Escherichia coli

Biochem Biophys Res Commun, 1987 Jan 15, 142(1), 194 - 9
Mass spectral analysis of complex lipids desorbed directly from lyophilized membranes and cells; Heller DN et al.; Three desorption ionization techniques--laser desorption, plasma desorption and fast atom bombardment mass spectrometry--have been applied to lyophilized cells, membranes, lysed cells and various extracts . It has been shown that intact polar lipids are selectively desorbed from biological membranes by these methods and that their mass spectra provide "fingerprints" which reflect the unique biochemical composition of each class of cell or membrane.

J Biol Chem, 1987 Jan 15, 262(2), 877 - 82
Scanning transmission electron microscopic study of alpha-ketoglutarate dehydrogenase complex from Escherichia coli; Wagenknecht T et al.; The alpha-ketoglutarate dehydrogenase complex was resolved into its three component enzymes: alpha-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase . Subcomplexes were prepared in vitro by incubating the resolved E2, a 24-subunit cube-shaped molecule, with E1 (dimeric) . The morphology and mass of the subcomplexes were determined by scanning transmission electron microscopy of negatively stained and of freeze-dried specimens . Images of both negative stained and freeze-dried subcomplexes were consistent with E1 binding at or near the midpoints of the edges of the E2 molecule . Mass analysis of the freeze-dried specimen showed that at least 95% of E1 remains in the dimeric state (or as two closely juxtaposed monomers) when it binds to E2.

J Biol Chem, 1987 Jan 15, 262(2), 622 - 9
Structural and catalytic characteristics of Escherichia coli adenylate kinase; Saint Girons I et al.; The adk gene encoding adenylate kinase in Escherichia coli was cloned in pBR322 . Adenylate kinase represented about 4% of total proteins in extracts of cells containing the pBR322:adk plasmid . This allowed preparation of more than 90% pure enzyme in a single-step purification procedure . Amino acid analysis, high performance liquid chromatography separation of trypsin digests, sequence analysis of most peptides, and determination of the N-terminal sequence of the whole protein confirmed the primary structure of E . coli adenylate kinase predicted from the nucleotide sequence of the adk gene (Brune, M., Schumann, R., and Wittinghofer, F . (1985) Nucleic Acids Res . 13, 7139-7151) . 2-Nitro-5-thiocyanatobenzoic acid reacted with the single cysteine residue of E . coli adenylate kinase . The cyanylated protein was cleaved upon exposure to alkaline pH, yielding two peptides corresponding to residues 1-76 and 77-214, respectively . A mixture of purified peptides tended to reassociate, recovering both catalytic activity and binding properties for adenine nucleotides . E . coli adenylate kinase has a broader specificity for nucleoside monophosphates than does the mammalian enzyme . In addition to 2'-dAMP, other nucleoside monophosphates such as 3'-dAMP, adenine-9-beta-D-arabinofuranoside 5'-monophosphate, and 7-deazaadenosine (tubercidine) 5'-monophosphate were able to replace AMP as substrate.

J Biol Chem, 1987 Jan 15, 262(2), 892 - 8
DNA functional groups required for formation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter . Identification via base analog substitutions; Dubendorff JW et al.; Synthetic 75-base pair promoters bearing base changes and/or base analog substitutions at selected positions were constructed . Using both abortive initiation and run-off transcription assays, the interaction of these altered promoters with Escherichia coli RNA polymerase was studied in order to determine the involvement of DNA functional groups in promoter recognition . Two adjacent thymines in the -35 region were identified whose 5-methyl groups play a crucial role . Additionally, the combined results from several substitution experiments showed that functional groups in the major groove of the strongly conserved T-A base pair at the -7 position are probable sites of direct interaction with RNA polymerase.

J Biol Chem, 1987 Jan 15, 262(2), 722 - 6
The energy utilized in protein breakdown by the ATP-dependent protease (La) from Escherichia coli; Menon AS et al.; A crucial enzyme in the pathway for protein degradation in Escherichia coli is protease La, an ATP-hydrolyzing protease encoded by the lon gene . This enzyme degrades various proteins to small polypeptides containing 10-20 amino acid residues . To learn more about its energy requirement, we determined the number of ATP molecules hydrolyzed by the purified protease for each peptide bond cleaved . The enzyme hydrolyzed about 2 molecules of ATP for each new amino group generated with casein, bovine serum albumin, glucagon, or guanidinated casein as substrates, even though these proteins differ up to 20-fold in size and 3-4 fold in rates of hydrolysis of peptide bonds . Similar values for the stoichiometry (from 1.9 to 2.4) were obtained using fluorescamine or 2,4,6-trinitrobenzene sulfonic acid to estimate the appearance of new amino groups . These values appeared lower at 1 mM than at 10 mM Mg2+ . The coupling between ATP and peptide bond hydrolysis appeared very tight . However, when the protease was assayed under suboptimal conditions (e.g . at lower pH or with ADP present), many more ATP molecules (from 3.5 to 12) were consumed per peptide bond cleaved . Our data would indicate that the early steps in protein degradation consume almost as much energy (2 ATPs for each cleavage) as does the formation of peptide bonds during protein synthesis.

Biochemistry, 1987 Jan 13, 26(1), 269 - 76
Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA; Evans RK et al.; A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps . The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide . Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP) . Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield . The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity . After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP . When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E . coli RNA polymerase . To prepare {gamma-32P}-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed . {gamma-32P}-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1987 Jan 12, 15(1), 313 - 32
Nucleotide sequence and expression of the gene encoding the EcoRII modification enzyme; Som S et al.; The gene coding for the EcoRII modification enzyme has been cloned and the nucleotide sequence of 1933 base pairs containing the gene has been determined . The gene codes for a protein of 477 amino acids . Two transcriptional start sites have been mapped by S1 mapping . One deletion that removes 34 N-terminal amino acids was found to have partial enzyme activity . Comparison of the EcoRII methylase sequence with other cytosine methylases revealed several domains of partial homology among all cytosine methylases . Cloning the gene in multicopy pUC vectors increased the expression by 6-18 fold . A 40 fold overproduction of the EcoRII methylase was obtained by cloning the gene in the expression vector carrying the lambda PL promoter.

