that changes in peripheral protein can alter the conformation of the sites at which K+ is bound prior to transport . The TrkD system has a relatively modest rate of transport, is dependent solely on the product of the trkD gene, and is the sole saturable system for Cs+ uptake in this species (D . Bossemeyer, A . Schlosser, and E . P . Bakker, J . Bacteriol . 171:2219-2221, 1989). J Bacteriol, 1991 Jan, 173(2), 636 - 41 Modification of the properties of a Ruminococcus albus endo-1,4-beta-glucanase by gene truncation; Ohmiya K et al.; An endo-1,4-beta-glucanase (EgI) gene isolated from Ruminococcus albus was deleted at the 5'-flanking region by gene truncation or at the 3'-flanking region by insertion of an omega (omega) fragment with a universal stop codon at the EcoRI or BamHI site . These modified genes were integrated into pUC vectors to construct chimera plasmids for Escherichia coli . The truncated EgIs were produced from transformants (E . coli) harboring the chimera plasmids . An EgI with a 15-amino-acid N-terminal deletion exibited higher activity at lower pH and temperature compared with the activity of the original EgI . The EgIs with 59- and 75-amino-acid deletions from the N and C terminals, respectively, had no activity, indicating that both terminal moieties are essential for enzyme activity. J Bacteriol, 1991 Jan, 173(2), 601 - 8 Suppression of a mutation in OmpR at the putative phosphorylation center by a mutant EnvZ protein in Escherichia coli; Brissette RE et al.; Phosphorylation of OmpR, a transcription activator for ompF and ompC expression, is essential for its function and has been shown to be mediated in vitro by EnvZ, a transmembrane sensory receptor protein . On the basis of the three-dimensional structure of CheY which has an extensive sequence similarity with OmpR, three aspartic residues, D11, D12, and D55, of OmpR are considered to form a triacidic pocket serving as the phosphorylation center . When these aspartic acid residues were replaced with asparagine (D11N) or glutamine (D12Q and D55Q), ompF and ompC expression was almost completely blocked . Two pseudorevertants of the D11N mutation were isolated: one of them is a mutation in EnvZ (G240E), and the other is a mutation in OmpR (S48F) . The envZ mutation (G240E) by itself was found to confer a phenotype very similar to that of the well known envZ11 mutation (T247R), suggesting that EnvZ (G240E) is an elevated kinase for OmpR . Consistent with this notion, EnvZ (T247R) was also able to suppress the D11N mutation in OmpR . An in vitro phosphorylation study showed that while the wild-type OmpR was phosphorylated by EnvZ, the D11N OmpR was not . These results suggest that the D11N mutation alters OmpR conformation in such a way that OmpR is very poorly phosphorylated by EnvZ . On the basis of the in vivo and in vitro analysis, the mechanisms by which the G240E mutation in EnvZ and the S48F mutation in OmpR suppress the D11N mutation in OmpR are discussed. J Bacteriol, 1991 Jan, 173(2), 549 - 58 Dual regulation of the ugp operon by phosphate and carbon starvation at two interspaced promoters; Kasahara M et al.; The ugp operon of Escherichia coli includes genes involved in the uptake of sn-glycerol-3-phosphate and glycerophosphoryl diesters and belongs to the pho regulon which is induced by phosphate limitation . This operon has two transcriptional initiation sites, as determined by S1 nuclease mapping of the in vivo transcripts . The downstream promoter has multiple copies of the pho box, the consensus sequence shared by the pho promoters; the upstream promoter has a consensus sequence for the promoters regulated by cyclic AMP and its receptor protein, CRP . PhoB protein, which is the transcriptional activator for the pho regulon, protected the regulatory region with the pho boxes in DNase I footprinting experiments and activated transcription from the downstream promoter in vitro . Studies with transcriptional fusions between ugp and a promoterless gene for chloramphenicol acetyltransferase show that the upstream promoter is induced by carbon starvation in a manner that required the cya and crp genes . PhoB protein may act as a repressor for this upstream promoter, which also overlaps the upstream third pho box . The downstream promoter was induced by phosphate starvation and requires the PhoB protein for its activation as do the other pho regulon promoters . These results suggest that the two promoters function alternately in responding to phosphate or carbon starvation, thus providing the cell with a means to adapt to these physiological stresses. J Bacteriol, 1991 Jan, 173(2), 514 - 20 Nucleotide sequence of Escherichia coli katE, which encodes catalase HPII; von Ossowski I et al.; A 3,466-bp nucleotide sequence containing the katE gene of Escherichia coli has been determined . An open reading frame of 2,259 bp was found and was preceded by a potential ribosome-binding site . The predicted N-terminal sequence agreed with the sequence determined by direct amino acid sequencing, and the predicted direction of transcription was confirmed by expression of the gene cloned in both directions behind a T7 promoter . The start site of transcription was determined to be 127 bp upstream from the start of the open reading frame, and a potential RNA polymerase-binding site similar to a sequence preceding the xthA gene, which is also controlled by the KatF protein, was identified . The predicted sequence of the 753-amino-acid protein was compared with known sequences of other catalases, revealing significant similarity to the shorter catalases, including the residues in the putative active site and residues involved in heme binding. J Bacteriol, 1991 Jan, 173(1), 73 - 9 Characterization of a phage-plasmid hybrid (phasyl) with two independent origins of replication isolated from Escherichia coli; Gielow A et al.; The phage-plasmid hybrid phasyl can replicate as a phage in the presence of a filamentous phage of Escherichia coli (M13, fl, fd) . The extragenic region of phasyl shows homology with the plus and the minus origins of filamentous phages . Insertion of a Cmr fragment into the plus origin or of a Kmr fragment into the minus origin resulted in a reduced transduction frequency, while insertion into other parts of the extragenic region did not . This suggests that phagelike replication of phasyl is mediated by an origin that coincides with the two homologous elements in the extragenic region . Autonomous replication of phasyl occurs from a second origin (oriA) that is located between positions 297 and 636 . This fragment mediates replication if the Arp protein is supplied in trans . Arp is the only phage-encoded protein and is essential for plasmidlike replication . No sequence homology to other known origins was found . Phasyl derivatives with either one of the two origins inactivated can be rescued via the alternative replication mode, suggesting that the two replication pathways are independent. J Bacteriol, 1991 Jan, 173(1), 59 - 66 The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro; Hudson JM et al.; The lactose promoter-operator region of Escherichia coli contains two binding sites for cyclic AMP receptor protein (CAP), two for the lactose repressor, and two for RNA polymerase . The high density of binding sites makes cooperative interactions between these proteins likely . In this study, we used the gel electrophoresis mobility shift assay and binding partition analysis techniques to determine whether the secondary CAP site influences the binding of CAP to the principal CAP site in the lactose promoter when both are present on a linear DNA molecule . Such an effect could occur through the formation of a bridged DNA-CAP-DNA structure, through the interaction of CAP molecules bound to each of the sites, or through allosteric effects caused by CAP-mediated DNA bending . We found, however, that the interaction of CAP with these sites was not cooperative, indicating that CAP sites 1 and 2 bind CAP in an independent manner. J Bacteriol, 1991 Jan, 173(1), 412 - 6 Temperature sensitivity caused by missense suppressor supH and amber suppressor supP in Escherichia coli; Thorbjarnardottir S et al.; The temperature-sensitive missense suppressor supH and amber suppressor supP in Escherichia coli are mutations of the serU and leuX genes, respectively . The supH tRNA, tRNA(SerCAA), is expected to recognize UUG codons, which are normally read by tRNA(LeuCAA) and tRNA(LeuUAA), coded for by the leuX gene and the leuZ gene, respectively . We show that supP and supH are incompatible and that strains carrying both supP and a restrictive rpsL allele are temperature sensitive . It is suggested that the temperature sensitivity of both supH and supP strains is caused by deficient reading of UUG codons by tRNA(LeuUAA). J Bacteriol, 1991 Jan, 173(1), 334 - 44 Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli; Dicker IB et al.; The Escherichia coli gene firA, previously reported to code for a small, histonelike DNA-binding protein, has been cloned and found to reside immediately downstream from skp, a gene previously identified as the firA locus . firA encodes a 36-kDa protein . The mutant firA200(Ts) allele was also cloned and shown to contain three mutations, each mutation giving rise to a single amino acid change . Partially purified wild-type FirA (from a firA+ strain) and mutant FirA {from a firA200(Ts) strain} proteins have amino-terminal sequences predicted from their common DNA sequences . Both proteins lack an N-terminal methionine . Modest overexpression of wild-type or mutant FirA restored wild-type growth to firA200(Ts) strains at 43 degrees C, whereas high-level expression of wild-type FirA was required for more complete suppression of the rifampin sensitivity of firA200(Ts) rpoB double mutants . High-level expression of mutant FirA did not suppress this rifampin sensitivity. J Bacteriol, 1991 Jan, 173(1), 229 - 33 Detergent (sodium dodecyl sulfate) shock proteins in Escherichia coli; Adamowicz M et al.; The protein composition of Escherichia coli W3110 grown in the presence and absence of 5% sodium dodecyl sulfate (SDS) was examined by two-dimensional gel electrophoresis . In SDS-grown cells, at least 4 proteins were turned on, 13 were turned off, 15 were elevated, and 15 were depressed . The 19 unique and elevated SDS-induced spots constituted 7.91% of the total 35S-labeled protein . There was no apparent overlap between these 19 detergent (SDS) stress proteins and those of other known bacterial stress responses . The detergent stress stimulon is a distinct and independent stimulon . Its physiological relevance probably derives from the presence of bile salts in animal gastrointestinal tracts. J Bacteriol, 1991 Jan, 173(1), 191 - 6 Synthesis and functioning of the colicin E1 lysis protein: comparison with the colicin A lysis protein; Cavard D; The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either {35S}cysteine or {3H}lysine . This 3-kDa protein was acylated, as shown by {2-3H}glycerol labeling, and seemed to correspond to the mature CelA protein . The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein . In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment . Thus, the rate of synthesis would not be specific to lysis proteins . Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure . This particular property would play a role in the mechanism of colicin export . The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal . Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells . They were minimal in pldA mutants. J Bacteriol, 1991 Jan, 173(1), 141 - 9 Lanthanide accumulation in the periplasmic space of Escherichia coli B; Bayer ME et al.; Treatment of growing Escherichia coli B with lanthanide ions {lanthanum(III), terbium(III), and europium(III)} and subsequent aldehyde-OsO4 fixation caused areas of high contrast to appear within the periplasm (the space between inner and outer membrane of the cell envelope) . X-ray microanalysis of ultrathin sections of Epon-embedded or acrylic resin-embedded cells revealed the presence of the lanthanide and of phosphorus in the areas, whose contrast greatly exceeded that of other stained structures . Comparatively small amounts of the lanthanide were also present in the outer membrane and in the cytoplasm . The distribution of the periplasmic areas of high contrast was found to be random and not clustered at areas of current or future septum formation . Irregular cell shapes were observed after lanthanide treatment before onset of fixation . In contrast to glutaraldehyde-OsO4 fixation, glutaraldehyde used as the sole fixer caused a scattered distribution of the lanthanide . Cryofixation (slam-freezing) and freeze substitution revealed a lanthanum stain at both the periplasm and the outer part of the outer membrane . Deenergization of the cell membrane by either phage T4 or carbonyl cyanide m-chlorophenylhydrazone abolished the metal accumulation . Furthermore, addition of excess calcium, administered together with the lanthanide solution, diminished the quantity and size of areas of high contrast . Cells grown in media of high NaCl concentration revealed strongly stained areas of periplasmic precipitates, whereas cells grown under low-salt conditions showed very few high-contrast patches in the periplasm . Terbium treatment (during fixation) enhanced the visibility of the sites of inner-outer membrane contact (the membrane adhesion sites) in plasmolized cells, possibly as the result of an accumulation of the metal at the adhesion domains . The data suggest a rapid interaction of the lanthanides with components of the cell envelope, the periplasm, and the energized inner membrane. J Bacteriol, 1991 Jan, 173(1), 108 - 15 Cloning, nucleotide sequence, and characterization of mtr, the structural gene for a tryptophan-specific permease of Escherichia coli K-12; Heatwole VM et al.; The mtr gene of Escherichia coli K-12 encodes an L-tryptophan-specific permease . This gene was originally identified through the isolation of mutations in the 69-min region of the chromosome, closely linked to argG . Cells with lesions in mtr display a phenotype of 5-methyltryptophan resistance . The mtr gene was cloned by using the mini-Mu system . The amino acid sequence of Mtr (414 codons), deduced by DNA sequence analysis, was found to be 33% identical to that of another single-component transport protein, the tyrosine-specific permease, TyrP . The hydropathy plots of the two permeases were similar . Possible operator sites for the tyrosine and tryptophan repressors are situated within the region of DNA that is likely to be the mtr promoter. J Bacteriol, 1991 Jan, 173(1), 101 - 7 Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12; Austin D et al.; Aerobic sn-glycerol 3-phosphate dehydrogenase, encoded by the glpD gene of Escherichia coli, is a cytoplasmic membrane-associated respiratory enzyme . The nucleotide sequence of glpD was determined . An open reading frame of 501 codons was preceded by a consensus Shine-Dalgarno sequence . The proposed translational start and reading frame of glpD were confirmed by determining the nucleotide sequence across the fusion joint of a glpD-lacZ translational fusion . The predicted molecular weight, 56,750, corresponds well with the reported value of 58,000 for purified sn-glycerol 3-phosphate dehydrogenase . The flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins from E . coli . Repetitive extragenic palindromic sequences were identified downstream of the glpD coding region . The site for transcription termination was located between 87 and 216 bp downstream of the translation stop codon. Infect Immun, 1991 Jan, 59(1), 357 - 64 Polymorphism in the aerobactin-cloacin DF13 receptor genes from an enteroinvasive strain of Escherichia coli and pColV-K30 is associated only with a decrease in cloacin susceptibility; Marolda CL et al.; We have cloned chromosomal genes mediating the aerobactin iron transport system from the enteroinvasive strain Escherichia coli 978-77 . The physical map of the region spanning the siderophore biosynthesis genes and the upstream portion of the receptor gene in strain 978-77-derived clones was identical to the corresponding regions in pColV-K30, while the downstream portion was different . Recombinant plasmids derived from strain 978-77 encoded a 76-kDa outer membrane protein, in contrast to the 74-kDa polypeptide encoded by similar clones derived from pColV-K30 . No differences were found in the uptake of ferric aerobactin mediated by either the 76-kDa- or the 74-kDa-encoding plasmids . In contrast, cells containing the 76-kDa-encoding plasmids showed a 16-fold decrease in susceptibility to cloacin compared with cells harboring the 74-kDa-encoding plasmids . Two classes of chimeric aerobactin receptor genes were constructed by exchanging sequences corresponding to the downstream portion from the aerobactin receptor gene of both systems . The pColV-K30-978-77 chimeric gene encoded a 76-kDa outer membrane protein which mediated a low level of cloacin susceptibility, whereas the 978-77-pColV-K30 type encoded a protein of 74 kDa determining a level of cloacin susceptibility identical to that mediated by pColV-K30. Infect Immun, 1991 Jan, 59(1), 168 - 71 Heat-labile enterotoxin can be released from Escherichia coli cells by host intestinal factors; Hunt PD et al.; The heat-labile enterotoxin (LT) of Escherichia coli is localized in the periplasm of the bacterial cell . Growth of an LT-producing strain of E . coli in the presence of bile salts, in concentrations within the range found in the human small intestine, caused leakage of LT into the culture medium . This leakage could be augmented by the presence in the medium of trypsin and by minimizing the concentration of free iron. Infect Immun, 1991 Jan, 59(1), 109 - 13 Resistance of Escherichia coli to osmotically introduced complement component C9; Dankert JR; Investigation into the action of osmotically introduced C9 in Escherichia coli (in the absence of any other complement components) revealed that C9 could inhibit inner membrane respiration and cause a decrease in the viability of cells that were normally complement sensitive . This effect is analogous to the loss of inner membrane function and viability due to the assembly of the C5b-9 complex on these cells . Complement-resistant cells showed no such inhibition of respiration or loss of viability when subjected to the osmotic introduction of C9 . The reason for this failure of C9 to affect complement-resistant cells was explored to determine whether this resistance to C9 was due to an inability of proteins in general to be osmotically introduced into the complement-resistant cells . The protein toxins melittin and colicin E1 were showed to be able to kill these complement-resistant cells (as well as complement-sensitive cells) when osmotically introduced into the periplasm . Therefore, cellular resistance to osmotically introduced C9 is not due to an inability of proteins to be introduced into the cells and may be related to a mechanism of cellular resistance to the C5b-9 complex. Am J Respir Cell Mol Biol, 1991 Jan, 4(1), 18 - 25 Synthesis and secretion of high- and low-molecular-weight forms of the enzyme-releasing peptide (ERP) from the macrophage-like cell line THP-1; MacArthur C et al.; Neutrophil enzymes have been implicated as a source of lung injury in patients with the adult respiratory distress syndrome (ARDS) and with emphysema . We studied a human alveolar macrophage-derived peptide messenger, the enzyme-releasing peptide (ERP), which causes neutrophils to secrete their enzymes . The secretion and synthesis of ERP was studied in human alveolar macrophages and in the macrophage-like cell lines THP-1, HL-60, and U937 . All four cell types secrete an ERP-like peptide . THP-1 cells secrete a higher concentration of the peptide than do macrophages . The secretion of ERP by THP-1 is suppressed by the protein synthesis inhibitors actinomycin D and cycloheximide . While the macrophages secrete ERP, they do not synthesize it . These studies suggest that ERP is synthesized by an alveolar macrophage precursor and stored in the mature macrophage for later release . 12-O-tetradecanoylphorbol-13-acetate (TPA) suppresses ERP secretion by THP-1 cells, but it does not modify secretion in macrophages . Escherichia coli-derived lipopolysaccharide and dimethyl sulfoxide do not modify secretion in either cell type . The THP-1 cells secrete a high- and low-mass-ratio (Mr) form of ERP-like proteins . The low Mr but not the high Mr form stimulates neutrophils to secrete their granule enzymes . We conclude that human alveolar macrophages secrete ERP but do not synthesize it . It is likely that ERP is made by an alveolar macrophage precursor in a high Mr form that is cleaved prior to secretion by the macrophages. Proc Natl Acad Sci U S A, 1991 Jan 1, 88(1), 291 - 5 Selection of suppressor methionyl-tRNA synthetases: mapping the tRNA anticodon binding site; Meinnel T et al.; Accurate aminoacylation of a tRNA by Escherichia coli methionyl-tRNA synthetase (MTS) is specified by the CAU anticodon . A genetic screening procedure was designed to isolate MTS mutants able to aminoacylate a methionine amber tRNA (CUA anticodon) . Selected suppressor MTS enzymes all possess one or several mutations in the vicinity of Trp-461, a residue that is the major contributor to the stability of complexes formed with tRNAs having the cognate CAU anticodon . Analysis of catalytic properties of purified suppressor enzymes shows that they have acquired an additional specificity toward the amber anticodon without complete disruption of the methionine anticodon site . It is concluded that both positive and negative discrimination toward the binding of tRNA anticodon sequences is restricted to a limited region of the synthetase, residues 451-467. Proc Natl Acad Sci U S A, 1991 Jan 1, 88(1), 209 - 13 Enzymatic aminoacylation of single-stranded RNA with an RNA cofactor; Musier-Forsyth K et al.; A chemically synthesized single-stranded ribonucleotide tridecamer derived from the 3' end of Escherichia coli alanine tRNA can be charged with alanine in the presence of short complementary RNA oligonucleotides that form duplexes with the 3' fragment . Complementary 5' oligomers of 9, 8, 6, and 4 nucleotides all confer charging of the 3' fragment . Furthermore, in the presence of limiting 5' oligomer, greater than stoichiometric amounts of the single-stranded 3' acceptor fragment can be aminoacylated . This is due to a reiterative process of transient duplex formation followed by charging, dissociation of the 5' oligomer, and then rebinding to an uncharged single-stranded ribotridecamer so as to create another transient duplex substrate . Thus, a short RNA oligomer serves as a cofactor for a charging enzyme, and it thereby makes possible the aminoacylation of single-stranded RNA . These results expand possibilities for flexible routes to the development of early charging and coding systems. Proc Natl Acad Sci U S A, 1991 Jan 1, 88(1), 189 - 93 Genetic disease detection and DNA amplification using cloned thermostable ligase; Barany F; Polymerase chain reaction, using thermostable DNA polymerase, has revolutionized DNA diagnostics . Another thermostable enzyme, DNA ligase, is harnessed in the assay reported here that both amplifies DNA and discriminates a single-base substitution . This cloned enzyme specifically links two adjacent oligonucleotides when hybridized at 65 degrees C to a complementary target only when the nucleotides are perfectly base-paired at the junction . Oligonucleotide products are exponentially amplified by thermal cycling of the ligation reaction in the presence of a second set of adjacent oligonucleotides, complementary to the first set and the target . A single-base mismatch prevents ligation/amplification and is thus distinguished . This method was exploited to detect 200 target molecules as well as to discriminate between normal beta A- and sickle beta S- globin genotypes from 10-microliters blood samples. Mutat Res, 1991 Jan, 254(1), 97 - 105 Ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts; Seetharam S et al.; In order to examine possible cell-type specificity in mutagenic events, a shuttle-vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in Epstein-Barr virus transformed lymphoblastoid cell lines from a patient, XP12BE, with xeroderma pigmentosum (XP), group A, and a normal control . XP is a skin-cancer-prone disorder with UV hypersensitivity and defective DNA repair . Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of E . coli . An earlier report on this data {Seetharam et al., (1990) J . Mol . Biol., 212, 433} indicated lower survival and higher mutation frequency with the UV-treated plasmid passed through the XP12Be(EBV) line . In the present report, sequence analysis of 198 mutant plasmids revealed a predominance of G:C----A:T transitions with both lymphoblastoid cell lines . This finding is consistent with the bias of polymerases toward insertion of an adenine opposite non-coding photoproducts (dinucleotides or other lesions) . Transversion mutagenesis, non-adjacent double mutations, and triple-base mutations may involve other mechanisms . These results were compared to similar data from a fibroblast line from the same patient {Bredberg et al., (1986) Proc . Natl . Acad . Sci . (U.S.A.), 83, 8273} . The frequency of G:C----A:T transitions was higher, and there were fewer plasmids with multiple-base substitutions and with transversion mutations with both XP lymphoblasts and fibroblasts than with the normal lymphoblasts and fibroblasts . There were no significant differences in classes or types of mutations in the UV-treated plasmid replicated in the XP lymphoblasts and the XP fibroblasts . This suggests that the major features of UV mutagenesis in different cell types from the same individual are similar. Mutat Res, 1991 Jan, 254(1), 37 - 44 Roles of two types of O6-methylguanine-DNA methyltransferases in DNA repair; Takano K et al.; Escherichia coli possesses 2 types of O6-methylguanine-DNA methyltransferases, one inducible and the other constitutive . These enzymes are coded by the ada and the ogt genes, respectively . Using a synthetic ogt-specific probe, we mapped ogt at 29.4 min, near the 5'-flanking region of the nirR gene, on the E . coli chromosome . To elucidate the roles of the 2 types of methyltransferases in DNA repair, we constructed mutant strains which lack either one or both of the genes . In either the ada+ or the ada- background, the ogt mutation had no effect on cell survival after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment . On the other hand, ada- ogt- cells were more prone to mutation as compared to the ada- ogt+ cells exposed to MNNG . The frequency of spontaneous mutation of cells defective in either one or both of the genes was the same, however, the introduction of the ogt+ plasmid into the cells produced a 2-3-fold decrease in the frequency of spontaneous mutation . O6-Methylguanine-DNA methyltransferases appear to eliminate premutagenic DNA lesions not only from cells exposed to alkylating agents but also from those grown in the absence of the agents. Mutat Res, 1991 Jan, 254(1), 27 - 35 Construction of Escherichia coli K12 phr deletion and insertion mutants by gene replacement; Akasaka S et al.; We replaced an Escherichia coli phr gene by a 1.4-kb fragment of DNA coding for resistance to chloramphenicol . Characterization of 2 deletions (phr-19 and phr-36) and 1 insertion (phr-34) in the phr gene revealed no photoreactivation . Photoreactivation-deficient strains of either recA56 or lexA1(ind-) were more sensitive to UV radiation in the dark than phr-proficient counterparts . The presence of the phr defect in uvrA6 strains increased by 1.5-2-fold his-4(Ochre) to His+ mutation induced by ultraviolet light compared to uvrA6 phr+ strains, although there was no difference in UV sensitivity between uvrA6 phr+ and uvrA6 phr- strains . 30-35% of the His+ mutations thus induced were suppressor mutations in uvrA6 phr+ and 49-55% in uvrA6 phr- strains . The UV mutagenesis results are consistent with the previous observations that suppressor mutations targeted by a thymine-cytosine pyrimidine dimer are reduced in the dark in cells with amplified DNA photolyase. Mutat Res, 1991 Jan, 254(1), 1 - 12 An endonuclease activity of Escherichia coli that specifically removes 8-hydroxyguanine residues from DNA; Chung MH et al.; An enzyme that specifically removes an 8-hydroxyguanine (8-OH-Gua) residue in DNA has been purified from Escherichia coli . To assay the enzymatic activity, a synthetic double-stranded DNA (dsDNA) containing 8-OH-Gua at a defined position was used as a substrate . The substrate DNA was simultaneously cleaved at 2 sites, i.e., the phosphodiester bonds 5' and 3' to 8-OH-Gua, leaving a phosphate at each of the neighboring deoxynucleosides . The cleavage was observed only in dsDNA, but not with single-stranded DNA containing 8-OH-Gua . This enzyme showed almost no activity on DNAs containing other kinds of modified bases such as 8-hydroxyadenine, O6-methylguanine and N7-methylguanine . Also DNAs containing mismatches (A/G or C/T) were not cleaved . Studies on several other properties of this enzyme indicate that it differs from endonucleases previously isolated from E . coli, indicating that it is likely to be an endonuclease which specifically recognizes 8-OH-Gua in dsDNA. Mutat Res, 1991 Jan, 246(1), 103 - 7 Caffeine-induced reduction of the survival of gamma-irradiated HeLa cells and the reversal of the caffeine effect by Escherichia coli RecA protein; Spivak IM et al.; It is confirmed that survival of gamma-irradiated HeLa cells is decreased by post-treatment with caffeine . The caffeine effect is believed to be the result of an inhibition of the repair of gamma-ray-induced DNA damage . In this work we show that the caffeine-induced reduction of the survival of gamma-irradiated HeLa cells is reversed when Escherichia coli RecA protein is introduced into the cells with the aid of liposomes. Mol Cell Biol, 1991 Jan, 11(1), 445 - 57 Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system; Jessberger R et al.; We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions . The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions . Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene . The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA . Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA . In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates . Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction . With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed. J Virol, 1991 Jan, 65(1), 499 - 504 The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces; Lai CF et al.; The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined . A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S . Maa, J . F . Rodriguez, and M . Esteban, J . Biol . Chem . 265:1569-1577, 1990) . To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32 . The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein . Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E . coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability . Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor . The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor. J Virol, 1991 Jan, 65(1), 445 - 9 Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein; Dillon PJ et al.; The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products . Site-directed mutations were made within the HIV-2 rev gene to identify functional domains . We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev . We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus . Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization . Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional. J Virol, 1991 Jan, 65(1), 155 - 61 Expression of yeast L-A double-stranded RNA virus proteins produces derepressed replication: a ski- phenocopy; Wickner RB et al.; The plus strand of the L-A double-stranded RNA virus of Saccharomyces cerevisiae has two large open reading frames, ORF1, which encodes the major coat protein, and ORF2, which encodes a single-stranded RNA-binding protein having a sequence diagnostic of viral RNA-dependent RNA polymerases . ORF2 is expressed only as a Gag-Pol-type fusion protein with ORF1 . We have constructed a plasmid which expresses these proteins from the yeast PGK1 promoter . We show that this plasmid can support the replication of the killer toxin-encoding M1 satellite virus in the absence of an L-A double-stranded RNA helper virus itself . This requires ORF2 expression, providing a potential in vivo assay for the RNA polymerase and single-stranded RNA-binding activities of the fusion protein determined by ORF2 . ORF1 expression, like a host ski- mutation, can suppress the usual requirement of M1 for the MAK11, MAK18, and MAK27 genes and allow a defective L-A (L-A-E) to support M1 replication . These results suggest that expression of ORF1 from the vector makes the cell a ski- phenocopy . Indeed, expression of ORF1 in a wild-type killer makes it a superkiller, suggesting that a target of the SKI antiviral system may be the major coat protein. J Virol, 1991 Jan, 65(1), 147 - 54 Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor; Liljestrom P et al.; The proteolytic processes involved in the cotranslational production of the Semliki Forest virus proteins p62, 6K, and E1 from a common precursor polypeptide were analyzed by an in vitro translation-translocation assay . By studying the behavior of wild-type and mutant variants of the polyprotein, we show that the signal sequences responsible for membrane translocation of the 6K and E1 proteins reside in the C-terminal regions of p62 and 6K, respectively . We present evidence suggesting that the polyprotein is processed on the luminal side by signal peptidase at consensus cleavage sites immediately following the signal sequences . Our results also lead us to conclude that the 6K protein is a transmembrane polypeptide with its N terminus on the luminal side of the membrane (type I) . Thus, the production of all three membrane proteins is directed by alternating signal and stop-transfer (anchor) sequences that function in translocation and cleavage of the virus precursor polyprotein . This also shows conclusively that internally located signal sequences can be cleaved by signal peptidase. Dev Biol, 1991 Jan, 143(1), 173 - 84 A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana; Thorsness MK et al.; The S-locus glycoprotein gene of Brassica is derived from the genetic locus that controls the self-incompatibility response and the specific recognition between pollen and stigma . The promoter of this gene was used to direct expression of the diphtheria toxin A chain gene and the Escherichia coli beta-glucuronidase gene in transgenic Nicotiana tabacum . Expression of the promoter in cells of the pistil and in pollen suggests that a single gene may direct the self-incompatibility response in the two interacting cell types . Additionally, the fusion genes were expressed gametophytically in the heterologous host species, Nicotiana, rather than sporophytically as expected for Brassica . Thus, although the genes involved in self-incompatibility in Brassica and Nicotiana are not homologous in their coding regions, signals for expression of these genes are apparently conserved between the two genera . Our analysis of toxic gene fusion transformants shows that genetic ablation is useful for probing developmental processes and for studying temporal and spatial patterns of gene expression in plants. Dig Dis Sci, 1991 Jan, 36(1), 75 - 81 Penetration of lanthanum through the main pancreatic duct epithelium in cats following exposure to infected human bile; Arendt T; The main pancreatic duct epithelium acts as a barrier to the diffusion of molecules from the duct lumen into pancreatic acinar and interstitial tissue . We studied sequential ultrastructural characteristics of the loss of epithelial barrier function in the cat using lanthanum, an electron-opaque tracer, following perfusion of the duct from the tail to the duodenum with infected human bile . Tight junctions between duct epithelial cells were found to become permeable to the tracer as early as after 15 min of exposure . Later, there was progressive disintegration of intercellular junctions and epithelial loss . Lanthanum penetrated the duct epithelium exclusively on an intercellular path . Loss of barrier function of the pancreatic duct epithelium was consistently associated with subsequent development of acute interstitial edematous pancreatitis . There was no association between the degree of duct epithelial damage and the severity of acute pancreatitis . Both bile and a suspension of bacteria alone were not harmful to the pancreas . Sequential perfusion produced acute pancreatitis only when at first bile and then the bacterial suspension was perfused . A reversed succession of perfusates produced no morphologic alterations . We conclude: (1) Increased tight junction permeability is an early lesion in acute bile-induced pancreatitis: (2) loss of duct epithelial barrier function is important for the initiation but not for the severity of the inflammation; and (3) bile renders duct epithelial intercellular junctions vulnerable to Escherichia coli bacteria. Pediatrics, 1991 Jan, 87(1), 18 - 27 Bismuth subsalicylate in the treatment of acute diarrhea in children: a clinical study; Soriano-Brucher H et al.; Bismuth subsalicylate (BSS) and placebo were evaluated in a double-blind, placebo-controlled study as adjunct to rehydration therapy in 123 children, aged 4 to 28 months, hospitalized with acute diarrhea . The dosing regimen was 20 mg/kg five times daily for 5 days . Significant benefits were noted in the BSS group compared with placebo as manifested by decreases in stool frequency and stool weights and an improvement in stool consistency, significant improvement in clinical well-being, and shortening of the disease duration . Patients treated with BSS had a significant reduction in duration of hospital stay (6.9 days) compared with placebo-treated patients (8.5 days) . Also, intravenous fluid requirements decreased significantly more rapidly and to a greater degree in the BSS-treated group . Bismuth subsalicylate was associated with clearance of pathogenic Escherichia coli from the stools in 100% of cases but was not different from placebo in rotavirus elimination . Bismuth subsalicylate was well tolerated with no reported adverse effects . Blood bismuth and serum salicylate levels were well below levels considered toxic . In this study, BSS provided effective adjunctive therapy for acute diarrhea, allowing children to get well sooner with less demand on the nursing and hospital staff. J Infect Dis, 1991 Jan, 163(1), 89 - 95 Pretreatment with ibuprofen augments circulating tumor necrosis factor-alpha, interleukin-6, and elastase during acute endotoxinemia; Spinas GA et al.; Plasma levels of tumor necrosis factor-alpha (TNF alpha), interleukin-1 (IL-1), and interleukin-6 (IL-6) were monitored after intravenous administration of Escherichia coli endotoxin with or without ibuprofen pretreatment to healthy volunteers . Intravenous endotoxin (n = 7) resulted in elevated plasma TNF alpha concentrations with maximal levels at 90 min (369 +/- 44 pg/ml, P less than .001 vs . saline controls, n = 7) . The rise in TNF-alpha was followed by a rise in plasma IL-6 (27 +/- 12.8 ng/ml), peaking 30-90 min thereafter . Pretreatment with ibuprofen (n = 6) caused a significant augmentation and temporal shift in cytokine elaboration with maximal TNF alpha levels (627 +/- 136 pg/ml) at 120 min and IL-6 peaks (113 +/- 66 ng/ml) at 180 min . In ibuprofen-treated volunteers, the additional increase in TNF alpha was paralleled by increased levels of circulating elastase . In vitro experiments suggest a causal relationship between these events . Thus, the cyclooxygenase inhibitor ibuprofen blunts the clinical response to endotoxin but augments circulating cytokine levels and leukocyte degranulation. Proteins, 1991, 11(1), 35 - 44 Thermodynamics of ligand binding and denaturation for His64 mutants of tissue plasminogen activator kringle-2 domain; Kelley RF et al.; The contribution of His64 to the function and stability of tissue plasminogen activator (t-PA) kringle-2 domain (His244 in t-PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild-type kringle-2 and mutants having His64 replaced with Tyr or Phe . This site was examined because modeling studies suggested that the His64 side chain could play an important role in ligand binding by forming an ion-pair with the carboxylate of the ligand, L-lysine . Kringle-2 domains were expressed by secretion of the 174-263 portion of t-PA in E . coli and purified as previously described for the wild-type domain . Both mutant proteins retain affinity for L-lysine, although reduced three- to four-fold relative to wild-type, demonstrating that His64 does not interact with the ligand carboxylate through an ion-pair interaction or by hydrogen bonding . The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 A chain length, whereas the wild-type domain prefers an 8.8 A long ligand . For both wild-type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (delta H = -6,000 to -7,000 cal/mol) and T delta S accounts for less than 15% of delta G . In addition, the H64Y mutant differs from wild-type in the effect of ligand alpha-amino group modification on binding affinity . Based on examination of the x-ray structure recently determined for wild-type kringle-2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site . Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild-type protein with the difference in stabilization free energy (delta delta G) equal to 2.7 kcal/mol at 25 degrees C and pH 3 . The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr. Proteins, 1991, 11(1), 13 - 28 Structural and functional relations among thioredoxins of different species; Eklund H et al.; Three-dimensional models have been constructed of homologous thioredoxins and protein disulfide isomerases based on the high resolution x-ray crystallographic structure of the oxidized form of Escherichia coli thioredoxin . The thioredoxins, from archebacteria to humans, have 27-69% sequence identity to E . coli thioredoxin . The models indicate that all the proteins have similar three-dimensional structures despite the large variation in amino acid sequences . As expected, residues in the active site region of thioredoxins are highly conserved . These include Asp-26, Ala-29, Trp-31, Cys-32, Gly-33, Pro-34, Cys-35, Asp-61, Pro-76, and Gly-92 . Similar residues occur in most protein disulfide isomerase sequences . Most of these residues form the surface around the active site that appears to facilitate interactions with other enzymes . Other structurally important residues are also conserved . A proline at position 40 causes a kink in the alpha-2 helix and thus provides the proper position of the active site residues at the amino end of this helix . Pro-76 is important in maintaining the native structure of the molecule . In addition, residues forming the internal contact surfaces between the secondary structural elements are generally unchanged such as Phe-12, Val-25, and Phe-27. Proteins, 1991, 11(1), 1 - 12 The electrostatic potential of Escherichia coli dihydrofolate reductase; Bajorath J et al.; Escherichia coli dihydrofolate reductase (DHFR) carries a net charge of -10 electrons yet it binds ligands with net charges of -4 (NADPH) and -2 (folate or dihydrofolate) . Evaluation and analysis of the electrostatic potential of the enzyme give insight as to how this is accomplished . The results show that the enzyme is covered by an overall negative potential (as expected) except for the ligand binding sites, which are located inside "pockets" of positive potential that enable the enzyme to bind the negatively charged ligands . The electrostatic potential can be related to the asymmetric distribution of charged residues in the enzyme . The asymmetric charge distribution, along with the dielectric boundary that occurs at the solvent-protein interface, is analogous to the situation occurring in superoxide dismutase . Thus DHFR is another case where the shape of the active site focuses electric fields out into solution . The positive electrostatic potential at the entrance of the ligand binding site in E . coli DHFR is shown to be a direct consequence of the presence of three positively charged residues at positions 32, 52, and 57--residues which have also been shown recently to contribute significantly to electronic polarization of the ligand folate . The latter has been postulated to be involved in the catalytic process . A similar structural motif of three positively charged amino acids that gives rise to a positive potential at the entrance to the active site is also found in DHFR from chicken liver, and is suggested to be a common feature in DHFRs from many species . It is noted that, although the net charges of DHFRs from different species vary from +3 to -10, the enzymes are able to bind the same negatively charged ligands, and perform the same catalytic function. Ciba Found Symp, 1991, 159, 103 - 12; discussion 112-7 Catalytic antibodies: contributions from engineering and expression in Escherichia coli; Pluckthun A et al.; Antibodies have been raised against the transition state of many reactions and shown to catalyse the relevant reaction . Their moderate catalytic efficiencies can be increased by protein engineering, if ways can be found to express the engineered antibody . We have developed a system by which fully functional Fv and Fab fragments can be expressed in Escherichia coli . The Fv fragment dissociates at low concentrations; we therefore devised methods to stabilize the fragment . We showed that the Fv fragment of the antibody McPC603, a phosphorylcholine-binding immunoglobulin A, binds the antigen with the same affinity as does the intact antibody isolated from mouse ascites . Phosphorylcholine is an analogue of the transition state for the hydrolysis of choline carboxylate ester . The Fv fragment of McPC603 catalysed this hydrolysis . Mutational analysis of the residues in the binding site of the antibody has shown which are essential for binding and for catalysis, and the importance of charged residues in certain positions . The E . coli expression system combined with protein engineering and screening methods will facilitate understanding of enzyme catalysis and the development of new catalytic antibodies. Nephrol Dial Transplant, 1991, 6(8), 554 - 6 Protective role of BCG in the rabbit model of mesangial proliferative glomerulonephritis; Xu JJ et al.; A study was designed to investigate whether BCG could play a protective role in a rabbit model of mesangial proliferative glomerulonephritis . Fifteen rabbits were immunised with multiple injections of bovine serum albumin and their mononuclear phagocytic system was depressed by endotoxin from E . coli . The rabbits were divided into two groups: Group 1 (n = 7) received intravenous BCG from 3 weeks prior to the pathogenic immunisation and