J Theor Biol, 1987 Jan 7, 124(1), 89 - 95
Codon usage in Homo sapiens: evidence for a coding pattern on the non-coding strand and evolutionary implications of dinucleotide discrimination; Alff-Steinberger C; This study reports the analysis of codon usage in 35 complete Homo sapiens genes . Both codon frequency and inter-codon interference exhibit patterns of evolutionary interest . There is a significant positive correlation between the frequency with which a given codon is used and the frequency with which its complement is used . Since the frequency of appearance of the complementary codon on the coding strand is equal to the frequency of appearance of the original codon on the non-coding strand, in the same phase, the non-coding strand is found to resemble the coding strand in triplet composition . The same effect has been observed in Escherichia coli . This preference for the use of certain complementary triplets as codons suggests that the evolution of the use of the genetic code depended to some extent upon the double-stranded nature of the coding material . In addition, the effect of discrimination against the use of two dinucleotides, CpG and UpA, is observed in codon usage and also in adjacent codon interference . Codons beginning with G, or A, are unlikely to be preceded by codons ending in C, or U, respectively . Consideration of codon assignment in the genetic code together with the observed CpG infrequency suggests that the evolution of the code may have been influenced by conditions in which the use of CpG dinucleotides was unfavorable . The infrequent use of UpA dinucleotides can be explained as the result of frameshift mutation during gene evolution.

J Theor Biol, 1987 Jan 7, 124(1), 43 - 55
Translation rate modification by preferential codon usage: intragenic position effects; Liljenstrom H et al.; We present a model for calculating the protein production rate as a function of the translation rate . The model takes into account that the elongation rate along an mRNA molecule is non-uniform as a result of different tRNA availabilities for different codons . Initiation of ribosomes on an mRNA is normally the rate-limiting step in the translation process, and blocking of the initiation site can be avoided if the codons closest to this site allow fast translation by the ribosome . Hence, different selective forces may act on the choice of synonymous codons in the initiation region than elsewhere on a given mRNA . We show that the elongation rate along the whole mRNA influences the production rate of abundant proteins, whereas only the elongation rate in the initiation region is of importance for the production rate of rare proteins . We also present an analysis of the codon distribution along known mRNAs coding for abundant and rare proteins.

J Biol Chem, 1987 Jan 5, 262(1), 492 - 8
Action mechanism of Escherichia coli DNA photolyase . III . Photolysis of the enzyme-substrate complex and the absolute action spectrum; Sancar GB et al.; The absolute action spectrum of Escherichia coli DNA photolyase was determined in vitro . In vivo the photoreactivation cross-section (epsilon phi) is 2.4 X 10(4) M-1 cm-1 suggesting that the quantum yield (phi) is about 1.0 if one assumes that the enzyme has the same spectral properties (e.g . epsilon 384 = 1.8 X 10(4) M-1 cm-1) in vivo as those of the enzyme purified to homogeneity . The relative action spectrum of the pure enzyme (blue enzyme that contains FAD neutral semiquinone radical) agrees with the relative action spectrum for photoreactivation of E . coli, having lambda max = 384 nm . However, the absolute action spectrum of the blue enzyme yields a photoreactivation cross-section (epsilon phi = 1.2 X 10(3) at 384 nm) that is 20-fold lower than the in vivo values indicative of an apparent lower quantum yield (phi approximately equal to 0.07) in vitro . Reducing the enzyme with dithionite results in reduction of the flavin semiquinone and a concomitant 12-15-fold increase in the quantum yield . These results suggest that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300-500 nm range, the photolyase chromophore (which consists of reduced FAD plus the second chromophore) donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines . The reduced chromophore is regenerated at the end of the photochemical step thus enabling the enzyme to act catalytically.+

J Biol Chem, 1987 Jan 5, 262(1), 486 - 91
Action mechanism of Escherichia coli DNA photolyase . II . Role of the chromophores in catalysis; Jorns MS et al.; DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light . Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution . Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox . Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity . The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis . Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis . On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity . This suggests that reduced flavin may also act as a sensitizer in catalysis . The blue color of the enzyme is lost upon reduction of the FAD radical . The fully reduced E . coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase . This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.

J Biol Chem, 1987 Jan 5, 262(1), 478 - 85
Action mechanism of Escherichia coli DNA photolyase . I . Formation of the enzyme-substrate complex; Sancar GB et al.; Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein . The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA . We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis . The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1 . The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1 . The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2 . From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.

J Biol Chem, 1987 Jan 5, 262(1), 455 - 9
Determination of the nucleotide sequence for the exonuclease I structural gene (sbcB) of Escherichia coli K12; Phillips GJ et al.; The complete nucleotide sequence of the structural gene for Escherichia coli exonuclease I has been determined . The coding region corresponds to a 465-amino acid protein with molecular weight of 53,174 . The partial amino acid sequence of purified exonuclease I agrees with that predicted by the DNA sequence . Two putative weak promoters have been localized by S1 nuclease analysis . The sbcB coding sequence contains many non-optimal codons, characteristic of many poorly expressed E . coli genes.

J Biol Chem, 1987 Jan 5, 262(1), 312 - 8
Catalytic-regulatory subunit interactions and allosteric effects in aspartate transcarbamylase; Ladjimi MM et al.; The x-ray structure of the unliganded aspartate transcarbamylase reveals that Arg-113 of the catalytic chain is involved in an important set of interactions at the interface between the catalytic and regulatory subunits (Honzatko, R.B., Crawford, J.L., Monaco, H.L., Ladner, J.E., Edwards, B.F.P., Evans, D.R., Warren, S.G., Wiley, D.C., Ladner, R.C., and Lipscomb, W . N . (1982) J . Mol . Biol . 160, 219-263) . In order to disturb this interaction, site-directed mutagenesis has been used to replace Arg-113 with glycine . This modification results in a substantial weakening of the interface between the catalytic and regulatory subunits leading to a high tendency for dissociation . The unliganded mutant enzyme exhibits a pH dependence and a sensitivity toward mercurials analogous to that obtained for the relaxed conformation of the wild-type enzyme . Moreover, the presence of saturating concentrations of aspartate is accompanied by only a slight shift in the optimal pH for activity . The bisubstrate analog N-(phosphonacetyl)-L-aspartate induces a 2-fold increase in the sulfhydryl reactivity as compared to the 4-fold increase observed for the wild-type enzyme . Despite this change in the interactions at the interface between the catalytic and regulatory subunits, the mutant enzyme still retains homotropic and heterotropic effects and exhibits a normal affinity for aspartate . Together these data show that a substantial weakening of the catalytic-regulatory interface can occur without altering the allosteric properties of the enzyme . These results also indicate that the intersubunit interactions involving Arg-113, between the polar domain of the catalytic chain and the zinc domain of the regulatory chain, do not participate in the homotropic cooperativity of the enzyme.

J Biol Chem, 1987 Jan 5, 262(1), 19 - 20
Crystallization of ricin A chain obtained from a cloned gene expressed in Escherichia coli; Robertus JD et al.; Ricin is a heterodimeric toxin of the form AB, where B is a lectin which binds cell surfaces, triggering endocytosis . The B chain then aids the A chain in escaping from the endosome . The A chain enzymatically attacks and inactivates ribosomes, thereby killing the intoxicated cell . We have recently solved the three-dimensional structure of whole ricin . Here we report that the A chain, expressed from a gene cloned into Escherichia coli has been crystallized in a suitable form for high resolution x-ray analysis . The crystals are monoclinic space group P2(1) with a = 42.6, b = 68.1, c = 50.2 A and beta = 112.9 degrees . There is evidence that the A chain undergoes a conformational change, resulting in activation, when it is released from the B chain . Comparison of the two structures should facilitate an analysis of this process.

J Mol Biol, 1987 Jan 5, 193(1), 81 - 95
Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA . Demonstration of competitive binding and the lack of a specific protein-protein interaction; Kowalczykowski SC et al.; The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E . coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments . The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied . The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive . In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein . In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration . When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed . Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex . Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex . However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA . Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA . Direct evidence for this conclusion is presented in the accompanying paper.

J Mol Biol, 1987 Jan 5, 193(1), 41 - 6
Expression of chimeric genes in Caenorhabditis elegans; Jefferson RA et al.; We have shown the expression of transformed genes in the nematode Caenorhabditis elegans using a new gene fusion system . Vectors consisting of the flanking regions of a collagen gene (col-1) or a major sperm protein gene of C . elegans fused to the Escherichia coli uidA gene, encoding beta-glucuronidase, were microinjected into worms and found to be propagated as high-copy extrachromosomal tandem arrays . We have detected beta-glucuronidase activity in transformed lines, and have shown that the activity is dependent upon the correct reading frame of the construction and on the presence of the worm sequences . The enzyme activity was shown to be encoded by the chimeric beta-glucuronidase gene by co-segregation analysis and by inactivation with specific antisera . Expression is at a very low level, and seems to be constitutive . We have used histochemical techniques to visualize the enzyme activity in embryos.

J Mol Biol, 1987 Jan 5, 193(1), 27 - 40
Differential repression of SOS genes by unstable lexA41 (tsl-1) protein causes a "split-phenotype" in Escherichia coli K-12; Peterson KR et al.; The lexA41 (formerly tsl-1) mutant was isolated as an ultraviolet light-resistant, temperature-sensitive derivative of its ultraviolet light-sensitive lexA3(Ind-) parent . Cells exhibit a so-called "split-phenotype", a phenomenon in which only a subset of the SOS responses can be detected physiologically following inducing treatments . lexA41 has been cloned and sequenced; the mutant gene retains the lexA3 mutation (Gly to Asp at position 85) and has a second mutation, lexA41 (Ala to Thr at position 131) . We show that LexA41 protein is not cleaved by the RecA protein-catalyzed pathway in vivo, but the mutant protein is degraded by the Lon protease at both 32 degrees C and 42 degrees C . beta-Galactosidase activities of lac fusions to 13 different SOS promoters were measured at 30 degrees C and 42 degrees C to determine levels of expression and were found to vary considerably . The temperature-sensitive phenotype is a result of increased expression of sulA, which encodes a division inhibitor, at 42 degrees C . Excision repair genes, including uvrA, uvrB and uvrD, are constitutively expressed at 30 degrees C accounting for the ultraviolet light resistance of the lexA41 mutant, but the SOS mutagenesis operon, umuD,C, is not adequately derepressed, thereby explaining the failure to induce mutagenesis in this background . This differential expression of SOS genes gives a plausible explanation of the split-phenotype associated with lexA41.