until the end of this period; Group 2 (n = 8) acted as a control and received normal saline . In the BCG group circulating immune complex (CIC) titres were significantly reduced, rabbit IgG deposition in glomeruli was significantly less, and mean glomerular cell counts were significantly less than those in the control group . We conclude that BCG stimulates the mononuclear phagocytic system to remove CIC and reduce the deposition of immune complexes in glomeruli, thereby mitigating the inflammatory response. Intensive Care Med, 1991, 17(5), 293 - 8 The consequences of continuous haemofiltration on lung mechanics and extravascular lung water in a porcine endotoxic shock model; Stein B et al.; Endotoxinaemia (E . coli endotoxin, 0.111.B4) and pulmonary hypertension were evoked in 20 swine, randomly assigned to receive either zero-balanced venovenous haemofiltration (HF) with an ultrafiltration and replacement rate of 600 ml/h (HF group, n = 10) or to undergo an uninfluenced spontaneous course (E group, n = 10) during a constant infusion of endotoxin until the end of the experiment . Endotoxin-induced pulmonary dysfunction was assessed on the basis of extravascular lung water (EVLW) using a thermo-dye technique via a fiberoptic intra-aortic probe, gas exchange and lung mechanics, the latter derived by a pressure-volume loop (P/V loop) of the respiratory system (super syringe, flow 30 ml/s, tidal volume 600 ml) . A comparable increase in alveolo-arterial oxygen difference and a constant EVLW was observed in both groups . The progressive deterioration of hysteresis area and compliance parameters by endotoxinaemia was significantly blunted by HF . Independent of an impact on pulmonary oedema zero-balanced HF modifies endotoxin induced lung injury, probably by the convective transport of mediator substances. J Immunoassay, 1991, 12(3), 293 - 304 Comparison of sandwich-ELISA and GM1-ELISA for the detection of Escherichia coli thermolabile enterotoxin; Drevet P et al.; Two different microtiter plate ELISA tests were devised for the detection of Escherichia coli thermolabile toxin (LTh) either free or extracted from isolated colonies . Both tests used as detection systems purified anti-LTh rabbit immunoglobulins conjugated to biotin, streptavidin peroxidase and TMB . The tests differed by their capture phase which was the GM1-ganglioside for GM1-ELISA and purified anti-LTh rabbit immunoglobulins for sandwich ELISA . The two methods were rapid since they could be performed in less than 2 hours . The detection limits for purified LT were 50 pg/ml and 1.3 ng/ml for sandwich ELISA and GM1-ELISA respectively . For the detection of toxinogenic isolates the extraction buffer containing Triton X-100 was always superior to polymyxin buffer . Using the polymyxin extraction buffer the sandwich ELISA was again more sensitive than the GM1-ELISA since a lower number of isolated colonies could be used for the detection of positive strains . With the Triton X-100 buffer both ELISAs could detect positive strains using a single colony but the sandwich ELISA gave the highest delta OD . We concluded that our sandwich ELISA can rapidly detect either the free Escherichia coli thermolabile toxin or LTh producing strains and could be applied routinely. Eur Biophys J, 1991, 20(2), 71 - 8 Lipid-protein surface films generated from membrane vesicles: selfassembly, composition, and film structure; Schurholz T et al.; Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts . A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity . In all instances, 10 microliters vesicle suspensions containing 25 micrograms protein yield at least 50 cm2 film area at a constant surface pressure of 12 mN/m within minutes . Upon formation, surface films are separated from vesicles by use of shear forces . Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams . The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer . Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure . Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure (greater than or equal to 15 mN/m) . Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (+/- 0.2) nm2. Cell Mol Biol, 1991, 37(5), 481 - 500 Further studies on the lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA; Bauluz C et al.; Our previous results on the genotoxic effect of 8-methoxypsoralen-induced lesions on pBR322 suggested an important involvement of an inducible error-free repair pathway in the repair of plasmid lesions . We present herein further results obtained in order to explore that possibility, together with a more general report on the subject . pBR322 treated with increasing concentrations of 8-MOP plus fixed UVA light irradiation was used to transform several E . coli strains differing in their repair capacities, and plasmid survival and mutagenesis were determined . Survival results suggested that crosslinks were completely lethal in pBR322 whereas monoadducts were partially removed from plasmid DNA mainly through an error-free excision pathway . A mutagenic repair pathway did not show a significant contribution to the total repair process . Cell preirradiation stimulated plasmid recovery in recA+ strains, including the umuC strain, thus confirming our previous results indicating that an inducible error-free repair had occurred . Globally, our results showed a strong requirement on the excision pathway for the repair of psoralen-damaged plasmid DNA . In contrast, the recA dependent pathway was needed only for SOS induction . After a theoretical correction of the data for estimating the effect only due to 8-MOP adducts, a different pattern of repair mechanisms appeared to be involved. Biomed Pharmacother, 1991, 45(4-5), 179 - 85 Differential utilization of 2',3'-dideoxyguanosine 5'-triphosphate as a substrate for various DNA polymerases; Ono K et al.; 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) was found to be an efficient substrate for DNA polymerase beta when activated DNA was used as the template.primer . Under the optimized reaction conditions with activated DNA, the rate of the incorporation of ddGTP into DNA was almost equal to that of the corresponding normal substrate dGTP . The Km value for ddGTP (1.8 microM) was smaller than that for dGTP (7.8 microM) . In contrast, ddGTP was not utilized as a substrate for DNA polymerase gamma with any of the activated DNA and (dC)n.(dG)12-18 as the template primer . Other DNA polymerases such as DNA polymerase alpha, E coli DNA polymerase I and retroviral reverse transcriptase could poorly utilize ddGTP as a substrate . Some of the kinetic properties of DNA polymerase beta revealed toward ddGTP are also described . Since DNA polymerase beta plays a role in DNA repair, the present results predict possible appearance of cytotoxicity or clinical side effect(s) of 2',3'-dideoxyguanosine (ddG), known as a potent inhibitor of human immunodeficiency virus, when ddG is administered to the patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex. Appl Theor Electrophor, 1991, 1(6), 339 - 41 An electrophoretic method to evaluate DNA polymerase activity; Ranganathan R et al.; We have devised an easy non-radioactive assay to evaluate the activity of six different DNA polymerases: the Klenow fragment of E.coli DNA Polymerase I, Taq DNA Polymerase, Sequenase, Moloney Murine Reverse Transcriptase, T7 DNA Polymerase and E.coli DNA Polymerase I . The method is based on the differential mobility of single stranded DNA compared to that of double-stranded DNA in agarose gel electrophoresis. Verh K Acad Geneeskd Belg, 1991, 53(3), 241 - 79 Role of prostaglandin-mediated mechanisms during experimentally induced endotoxin fever in the lactating goat; Massart-Leen AM et al.; The effects of endotoxin (LPS) on the cortisol, glucose, NEFA (non-esterified fatty acids), STH (somatotropin) and oxytocin levels in plasma of goats are described . The changes in plasma cortisol, STH and NEFA, as well as in RT (rectal temperature) were compared after i.v . and i.mam . administration of endotoxin . The other parameters, glucose and oxytocin, were followed only after i.v . endotoxin administration . The observed metabolic and hormonal alterations in plasma were also studied after pretreating the goats with the non-steroidal anti-inflammatory and antipyretic drug flurbiprofen in order to evaluate the possible involvement of prostaglandin in these phenomena . After i.v . administration of LPS a biphasic temperature curve for the highest dose of LPS with peak maxima at 1h and 4h after LPS challenge, was observed . Intramammary administration of endotoxin induces a monophasic fever response, with a latency time of approximately 3h, and peak values after 6h . The onset of the fever response in the i.v . experiments coincided with the oxytocin maximum and with early hyperglycemia . Intravenous endotoxin in goats also induces an increase in plasma NEFA, cortisol and STH . The early increase in NEFA, with a maximum after 2h and occurring before the fever peak, is followed by a significant rise in cortisol with peak effects after 3 h . The increase in plasma STH coincided with the decrease in plasma NEFA returning to control levels again . Peak concentrations in plasma STH occurred after 4 h . All the changes observed after the i.v . administration of endotoxin are dose-dependent . Pretreating goats with flurbiprofen completely abolished fever response, as well as the early hyperglycemia and the oxytocin release to i.v . LPS, indicating that these changes were prostaglandin-mediated and might be a reflexion of an activation of the sympathetic adrenomedullary system . The LPS-induced changes in plasma cortisol, NEFA and STH are only partly depressed and delayed by flurbiprofen . The residual hormonal responses to high doses of endotoxin suggest that an additional direct action of circulating endotoxins on the hypothalamus cannot be excluded . Intramammary LPS administration in goats only induced a very weak increase in plasma cortisol . The complex interplay of hormones and metabolic substances in the homeostasis of the inflammation reaction is discussed. Tsitologiia, 1991, 33(2), 103 - 9 {The restoration by Escherichia coli RecA protein of the survival of gamma-irradiated HeLa cells reduced by the DNA repair inhibitors caffeine and 3-aminobenzamide}; Spivak IM et al.; It is confirmed that inhibitors of DNA repair caffeine and 3-aminobenzamide decrease the survival of gamma-irradiated HeLa cells . It is shown that the decreased survival of irradiated cells is reversed when Escherichia coli RecA protein is introduced into cell nucleases with the aid of liposomes . This effect is more expressed in caffeine-treated (before or after irradiation) than in 3-aminobenzamide-treated (before irradiation) cells . It is suggested that E . coli 38 kD RecA protein may compensate the function of HeLa RecA-like protein, inhibited by DNA repair inhibitors, which is necessary for the repair of single-strand breaks and double-strand breaks of DNA. Chem Biol Interact, 1991, 80(1), 73 - 88 Genotoxicity of 1,3-dichloro-2-propanol in the SOS chromotest and in the Ames test . Elucidation of the genotoxic mechanism; Hahn H et al.; 1,3-Dichloro-2-propanol (1,3-DCP-OH, glycerol dichlorohydrin) is of great importance in many industrial processes and has been detected in foodstuffs, in particular in soup spices and instant soups . It has been shown to be carcinogenic, genotoxic and mutagenic . Its genotoxic mechanisms are, however, not yet entirely understood . We have investigated whether alcohol dehydrogenase (ADH) catalysed activation to the highly mutagenic and carcinogenic 1,3-dichloroacetone or formation of epichlorohydrin or other genotoxic compounds play a role for mutagenicity and genotoxicity . In our studies, no indications of ADH catalysed formation of 1,3-dichloropropane could