J Mol Biol, 1987 Jan 5, 193(1), 201 - 11
Sequences that adopt non-B-DNA conformation in form V DNA as probed by enzymic methylation; Brahmachari SK et al.; pBR322 form V DNA is a highly torsionally strained molecule with a linking number of zero . We have used sequence-specific DNA methylases as probes for B-DNA in this molecule, exploiting the inability of methylases to methylate single-stranded DNA and Z-DNA, both of which are known to occur in form V DNA . Some sequences in form V DNA were shown to be totally in the B-form, others were totally in an altered, unmethylatable conformation, while still other sites appeared to exist partly in altered and partly in normal B-conformation . Some potential Z-forming sequences (alternating pyrimidine/purine) of less than seven base-pairs were not in the Z conformation in form V DNA, whereas others did adopt an altered structure, indicating a modulating influence of flanking sequences . Furthermore, regions of imperfect alternating pyrimidine/purine structure were sometimes capable of adopting an altered structure . In addition, some regions of altered structure had no apparent Z-forming sequences, nor were they in polypurine stretches, which have also been proposed to form left-handed DNA . These non-B-DNA conformations may represent novel left-handed helical structures or sequences that become single stranded under torsional strain . Long regions of either altered (unmethylatable) DNA or B-DNA were not always observed . In fact, one region showed three transitions between B-like DNA and altered structure within 26 base-pairs.

J Biol Chem, 1987 Jan 5, 262(1), 152 - 7
In vitro expression of the Escherichia coli nusA-infB operon; Cenatiempo Y et al.; The expression of the nusA-infB operon has been investigated using an in vitro system based on the formation of the first dipeptide of the gene product . A series of plasmids containing various deletions of the operon were used as templates in this study . Of the four genes coding for protein products, 15Ka, nusA, infB, and 15Kb, only 15Ka was not expressed in this dipeptide system . The initial dipeptides for the other gene products, fMet-Asn (pnusA), fMet-Thr (IF-2 alpha), and fMet-Ala (p15Kb), were synthesized even from plasmids lacking the primary promoters . It appears that secondary (internal) promoters in the operon can efficiently direct the expression of these genes . No regulation of the expression was observed with IF-2 alpha, but pnusA inhibited the expression of the nusA gene (autoregulation) as well as the p15b gene . Experiments using an uncoupled system indicated that the effect of pnusA on nusA expression was at the level of transcription, but that both a transcriptional and a post-transcriptional effect of pnusA was seen on 15Kb expression.

J Biol Chem, 1987 Jan 5, 262(1), 97 - 102
Biosynthetic thiolase from Zoogloea ramigera . III . Isolation and characterization of the structural gene; Peoples OP et al.; The gene coding for the biosynthetic thiolase from Zoogloea ramigera has been isolated by using antibody screening methods to detect its expression in Escherichia coli under the transcriptional control of the lac promoter . We have located and determined the nucleotide sequence of the gene . The structural gene is 1173 nucleotides long and codes for a polypeptide of 391 amino acids; 282 nucleotides 5' and 58 nucleotides 3' to the coding sequence are also reported . By comparing the amino acid sequence data predicted from the gene with data determined experimentally, we have derived the complete primary structure of thiolase . A catalytically essential cysteine is located at residue 89 . The DNA sequence presented has a very high G/C content, 66.2%, typical of the Z . ramigera genome . In the coding region, this increases to 68.2% and is strongly reflected in the codon usage which demonstrates a strong preference for G or C in the third position . Examination of the 5'-flanking sequence establishes that the NH2-terminal methionine is specified by an ATG codon, 7 nucleotides downstream from a Shine-Dalgarno sequence.

J Biol Chem, 1987 Jan 5, 262(1), 411 - 8
Sequences distal to the mitochondrial targeting sequences are necessary for the maturation of the F1-ATPase beta-subunit precursor in mitochondria; Vassarotti A et al.; The beta-subunit of the mitochondrial F1-ATPase is synthesized as a precursor in the cytoplasm which is delivered through two bilayers bounding the mitochondria prior to its assembly with other proteins into a functional complex . In order to determine the role of the amino-terminal 50 residues of the precursor on its localization, maturation, and assembly, a set of deletions within this region of the ATP2 gene encoding the beta-subunit has been analyzed . These studies reveal that deletions between residue 10 of the F1 beta-presequence and residue 36 can still direct in vivo mitochondrial import and assembly of the mutant subunit into a functional complex . Deletions within ATP2 which contain less than the first 10 residues of the precursor are not imported . Thus, the extreme amino terminus (about half of the transient presequence) of the F1 beta-subunit can direct its mitochondrial import . The wild-type F1 beta-subunit precursor is matured by the matrix-located metalloprotease at Lys19-Gln20; however, small in-frame deletions up to 17 residues distal to this site fail to be matured either in vitro or in vivo . This nonmatured F1 beta-subunit is also assembled into a functional enzyme and supports growth of its host on a nonfermentable carbon source . These data indicate that maturation of the F1 beta-subunit precursor is dependent on a protein sequence located distal to the proteolytic maturation site which is distinct from the mitochondrial targeting sequence.

J Biol Chem, 1987 Jan 5, 262(1), 63 - 8
Nucleotide sequence of the pnp gene of Escherichia coli encoding polynucleotide phosphorylase . Homology of the primary structure of the protein with the RNA-binding domain of ribosomal protein S1; Regnier P et al.; The pnp gene is located at 69 min on the Escherichia coli chromosome adjacent to the rpsO gene which encodes the ribosomal protein S15 . In this paper, we present the sequence of a 3030-nucleotide DNA fragment containing the open reading frames coding for ribosomal protein S15 and polynucleotide phosphorylase . Translation of pnp is initiated by 5'-UUG-3' codon separated by 7 nucleotides from a good ribosome binding site . Codon usage in this gene is typical of highly expressed proteins of E . coli . Some of the transcripts of the pnp gene terminate just after the stem of the terminator t2 visible in the nucleotide sequence . However, a very strong read-through occurs at this site, thus permitting many of the pnp transcripts to extend beyond this transcription terminator . We also describe the primary structure homologies between a 69-amino-acid stretch of polynucleotide phosphorylase and the four homologous stretches of ribosomal protein S1 which form its RNA binding site . The possibility that this 69-amino-acid stretch constitutes the polynucleotide binding domain of polynucleotide phosphorylase is discussed.

J Biol Chem, 1987 Jan 5, 262(1), 389 - 93
Cotranscription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes . Fine-structure mapping of the lsp internal promoter; Miller KW et al.; We have applied the technique of Northern blotting hybridization analysis to examine the transcription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes . RNA samples from the wild-type strain CS412 and CS412 containing the plasmids pMT521 and pSYC890 were examined with ileS- and lsp-specific 32P-labeled probes . pMT521 is a pBR322 derivative into which a chromosomal DNA fragment containing the ileS and lsp genes has been cloned downstream of the pBR322 tet promoter . pSYC890 is a derivative of pMT521 which lacks the tet promoter, but nevertheless is active in lsp mRNA transcription due to the presence of a weak lsp internal promoter located in the distal portion of the upstream ileS DNA, RNA from CS412(pMT521) exhibited a high level of a 5500-nucleotide ileS-lsp cotranscript that hybridized to both probes . RNA from CS412(pSYC890) contained a high level of low molecular weight lsp-specific transcripts from the lsp internal promoter . However, only high molecular weight (5000-6500 nucleotides) cotranscripts were detected in RNA from CS412 by Northern blotting analysis . A trace level of lsp-specific mRNA was detected in CS412 by S1 nuclease mapping . The 5'-end of this transcript was mapped using RNA from CS412(pSYC890) . The mRNA begins 192/193 base pairs upstream of the lsp translation initiation codon at a site within ileS DNA that codes for the aspartate residue located 61 amino acids from the carboxyl-terminal of ILES . In conclusion, most of the transcription of lsp in wild-type E . coli originates from upstream of ileS and yields ileS-lsp cotranscripts.

Science, 1987 Jan 2, 235(4784), 83 - 5
Cloned gene of Rickettsia rickettsii surface antigen: candidate vaccine for Rocky Mountain spotted fever; McDonald GA et al.; Two major protective antigens of Rickettsia rickettsii have been previously described . In this study, we cloned the gene encoding one of these antigens into Escherichia coli and tested the effectiveness of the recombinant-made product as a vaccine for Rocky Mountain spotted fever . A clone bank of R strain R . rickettsii DNA was made in E . coli K-12 by using the plasmid vector pBR322 . Transformants were screened for their ability to make rickettsial antigens by reactivity with rabbit antibodies to R . rickettsii . One of the transformants, EM24(pGAM21), made a product reactive with two monoclonal antibodies that recognize a 155-kilodalton protein of R . rickettsii . One of the monoclonal antibodies was a member of a class of antibodies that react to heat-sensitive epitopes and protect mice injected with a potentially lethal dose of viable R . rickettsii . The cloned product contained this protective heat-sensitive epitope . In order to obtain enhanced expression, the gene was subcloned downstream of the lactose promoter on the plasmid vector pUC8 . A sonic lysate of E . coli harboring the pUC8 subclone was used successfully as a vaccine to protect mice injected with a lethal dose of the viable R . rickettsii.

Eur J Biochem, 1987 Jan 2, 162(1), 31 - 6
Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli; Voordouw G et al.; Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa . Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9 . Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (approximately 0.1% w/w of total protein) . Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50-fold increased expression of hydrogenase polypeptides in E . coli . The protein formed is inactive and was purified in order to delineate its defect . Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D . vulgaris H . The derived protein is also an alpha beta dimer and electron-paramagnetic resonance studies indicate the presence of the electron-transferring ferredoxin-type iron-sulfur clusters . In contrast to the native protein from D . vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen-binding active centre which also contains an iron-sulfur cluster is missing.

Mol Biochem Parasitol, 1987 Jan 2, 22(1), 79 - 87
Isolation of lambda amp3 genomic recombinants coding for antigens of Eimeria tenella; Clarke LE et al.; Eimeria are the causative agents of coccidosis, a disease which is of world wide economic importance in the poultry industry . Immunity resulting from infection is species-specific and both antibody and cell-mediated responses have been implicated . As an initial step in the development of a genetically-engineered vaccine against coccidiosis, libraries of EcoRI-digested genomic DNA from E . tenella have been constructed in Escherichia coli using the expression vector lambda amp3 . Screening of the libraries with serum from chickens immunized by infection has identified at least 24 different recombinant phage which produce eimeria antigens fused to beta-galactosidase . A significant proportion of the Eimeria DNA inserts cross-hybridise with each other and contain sequences which are highly represented in the genome . The identification of these clones will enable the isolation of intact genes from E . tenella DNA and facilitate detailed analysis of the antigens and immune responses.

Science, 1987 Jan 2, 235(4784), 77 - 80
Mapping the main immunogenic region and toxin-binding site of the nicotinic acetylcholine receptor; Barkas T et al.; The alpha-chain of the nicotinic acetylcholine receptor carries the binding sites both for cholinergic ligands and for most experimentally induced or naturally occurring antibodies to the native receptor . By means of expression cloning in Escherichia coli, fusion proteins were derived from specific fragments of a complementary DNA encoding the mouse alpha-chain, allowing the mapping of the toxin-binding site to residues 160-216 and the main immunogenic region to residues 6-85 . This approach permits the independent study of different functional domains of a complex receptor molecule and should be generally applicable to other proteins for which complementary DNA clones are available.

Arch Biol Med Exp (Santiago), 1987, 20(3-4), 371 - 8
Function, structure and evolution of fructose-1,6-bisphosphatase; Marcus F et al.; The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate is a key reaction of carbohydrate metabolism . The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms . Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms . These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast) . In this short review, we have analyzed the function of several fructose-1,6-bisphosphatases and we have made a comparison of partial amino acid sequences obtained from the enzymes of the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplasts with the known entire amino acid sequence of a mammalian gluconeogenic fructose-1,6-bisphosphatase . These results demonstrate a very high degree of sequence conservation, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.