be found, although we could demonstrate a clear activation by ADH in the case of 2-chloropropenol . Formation of allyl chloride could also be excluded . We found, however, clear evidence that epichlorohydrin formed chemically in the buffer and medium used in the test is responsible for genotoxicity . No indication was found that enzymatic formation of epichlorohydrin plays a role . Additional mutagenicity and genotoxicity studies with epichlorohydrin also confirmed the hypothesis that genotoxic effects of 1,3-DCP-OH depend on the chemical formation of epichlorohydrin. Eicosanoids, 1991, 4(2), 99 - 105 Beneficial effects of the prostacyclin analogue taprostene on cardiovascular, pulmonary and renal disturbances in endotoxin-shocked rabbits; Schneider J; The prostacyclin analogue taprostene protects against lethal endotoxemia in rats . In the present study, the effects of taprostene on endotoxin-induced cardiovascular, pulmonary and renal alterations have been investigated . In anesthetized rabbits, infusion of 0.5 mg/kg Escherichia coli lipopolysaccharide i.v . over 30 min produced systemic hypotension, pulmonary hypertension, and decreases in cardiac output, peripheral oxygen delivery and renal glomerular filtration rate . In endotoxemic rabbits treated with taprostene (0.2 micrograms.kg-1.min-1 i.v . over 180 min), the blood pressure tended to be lower than in untreated endotoxemic controls . Taprostene reduced the total peripheral resistance and abolished the endotoxin-induced increases in pulmonary artery pressure and resistance . Taprostene prevented the decreases in cardiac output and peripheral oxygen supply . At the end of the experiment the glomerular filtration rate was higher in taprostene-treated than in untreated endotoxemic rabbits and did not differ significantly from that in non-endotoxemic controls . The results show that taprostene prevents the pulmonary hypertension, preserves cardiac output and peripheral oxygen delivery, and substantially maintains the glomerular filtration rate in endotoxin-shocked rabbits. Tsitologiia, 1991, 33(1), 88 - 96 {The adaptive response to mitomycin C exposure in the hyper-radioresistant mutant Escherichia coli Gamr444}; Zhestianikov VD et al.; Adaptive response to mitomycin C (MC) (lethal effect and recovery of molecular mass of DNA) in hyper-radioresistant mutant Escherichia coli Gamr444 have been investigated . This mutant is more resistant to MC than parent strain E . coli K12 AB1157 . Adaptation of Gamr444 mutant to MC in nonlethal concentrations increases its resistance to MC in lethal concentrations with dose modification factor (DMF) 2.4 at the LD90 level . During the adaptation of this mutant to methyl-methane sulfonate (MMS) its resistance to this agent increases with DMF by 2.2 and resistance to MC with DMF by 1.5 times . During the adaptation of Gamr444 mutant to MC its resistance to MMS increases with DMF by 1.5 times . Adaptive response to MC abolishes by chloroamphenicol treatment during the adaptation . Adaptive response to nitrogen mustard (HN2) in E . coli Gamr444 is absent (HN2 induces cross-links in DNA as MC) . Degradation of DNA following the formation of cross-links in DNA takes place . Adaptation to MC in Gamr444 mutant leads to restoration of DNA molecular mass which is more quicker than in the case without adaptation . Adaptive restoration of DNA molecular mass after the MC treatment is absent in E . coli K12 AB1157 . The repair of cross-links in DNA after the treatment of HN2 in Gamr444 mutant takes place with equal rate both in the case of adaptation to HN2 and in the case without adaptation . It is proposed, that under the treatment of MC in E . coli Gamr444 the ada-alkA-dependent adaptive response takes place . This adaptive response is connected with alkylation of O6-guanine and elimination of the product by O6-alkyl-DNA-alkyltransferase . Partial recA-dependency of the adaptive response to MC allows to suggest the participation of another inducible system . The nature of this system is unknown. Int J Biochem, 1991, 23(7-8), 695 - 702 Molecular cloning of human beta 1,4-galactosyltransferase and expression of catalytic activity of the fusion protein in Escherichia coli; Chatterjee SK; 1 . Three groups of cDNA clones (total of six) for human UDP-galactose: beta N-acetylglucosamine galactosyltransferase (4 beta GT) were obtained by screening of a fetal liver library in lambda gt11 with an affinity purified anti4 beta GT antibody . 2 . One group of clones (three clones) reacted with two distinct anti4 beta GT murine monoclonal antibodies . 3 . Nucleotide sequence of this group of clones were similar to published sequence for human 4 beta GTcDNA, except the 74 nucleotides at the 5'-end . 4 . Partially purified fusion protein encoded by this group of clones showed all the catalytic properties of 4 beta GT, although the cDNA was partial and the protein was probably unglycosylated. Arch Microbiol, 1991, 155(5), 449 - 52 Electroporation and conjugal plasmid transfer to members of the genus Aquaspirillum; Eden PA et al.; Electroporation methods and conjugal matings were used to transfer several plasmid vectors to Aquaspirillum dispar and Aquaspirillum itersonii . The incompatibility P class plasmid RP4 was conjugally transferred from Escherichia coli HB101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free E . coli and A . dispar strains via conjugal matings . High-voltage electrotransformation was used to transfer plasmids pUCD2, pSa151 and RP4 to A . dispar and A . itersonii, at efficiencies as high as 3 x 10(4) transformants per micrograms plasmid DNA . RP4 DNA isolated from spirillum hosts, but not RP4 from E . coli cells was successfully transferred to A . dispar and A . itersonii by electrotransformation, suggesting that modification and/or restriction activity may be present in these Aquaspirillum species. Microbiologica, 1991 Jan, 14(1), 1 - 7 Detection of antibodies to p24 and gp41 epitopes of HIV by ELISA using the recombinant core protein (p24) and envelope synthetic protein (gp41); Filice G et al.; We have developed a system consisting of two separate ELISA, one designed to detect antibodies to HIV gag gene (p24) and the other to detect antibodies to HIV env gene (gp41) . The antigen used in these ELISA was produced as recombinant DNA-derived proteins expressed in E . coli for HIV gag gene (p24) and synthetic peptide for the HIV env gene (gp41) . These HIV (env-gag) ELISA, that provide independent determinations of the antibody response to the core and envelope proteins, are highly specific and sensitive . In this work we have demonstrated that determinations of antibodies such as those to p24 and gp41 by HIV (env-gag) ELISA are among the criteria for a confirmation procedure, and sensitivity one (gp41) and/or both these determination should be equal or greater than the sensitivity of W.B . In addition, the procedure should be objective and standardized and the antigen source used should be different from that adopted in the "classical" W.B . and screening test . In view of these considerations, this HIV (env-gag) ELISA could be used as a reliable alternative to W.B . for confirmation of antibody detection. Cytometry, 1991, 12(4), 291 - 301 Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs; Fiering SN et al.; The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E . coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications . Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and retained intracellularly . In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated sorting of cells based on their lacZ expression level . This report details the following improvements of the original assay: 1) use of phenylethyl-beta-D-thiogalactoside, a competitive inhibitor, to inhibit beta-galactosidase activity; 2) reduction of false positives by two-color measurements; and 3) inhibition of interfering mammalian beta-galactosidases by the weak base chloroquine . We found an exponential relationship between fluorescence generated by beta-galactosidase in this assay and the intracellular concentration of beta-galactosidase molecules . Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein . Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments . Together, these improvements make FACS-Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells. Arch Microbiol, 1991, 155(4), 360 - 5 Molecular cloning and expression of Spirulina platensis acetohydroxy acid synthase genes in Escherichia coli; Riccardi G et al.; The coding sequence for Spirulina platensis acetohydroxy acid synthase (AHAS, EC 4.1.3.18) is shown to be contained within a 4.2 Kb ClaI fragment (ilvX) that has been cloned from a recombinant lambda library . This fragment was able to complement a suitable mutant of Escherichia coli when inserted into the ClaI site of plasmid pAT153 in either orientation, demonstrating that transcription of ilvX originated within the cloned fragment . The probe used for hybridization experiments was the corresponding gene from Anabaena sp . PCC7120 . The same probe allowed us to identify a second putative gene encoding AHAS in the S . platensis genomic library. Zentralbl Hyg Umweltmed, 1991 Jan, 191(1), 36 - 45 The SOS-Chromo-spottest: evaluation of a short-term test for the determination of genotoxic compounds in contaminated environmental samples; Mersch-Sundermann V; To evaluate the sensitivity of the SOS-Chromo-spottest towards genotoxic compounds 5 reference chemicals (4-nitroquinoline-1-oxide (4-NQO), methylmethansulfonate (MMS), 2,4,7-trinitro-9-fluorenone (TNF), sodium azide (SA) and daunomycin (DM) were tested by 3 different agar plate media (STA-plates: synthetic media containing Xgal, B-plates: synthetic media containing 1% lactose and bromocresolpurple, C-plates: 1% lactose bromocresolpurple media containing complex nutrients) . Even 1 ng of 4-NQO showed genotoxic effects by using STA-plates . The threshold value for MMS was 80 nl, for TNF 160 ng and for DM 80 ng . Similarly the spottest with B-plates are positive results, but the sensitivity of this test procedure was 80 to 250 times lower than the STA-plate test . The C-plate test only reacted with high amounts of 4-NQO (1000 ng) . Therefore, the SOS-chromo-spottest with STA-media described by Quillardet and Hofnung seems to be a sufficient procedure to detect genotoxic compounds in contaminated environmental samples directly without previous extraction procedures . The simpler B-plates can be used to examine the genotoxicity of certain compounds like industrial or household chemicals where the genotoxicants can be expected to be present in high doses. Mol Gen Genet, 1991 Jan, 225(1), 113 - 20 Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis; Maldener I et al.; It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N2 . Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp . strain PCC 7120 were identified via their expression in Escherichia coli . The prcA gene from A . variabilis was sequenced . The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids . The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms . The resulting mutants, which lacked Ca2(+)-dependent protease activity, were not impaired in heterocyst formation and grew on N2 as sole nitrogen source. Eur J Biochem, 1991 Jan 1, 195(1), 191 - 4 Linker mutagenesis in the lacZ gene of Escherichia coli yields variants of active beta-galactosidase; Breul A et al.; Synthetic octameric oligonucleotides that code for a unique restriction site were cloned into a randomly linearized plasmid that carries the lacZ gene . The insertions were mapped by digestion with appropriate restriction endonucleases . 12 mutants were identified which carry an insertion within the lacZ gene and still express active beta-galactosidase . Small deletions or duplications of the wild-type sequence occurred at these positions which restore the correct reading frame . The insertions occurred in the first and the last third of the internal duplication of the lacZ gene and within the domain homologous to dihydrofolate reductase. J Gen Virol, 1991 Jan, 72 ( Pt 1), 37 - 49 Search for a putative scrapie genome in purified prion fractions reveals a paucity of nucleic acids; Meyer N et al.; Scrapie can be transmitted by novel infectious pathogens termed prions . No evidence for a scrapie-specific nucleic acid has been detected to date . To investigate amounts, types and sizes of nucleic acid molecules associated with prions in purified preparations, aliquots were deproteinized, and the nucleic acids analysed by PAGE and silver staining . Digestion with nucleases and exposure to Zn2+ prior to analysis substantially diminished the content of nucleic acids, but did not alter the prion titre indicating that those nucleic acids which were removed are not essential for infectivity . Since a single species of scrapie-specific nucleic acid could not be identified, we explored the unprecedented possibility of scrapie-specific nucleic acids of variable length which are biologically active . If such molecules of variable length exist then they might be hidden within the background smear on silver-stained gels after PAGE . A new procedure designated return refocusing gel electrophoresis (RRGE) was developed to identify heterogeneous nucleic acids in purified prion fractions . The content of variable length nucleic acids was reduced by a factor of 10 by exhaustive Bal 31 exonuclease digestion after dispersion of purified prions into detergent-lipid-protein complexes . For example, a typical sample after Bal 31 digestion contained approximately 4 ng of nucleic acid of variable length and 10(8.7) ID50 units of scrapie prion infectivity . Consideration of different models for a hypothetical scrapie-specific nucleic acid suggests that such a molecule would have to be: (i) quite small (less than 100 nucleotides), (ii) possess a particle-to-infectivity ratio near unity or (iii) heterogeneous in size . Although our results do not eliminate the possibility that prions possess a scrapie-specific nucleic acid of variable length, they narrow considerably the spectrum of features specifying such a candidate molecule. J Acquir Immune Defic Syndr, 1991, 4(2), 165 - 72 Contribution of antibody response to recombinant HIV-1 gene-encoded products nef, rev, tat, and protease in predicting development of AIDS in HIV-1-infected individuals; Reiss P et al.; The relation between antibody-response profiles to Escherichia coli-produced HIV-1 nef, rev, tat, and protease proteins and the risk of developing AIDS was studied using stored serum samples taken sequentially from a cohort of 195 initially symptom-free men who were seropositive for antibodies to HIV-1 structural proteins and 72 men who seroconverted for such antibodies . The AIDS attack rates at 39 months follow-up were significantly higher in the men with negative versus positive antibody profiles to nef, tat, and protease, respectively . {Difference (D) between attack rates = 11.279, 5.884, and 8.322, respectively} . No significant difference was found between men with negative versus positive antibody profiles to rev . The above differences between AIDS attack rates were clearly lower than those reported from the same cohort for men who were serum HIV-1 antigen positive versus negative, and for men with low versus normal CD4+ lymphocyte counts, but with respect to nef antibody-response profiles, resembled the difference reported between anti-HIV-1 core antibody-negative versus antibody-positive men . In the subgroup of men without any of the markers previously found to be predictive of progression to AIDS in the cohort (persistent HIV-1 p24 antigenemia, low anti-HIV-1 anti-core antibody reactivity, and low CD4+ cell counts), antibody profiles to nef, rev, tat, and protease did not contribute to the prediction of outcome of infection . When used in combination with persistent HIV-1 p24 antigenemia and low CD4+ cell counts, negative antibody profiles to nef and protease, respectively, were equally sensitive and specific in predicting progression to AIDS, as was low anti-HIV-1 anti-core antibody reactivity. J Bacteriol, 1991 Jan, 173(2), 727 - 33 Positive regulation of the pts operon of Escherichia coli: genetic evidence for a signal transduction mechanism; De Reuse H et al.; The pts operon of Escherichia coli is composed of the genes ptsH, ptsI, and crr, which code for three proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS): the HPr, enzyme I (EI), and EIIIGlc proteins, respectively . These three genes are organized in a complex operon in which the major part of expression of the distal gene, crr, is initiated from a promoter region within ptsI . Expression from the promoter region of the ptsH and ptsI genes has been studied in vivo by using gene fusions with lacZ . Transcription from this promoter region is under the positive control of catabolite activator protein (CAP)-cyclic AMP (cAMP) and is also enhanced during growth in the presence of glucose (a PTS substrate) . This report describes a genetic characterization of the mechanism by which growth on glucose causes transcriptional stimulation of the pts operon . This regulation is dependent on transport through the glucose-specific permease of the PTS, EIIGlc . Our results strongly suggest that transcriptional regulation of the pts operon is the consequence of an increase in the level of unphosphorylated EIIGlc which is produced during glucose transport . Furthermore, overproduction of EIIGlc in the absence of transport was found to stimulate expression of the pts operon . We also observed that CAP-cAMP could cause stimulation independently of the EIIGlc and that glucose could activate in the absence of cAMP in a strain overproducing EIIGlc . Our results indicate that glucose acts like an environmental signal through a mechanism of signal transduction . A sequence similarity between the C terminus of EIIGlc and the consensus of transmitter modules of the sensor proteins defined by E . C . Kofoid and J . S . Parkinson (Proc . Natl . Acad . Sci . USA 85:4981-4985, 1988) suggests that EIIGlc might have properties in common with the sensors of the two-component systems. J Bacteriol, 1991 Jan, 173(1), 16 - 22 Fnr mutants that activate gene expression in the presence of oxygen; Kiley PJ et al.; The regulatory protein Fnr is required for anaerobic expression of several anaerobic respiratory enzymes in Escherichia coli . To gain insight into how Fnr activity is regulated by oxygen, we have isolated Fnr mutants that increase expression of the nitrate reductase operon in the presence of oxygen (Fnr* mutants) . Seven single-amino-acid substitutions that mapped within two regions of Fnr have been characterized . Two mutants mapped adjacent to two Cys residues in the N-terminal Cys cluster . Five Fnr* substitutions mapped to a region of Fnr that is similar to the cyclic AMP-binding domain of the catabolite activator protein (CAP) . Within this group, four mutants were clustered in a region analogous to the CAP C helix, which is important in CAP dimer subunit interactions . Taken together, these data implicate regions in Fnr that may be important either in sensing oxygen deprivation or in the conformational change proposed to be necessary for Fnr activation under anaerobic conditions. Infect Immun, 1991 Jan, 59(1), 131 - 6 Diphosphoryl lipid A derived from lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides inhibits activation of 70Z/3 cells by LPS; Kirkland TN et al.; Diphosphoryl lipid A derived from nontoxic lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides ATCC 17023 did not stimulate the murine pre-B cell line 70Z/3 to synthesize surface immunoglobulin or kappa mRNA . However, it effectively blocked Escherichia coli LPS-induced activation of 70Z/3 cells in a concentration-dependent manner . This inhibition was specific only to cells activated by LPS, since it did not inhibit activation of 70Z/3 cells by gamma interferon . Maximal inhibitory effect occurred when the antagonist was added within 2 h before adding the LPS . These results strongly suggested that R . sphaeroides diphosphoryl lipid A is competing with E . coli LPS for physiological lipid A receptors on the 70Z/3 cells. Mutat Res, 1991 Jan, 254(1), 71 - 7 'Rec-lac test' for detecting SOS-inducing activity of environmental genotoxic substance; Nunoshiba T et al.; beta-Galactosidase activities (beta-GA) of E . coli strains carrying the fusion gene of recA and lacZ, GE94 and its DNA repair-deficient derivatives such as KY946{uvrA}, KY945{recA} and KY943{lexA} treated with UV, 4NQO, MNNG and MMC were examined . The beta-GA, reflecting the SOS-inducing activity, of GE94 and KY946 treated with these compounds increased significantly with a clear dose-response relationship, and reached a maximum level within 60 min, while no response was seen in KY945 and KY943 . Using KY946 and KY945 as a positive and a negative indicator, respectively, the SOS-inducing activity of oxidative mutagens, i.e., hydrogen peroxide (H2O2), formaldehyde, tert-butyl hydroperoxide, cumene hydroperoxide and streptonigrin, was investigated . Clear dose-dependent increases in beta-GA were observed in KY946 treated with all oxidative mutagens tested, but not in KY945 . Significant increases in beta-GA were observed with a lower concentration of H2O2 and a shorter incubation time of 4NQO in this assay than in the umu-test . The assay, called 'Rec-lac test' by us, may be useful to detect environmental genotoxic substances. Arch Surg, 1991 Jan, 126(1), 84 - 8 Lipid-free total parenteral nutrition and macrophage function in rats; Nussbaum MS et al.; Certain lipids are immunosuppressive when used for nutritional support, while other lipids and nutritional additives may enhance immunologic function . We hypothesized that total parenteral nutrition (TPN) may be immunosuppressive irrespective of lipids . Twenty-four rats underwent central vein catheterization and received either intravenous saline solution and oral chow or TPN alone . At 7 or 14 days, the animals were killed . Splenic and bone marrow macrophages were isolated and cultured in either M199 medium alone or were stimulated with Escherichia coli lipopolysaccharide . The supernatants were tested for prostaglandin E2 and C3 . The splenic prostaglandin E2 levels were significantly higher in the TPN group following lipopolysaccharide stimulation at 7 days but not at 14 days . Administration of TPN to rats, even without lipids, may be immunosuppressive through the release of prostaglandin E2 from splenic macrophages following a septic challenge . This effect appears to be abolished after 14 days of TPN infusion. Arch Surg, 1991 Jan, 126(1), 100 - 3 Interferon-gamma reverses bone marrow inhibition following hemorrhagic shock; Livingston DH; Hemorrhagic shock has been demonstrated to alter the myelopoietic response to bacterial lipopolysaccharide . Interferon-gamma has been shown to improve the immune response following experimental shock and injury; however, its effect on myelopoiesis is controversial . This study was performed to determine whether treatment with interferon-gamma will improve the bone marrow response to lipopolysaccharide after hemorrhagic shock . Rats subjected to either shock or a sham procedure were allocated into three groups: (1) control rats received no further treatment; (2) lipopolysaccharide-treated rats received saline for 3 days and then were challenged with lipopolysaccharide to stimulate myelopoiesis; and (3) interferon-treated rats received interferon-gamma (7500 U subcutaneously 1 hour after shock and then every day for 3 days) and lipopolysaccharide as in group 2 . Serum