Acta Anaesthesiol Scand, 1987 Jan, 31(1), 100 - 3
Granulocyte microbicidal function in patients undergoing major abdominal surgery under balanced anaesthesia; Perttila J et al.; Granulocyte microbicidal functions were studied in 11 major abdominal surgery patients with the luminol-enhanced chemiluminescence method . The responses of granulocytes in phagocytosis of zymosan, S . aureus and E . coli and responses to N-formylmethionyl-leucylphenylalanine (FMLP) were unaffected by surgery . The percentage of high-peroxidase-activity cells among neutrophils was increased on postoperative days 1 (P less than 0.01) and 3-4 (P less than 0.05) . We propose that microbicidal-related oxidative phagocytic functions of granulocytes are well maintained after major abdominal surgery under balanced anaesthesia.

Circ Shock, 1987, 21(1), 65 - 78
Respiratory compensation and acidosis in endotoxin shock: effects of naloxone in conscious rats; Law WR et al.; Naloxone treatment of endotoxin shock has been shown to alter many cardiovascular parameters . However, since opioids can affect ventilatory function we thought it important to assess the effects of naloxone on some respiratory variables during endotoxin shock . Male Sprague-Dawley rats (300-400 g) were surgically prepared with carotid artery and jugular vein cannulas 24 hours before experiments were begun . Conscious, unrestrained rats were challenged with 10 mg/kg E . coli endotoxin or saline . Measurements of blood gases, pH, respiratory rate, serum lactate, and medullary/pontine blood flow (radio labelled microsphere method) were made 0, 10, 30, and 60 minutes postchallenge . Rats were treated with either 2 mg/kg naloxone or saline at 25 minutes postchallenge . Arterial PO2 rose and PCO2 fell in a stepwise fashion in saline-treated endotoxic rats . These changes were unrelated to medullary/pontine perfusion or to arterial pH (up to 30 minutes postchallenge) . In naloxone-treated endotoxic rats the increased ventilatory drive at 60 minutes was attenuated, apparently as a result of prevention of acidosis at this time . These data further support uncoupling of ventilatory drive and arterial pH reported earlier and also indicate that naloxone's ability to prevent acidosis in these animals is not mediated through respiratory compensation.

Ukr Biokhim Zh, 1987 Jan-Feb, 59(1), 55 - 61
{Interaction of glucocorticoid hormones with mitochondrial membranes}; Vol'skii GG; The mechanism of 3H glucocorticoid binding with the rat liver mitochondria in vitro is investigated . The linear dependence of the amount of bound hormones on the concentration of the free ones is shown and no saturation in the region of the physiological concentrations is observed . A very low specific binding in the presence of a 100-fold excess of an unlabelled hormone is found . The outer mitochondrial membranes binds a considerably higher amount of steroids, than the inner one . The binding of steroids with the intact liver mitochondria is 2-3 times higher as compared to the binding with spheroplasts of Escherichia coli . Delipidization of mitochondria by diverse lipotropic agents differently influences the binding of steroids with the different functional groups . The interaction of steroids with mitochondria depends on the osmolarity of the incubation medium: the binding is 1.5-3 times higher in the isotonic sucrose solution, that in the hypo- or hypertonic ones . A conclusion is made about the nonspecific character of glucocorticoid binding with mitochondria caused by the interaction with hydrophobic compounds of the mitochondrial membranes . The possible chemical mechanisms for such an interaction are discussed.

Clin Endocrinol (Oxf), 1987 Jan, 26(1), 125 - 8
An open-labelled study of the safety, acute metabolic activity and pharmacokinetic profile of a short-term course of recombinant human growth hormone in healthy volunteers; Wilton P et al.; The safety and short-term biological effects of recombinant human GH (Genotropin, KmbiVitrum AB, Stockholm, Sweden), given i.m . (8 IU/d) for 4 consecutive days to 10 healthy male volunteers, have been evaluated . No adverse reactions were detected by clinical investigation . Haematological parameters, clinical chemistry and the acute phase inflammatory profile showed no negative effects of the drug . The well-known metabolic effects of GH were seen as an increase in free fatty acids and in the somatomedin level (IGF-1) . No antibodies to GH or periplasmic Escherichia coli peptides developed during the first month after treatment . The peak plasma level of hGH (62 +/- 20 mU/l) occurred between 3.0 and 3.5 h . This study shows that recombinant hGH induces no toxic or other adverse reactions and that the acute metabolic effects and the pharmacokinetic profiles are comparable to those reported after injections of GH extracted from pituitary glands.

Surg Gynecol Obstet, 1987 Jan, 164(1), 49 - 56
Skin microvascular permeability after resuscitation with Ringer's lactate solution from endotoxin shock; Mullins RJ et al.; Skin microvascular membrane permeability was assessed in anesthetized dogs after resuscitation from endotoxin shock . Infusion of Ringer's lactate solution, in a volume equal to 7 per cent of the body weight, reversed hypotension produced by an intravenous injection of Escherichia coli endotoxin (0.5 milligrams per kilogram) . The shock group was compared with a group of dogs not given endotoxin and infused with a similar dose of Ringer's lactate solution and a with control group of dogs . Prenodal lymph was collected from both hindpaws and three to four hours before ending an increase of 20 to 28 millimeters of mercury in venous pressure of the hindpaw was produced in one limb by tightening a tourniquet . This maneuver produced an increase in lymph flow associated with an increase in lymph protein flux that was similar in all three groups . The wet to dry tissue weight ratio of skin samples did not increase in the endotoxin group . Skin microvascular membrane permeability did not increase . The safety factors that helped to prevent edema after infusion of Ringer's lactate solution included an increase in lymph flow and sieving by the intact microvascular membrane which diluted the interstitial protein concentration.

IARC Sci Publ, 1987, (84), 340 - 4
N-nitrosamine formation by macrophages; Miwa M et al.; Nitrate biosynthesis is a known mammalian process, and macrophages from mice treated with Escherichia coli lipopolysaccharide (LPS) have been shown to be capable of nitrate synthesis . Cell culture studies showed that macrophages produce nitrite as well as nitrate . We report here N-nitrosamine formation by stimulated macrophages . Experiments were carried out with the macrophage cell lines, J774.1, WEHI-3 and RAW 264 . Macrophages were cultured in Dulbecco's modified Eagle's medium (pH 7.5) supplemented with calf serum (10%) . The concentration of nitrate in the supernatant was measured . N-nitrosamines were extracted with dichloromethane and the extracts were analysed by gas chromatography-thermal energy analysis . When J774.1 (1.5 X 10(6) cells/ml) were incubated with LPS (10 micrograms/ml) and morpholine (15 mM) for 72 h at 37 degrees C, N-nitrosomorpholine (NMOR) was produced (0.8 microM) . The amount of nitrite produced was 50 microM . RAW 264 and WEHI-3 also produced NMOR; LPS was required for nitrite and NMOR formation . gamma-Interferon (IFN) promoted both NMOR (2.5 microM) and nitrite (70 microM) formation . Nitrite (150 microM) incubated with morpholine and the medium did not form NMOR . Kinetics of LPS-induced nitrite and NMOR formation in J774.1 showed that the rate of NMOR formation was highest in the middle incubation period (24-36 h), although the nitrite concentration was highest in the latter incubation period (48-60 h) . Our results showed that macrophages may be capable of nitrosamine formation under physiological conditions that do not normally permit this reaction.

IARC Sci Publ, 1987, (84), 30 - 4
Specificity of O6-alkylguanine-DNA alkyltransferase; Pegg AE et al.; Extensive investigations of the specificity of O6-alkylguanine-DNA alkyltransferase (AAT) have been carried out . These studies have shown that: (i) the mammalian protein differs from that of Escherichia coli in lacking the ability to remove methyl groups from O4-methylthymine; (ii) the protein can remove longer alkyl groups from the O6 position but the rate of repair declines as the chain length increases; (iii) O6-methylguanine in RNA is much less active as a substrate for the protein than O6-methylguanine in double-stranded DNA; (iv) the free-base O6-alkylguanine is a very weak substrate for the protein so that reaction with it leads to the loss of alkyltransferase activity . (This property can be used to deplete AAT in cultured cells and in tissues and tumours after administration of O6-methylguanine); and (v) oligodeoxynucleotides containing O6-methylguanine are substrates for AAT . Such oligodeoxynucleotides can be labelled with 32P at very high specific activity and can be used in an ultrasensitive assay for AAT activity.

Adv Enzyme Regul, 1987, 26, 319 - 33
Chemical characterization of phosphoribosylamine, a substrate for newly discovered trifunctional protein containing glycineamide ribonucleotide synthetase activity; Cheng YS et al.; PRA has been characterized for the first time using 13C-NMR spectroscopy . Incubation of {1-13C}ribose-5-phosphate with NH3 results in the production of 38:62 alpha:beta anomeric mixture of PRA, alpha,beta ribose-5-phosphate and variable amounts of dimeric materials . NMR studies at various pHs allowed determination of the pH independent Kequi = 0.95 +/- 0.14 M-1 for this reaction . In addition, using magnetization transfer NMR methodology the rate of conversion of alpha to beta PRA was determined to be 44 sec-1 at 37 degrees C (pH 8.0) . The rates of formation (from ribose-5-phosphate and NH3) and degradation of PRA were also measured using E . coli GAR synthetase (recently cloned, overproduced and purified to homogeneity) as a trap . Determination of these rates allowed an independent and accurate measurement of Kequi = 2.7 M-1 . In addition, in close agreement with early studies of Nierlich and Magasanik, the half life of PRA at 37 degrees C and pH 7.5 was determined to be 35 sec . Characterization of the chemical stability of PRA and Kequi for ribose-5-phosphate, NH3 with PRA will now allow detailed kinetic analysis of the newly discovered trifunctional protein containing GAR synthetase activity in addition to AIR synthetase and GAR transformylase activities . Comparison of the properties of the 110 kd GAR synthetase and an independently isolated 54 kd GAR synthetase are reported . Experiments are underway to investigate the possibility that unstable intermediates such as PRA are not released into solution, but that the transfer is mediated by specific protein-protein interactions between GAR synthetase and PRPP amidotransferase.

Int J Immunopharmacol, 1987, 9(3), 349 - 53
Selective inhibition of antibody-dependent allergic autocytotoxicity by OM-89, in patients with bronchial asthma and urticaria having food allergy toward wheat; Podleski WK; Twenty-five patients with bronchial asthma and urticaria expressing hypersensitivity to ingested wheat were examined and compared with fourteen controls using the allergic autocytotoxicity assay (ACT) . To establish a possible interaction between wheat antigen and antibody with OM-89 (immunostimulating fraction extracted from E . coli), peripheral WBC of the above individuals were tested in vitro . Preincubation with OM-89 and subsequent exposure toward wheat antigen in the direct ACT assay and antibody-dependent ACT assay using specific wheat antibody were performed . A separate study of spontaneous ACT was conducted in which unprimed white blood cells were pre-incubated with OM-89 alone . Our results show that OM-89 exerted its most potent inhibitory activity in the antibody-dependent ACT assay when the wheat antigen-antibody test system was used . Only borderline inhibition of the direct ACT response was observed, while OM-89 did not exert any effect on spontaneous ACT . This observation may be of importance in future clinical trials since patients sensitive to wheat with high antibody levels toward this common ingestant antigen may benefit from oral therapy with OM-89.