colony-stimulating factor levels were measured 6 hours and bone marrow white blood cell count and granulocyte-macrophage colony-forming units (CFU-GM) were measured 24 hours following lipopolysaccharide administration . In sham-treated rats, lipopolysaccharide increased CFU-GM 77% compared with controls . In contrast, treatment with lipopolysaccharide decreased CFU-GM 43% following shock . Treatment with interferon-gamma increased CFU-GM in all animals and reversed the decline in CFU-GM seen in shocked lipopolysaccharide-treated animals . Serum colony-stimulating factor levels were unaffected by either shock or interferon-gamma administration . These data demonstrate that interferon-gamma exerts a stimulatory effect on bone marrow following shock and restores the myelopoietic response to lipopolysaccharide. Z Med Lab Diagn, 1991, 32(3-4), 167 - 72 {Agent-conditioned influence on carbohydrate metabolism and energy state in liver and lung of rats in endotoxic shock}; Kopprasch S et al.; The pretreatment of rats with the substance LPH and LXU caused decrease of lethality in endotoxin shock and suppression of the following changes 6 hours post endotoxin: Increase of plasma ICDH activity, decrease in platelet count, decrease of the hepatic energy state and increase of the ATP content in the lung . The catabolic glucose metabolism during endotoxin shock was not influenced by LPH and LXU administration. Rev Stomatol Chir Maxillofac, 1991, 92(4), 273 - 6 {Osteomyelitis of the jaw and osteopetrosis}; Khochtali H et al.; Osteopetrosis (Albers-Schonberg disease) is a rare sclerosing bone disorder in which osteomyelitis of the jaws is a frequent complication . Treatment of osteomyelitis is difficult and may lead to large resection . We report three new cases and we review the literature. Mol Biol (Mosk), 1991 Jan-Feb, 25(1), 153 - 61 {Site-directed mutagenesis in the uracil-repair system . Preparation of mutated forms of human alpha 2 interferon}; Shekhter II et al.; The mutant forms of human IFN-alpha 2 gene are obtained by oligonucleotide-directed mutagenesis with the use of uracil-repair system . To intensify the process the procedure of the uracil-containing DNA template preparation is modified . It was determined that when mutagenesis is performed in the uracil-repair system the yield of the process depends on the mutant DNA-strand in vitro synthesis efficiency . It is shown that the stability of the 5'-end primer-template complex and the level of the endogenic primers elongation are the basis factors, that determine induction mutations. Haemostasis, 1991, 21 Suppl 1, 99 - 106 Comparative study of three recombinant hirudins with heparin in an experimental venous thrombosis model; Doutremepuich C et al.; Three recombinant hirudins (r-hirudins) produced by genetic processes from Escherichia coli and yeast were studied . r-Hirudins could be an alternative treatment to heparin; so, the antithrombotic activity of these drugs should be compared to heparin, the reference substance, in an experimental venous thrombosis model . In this model, the effect of these r-hirudins on thrombus weight reduction were not identical . They varied depending on the original product (E . coli or yeast) . The growth-inhibiting activity of r-hirudins on existing thrombi is not dose dependent, whereas that of heparin is . Moreover, in the conditions of this study, higher doses of heparin, but not of hirudins, increased the bleeding time . Although hirudin has limited applications for the time being, it seems an interesting anticoagulant drug, and the availability of r-hirudin opens new therapeutic anticoagulation perspectives. Parasitol Res, 1991, 77(5), 369 - 73 Studies on the lethal effect of ultraviolet light on Trichomonas vaginalis; Karanis P et al.; Following cultivation in Asami medium, centrifugation and resuspension in saline or in water from a medicinal spring, Trichomonas vaginalis trophozoites were exposed to well-defined doses of ultraviolet (UV) light (254 nm) . We used 24- and 48-h-old trichomonads at concentrations of 1 x 10(5) and 5 x 10(4) trophozoites/ml in a total volume of 20 ml for these studies . The apparatus for UV irradiation was especially constructed for batch experiments . After irradiation at doses ranging from 80 to 160 mJ/cm2, the mobility of the parasites was reduced and morphological alterations appeared: rounding of the cells, vacuolization of the cytoplasm and even cytolysis . A dose of 401.7 mJ/cm2 killed 99.8% of the 48-h-old trichomonads when irradiation occurred in saline at a cell density of 1 x 10(5) trichomonads/ml and 98.9% when irradiation was done at a cell density of 5 x 10(4) trichomonads/ml . A dose of 362.1 mJ/cm2 killed only the more sensitive 24-h-old trichomonads . In mineral water, 241 mJ/cm2 was sufficient to kill up to 99.5% of the 48-h-old trichomonads . When 48-h-old trichomonads that had been exposed to a radiation dose of 160-240 mJ/cm2 were subcultured, they lost their ability to propagate . At a dose of 80 mJ/cm2, both the trichomonads that had been harvested during the log phase and the 48-h-old organisms suspended in mineral water lost their ability to propagate on subculture . These results indicate that 24-h-old trichomonads were more sensitive than 48-h-old organisms . Furthermore, the experiments demonstrated that a higher dose of UV radiation must be applied to T . vaginalis trophozoites than to the more sensitive bacterial strain Escherichia coli ATCC 11229 so as to achieve comparable killing results. J Med Vet Mycol, 1991, 29(3), 211 - 4 Black grain eumycetoma (Madurella mycetomatis) in the abdominal cavity of a dog; Lambrechts N et al.; A uterine stump granuloma was surgically removed from a sterilized bitch . Histopathology and fungal culture revealed Madurella mycetomatis eumycetoma . Infection may have occurred through a cesarean wound dehiscence . Long-term fluconazole therapy was instituted but failed to arrest and eliminate the infection. Plant Mol Biol, 1991 Jan, 16(1), 129 - 39 Molecular analysis of two PR-1 pseudogenes from tobacco; Pfitzner AJ et al.; Two independent PR-1 lambda genomic clones (W38/1 and W38/3) were isolated and characterized from a tobacco (Nicotiana tabacum cv . Wisconsin 38) library . Neither clone is identical to the previously described PR-1 cDNA clones, and both clones carry mutations within the highly conserved PR-1 protein coding region . For example, clone W38/1 has a GAA Glu codon instead of the translation stop codon thus harbouring an open reading frame extended by 16 additional amino acids . Furthermore, both clones display considerable variations in the genomic flanking sequences when compared to the PR-1a gene . In order to test whether the encoded genes are active, their upstream sequences were fused to the E . coli beta-glucuronidase (GUS) reporter gene . While significant GUS activities as compared to the 35S RNA promoter from cauliflower mosaic virus (CaMV) were obtained with the W38/1 and W38/3 sequences in transient gene expression assays, no transcriptional activities could be observed upon stable transformation of the same constructs . In addition, the protein coding region of W38/1 was joined to the CaMV 35S RNA promoter and transgenic tobacco plants were generated . However, neither transcripts nor a protein could be detected deriving from the W38/1 structural gene with this chimaeric construct in the transformants . Taken together, these data indicate that the genes contained in lambda clones W38/1 and W38/3 are not active in planta. Yao Xue Xue Bao, 1991, 26(1), 6 - 9 Effects of platelet-activating factor antagonist SRI 63-441 on endotoxin-induced changes in rat mesenteric microcirculation; Li SH et al.; Intravenous injection of E . coli endotoxin 30 mg/kg to rats resulted in a systemic hypotension and rapid decrease in mesenteric arteriolar blood flow velocity (MABFV) . The mesenteric arterioles constricted within 3 min followed by a short period of vasodilation after endotoxin administration . Pretreatment with the platelet-activating factor receptor antagonist SRI 63-441 attenuated the endotoxin-induced systemic hypotension and the decrease in MABFV, and abolished the mesenteric vasodilation effect of endotoxin infusion . The results suggest that PAF may be a mediator of endotoxic shock. J Cell Sci Suppl, 1991, 14, 79 - 82 Nuclear envelope dynamics and nucleocytoplasmic transport; Stewart M et al.; We have combined structural, biochemical and recombinant DNA methods to explore molecular interactions involved in nuclear envelope assembly dynamics and nucleocytoplasmic transport . Electron microscopy has established the overall architecture of the envelope and the relationship between nuclear pores, lamina fibres and pore-connecting fibrils . The lamin proteins that constitute the lamina resemble intermediate filament proteins, and assemble and disassemble during mitosis in response to phosphorylation . Lamins have been expressed in E . coli to facilitate structural investigations and the exploration of interaction sites with other envelope components . Disruption of envelopes has shown that nuclear pores are constructed from a central cylinder with cytoplasmic and nucleoplasmic rings . Examination of envelopes transporting gold-labelled nucleoplasmin has indicated that the transport pathway is complex and probably involves ring components in addition to the central cylinder . Molecular motors may be involved in changes in pore shape to enable transport and in the translocation mechanism. Cytokine, 1991 Jan, 3(1), 1 - 4 Interleukin-6 administration has no acute hemodynamic or hematologic effect in the dog; Preiser JC et al.; To investigate the possible hemodynamic effects of interleukin-6 (IL-6), a single dose of 15 mcg/kg of recombinant IL-6 isolated from Escherichia coli was injected intravenously in six pentobarbital-anesthetized dogs . After 30 min, saline infusion was performed to maintain the pulmonary artery balloon-occluded pressure at baseline level . The animals were observed for up to 5 hours . No other hemodynamic alteration was observed than a gradual decline in cardiac output attributed to anesthesia . Hematologic variables, blood glucose, and total serum proteins were also constant . IL-6 levels were markedly elevated in the blood, but no tumor necrosis factor activity was detected . Thus a primary role for IL-6 in the early cardiovascular alterations associated with septic shock seems unlikely. Vet Res Commun, 1991, 15(3), 227 - 38 Endotoxin in the conscious piglet: its effects on some general and gastrointestinal myoelectrical parameters; De Saedeleer V et al.; The effect of an intravenous bolus injection of endotoxin, 0.1, 1 or 10 micrograms/kg, on rectal temperature, clinical appearance, haematological parameters, and on gastrointestinal electrical activity was examined in 11 conscious piglets of 4-5 weeks of age, with implanted electrodes in the antrum pylori, duodenum, jejunum and ileum . All doses resulted in a significant and dose-dependent increase in rectal temperature, in pronounced clinical signs and in distinct changes in haematological values . These included shivering, depression, respiratory distress, a leukopenia (0.1 micrograms/kg) or a leukocytosis (1 microgram/kg) with a shift to the left, an accelerated sedimentation rate and a decreased packed cell volume . Doses of 1 and 10 micrograms/kg induced a transient inhibition of gastroduodenal electrical activity . These results suggest that, in the piglet, endotoxin primarily manifests general clinical signs and that the gastrointestinal effects coincide with these. Nephrol Dial Transplant, 1991, 6(4), 232 - 7 Prognostic markers in diarrhoea-associated haemolytic-uraemic syndrome: initial neutrophil count, human neutrophil elastase and von Willebrand factor antigen; Milford DV et al.; The observed increase in pla