Gene, 1987, 53(2-3), 265 - 73
Replication origin (oric) on the complementary DNA strand of Escherichia coli phage G4: biological properties of mutants; Sakai H et al.; Phage G4 origin of complementary DNA strand synthesis (oric) consists of three stable secondary loop structures . In a cloned 274-bp DNA fragment that is active as an ori in the filamentous phage cloning vector R199, insertion mutants have been constructed by introducing EcoRI and HindIII linkers at the base of loop III . The in vivo activity of these oric mutants (conversion of single-strand form to replicative form in the presence of rifampicin) was significantly reduced (50-70%) but not completely abolished . Nucleotide sequences and/or potential secondary structure of loop III centered at the AvaII site are therefore an important functional part of oric.

Gene, 1987, 53(2-3), 201 - 9
The ura5 gene of the filamentous fungus Podospora anserina: nucleotide sequence and expression in transformed strains; Turcq B et al.; We have sequenced the ura5 gene of the filamentous fungus Podospora anserina . The deduced sequence for the orotidylic acid pyrophosphorylase (OMPppase) has been compared with the Escherichia coli enzyme which is the only known sequence for this enzyme . This comparison shows extensive blocks of homology . The expression of the ura5 gene has been studied in a ura5 mutant which has been transformed by a recombinant plasmid carrying the ura5 gene . We observed that strains carrying integrated multicopies of the transforming vector exhibit higher specific activity for OMPppase than wild type (wt) . By recombination we have constructed a strain in which the level of this enzyme is 32 times higher than in the wt strain.

Eur Surg Res, 1987, 19(4), 207 - 16
Measurement of operative plasma endotoxin levels in jaundiced and non-jaundiced patients; Pain JA et al.; A study of portal plasma endotoxin levels was performed using a chromogenic limulus amoebocyte lysate (LAL) assay . The assay proved sensitive and reproducible . In only 1 of 25 healthy subjects was the systemic plasma endotoxin level above 100 pg/ml (equivalent Escherichia coli 0111B4) . In 30 non-jaundiced patients undergoing surgery the mean (+SEM) portal plasma endotoxin level (60 + 9 pg/ml) was significantly higher (p less than 0.05) than the mean level in the systemic blood (46 + 6 pg/ml), supporting the concept of endotoxin absorption from the intestine into the portal blood . In 20 patients with obstructive jaundice undergoing surgery 42% of portal, 45% of inferior mesenteric and 35% of systemic venous plasma endotoxin levels were above 100 pg/ml . There were significantly higher levels in the portal (p less than 0.05) and inferior mesenteric (p less than 0.05) compared with the systemic blood . Neither the presence of malignancy nor the duration of surgery appeared to influence endotoxin absorption . The significance of raised plasma endotoxin levels in obstructive jaundice is discussed.

Gene, 1987, 53(1), 85 - 96
Improved single and multicopy lac-based cloning vectors for protein and operon fusions; Simons RW et al.; We describe several new vectors for the construction of operon and protein fusions to the Escherichia coli lacZ gene . In vitro constructions utilize multicopy plasmids containing suitable cloning sites located between upstream transcription terminators and downstream lac operon segments whose lacZ genes retain or lack translational start signals . Single-copy lambda prophage versions of multicopy constructs can be made genetically, without in vitro manipulation . The new vectors, both single and multicopy, are improved in that they have very low levels of background lac gene expression, which makes possible the easy detection and accurate quantitation of very weak transcriptional and translational signals . These vectors were developed for analysis of the expression of IS10's transposase gene, which is transcribed less than, once per generation, and whose transcripts are translated on average less than once each . Both single and multicopy constructs can also be used to select mutations affecting fusion expression, and mutations isolated in single-copy constructs can be crossed genetically back onto multicopy plasmids for further analysis.

Circ Shock, 1987, 22(2), 115 - 25
Central nervous system is involved in the cardiovascular responses to naloxone in canine endotoxic but not hemorrhagic shock; Gurll NJ et al.; We used naloxone to investigate the role of central nervous system opiate receptors in the cardiovascular depression of canine hemorrhagic and endotoxic shock . Shock was induced by bleeding dogs into a reservoir to achieve and maintain a mean arterial pressure (MAP) of 45 mmHg for 30 min; at 30 min the reservoir was clamped and the animals were treated with intracerebroventricular (ICV) perfusion of naloxone 0.1 mg/kg (n = 5) or artificial CSF (n = 5) for 30 min . Endotoxemic shock was induced by the iv injection of E . coli endotoxin 1 mg/kg; 15 min later the animals were given naloxone 0.1 mg/kg (n = 5) or artificial CSF (n = 5) ICV for 30 min . ICV naloxone significantly increased MAP, cardiac output (CO), and left ventricular performance (LV dP/dt max) compared to artificial CSF in canine endotoxic shock but not hemorrhagic shock . Naloxone 0.1 mg/kg (n = 5) given into the cisterna magna failed to significantly improve MAP, CO, or LV dP/dt max in dogs subjected to reservoir hemorrhagic shock for 60 min compared to artificial CSF (n = 5) . These results are compatible with opiate-receptor-mediated central cardiovascular depression in endotoxic shock and peripheral cardiovascular depression in hemorrhagic shock . Accordingly, the sites of action of naloxone are mainly central in endotoxic shock and peripheral in hemorrhagic shock.

Res Exp Med (Berl), 1987, 187(2), 87 - 94
Cardiac output and its distribution in peritonitis . (Septic) shock in the rat; Martinell S et al.; Cardiac output and its distribution were studied in rats made septic by an i.p . injection of live E . coli bacteria and in controls given an equivalent amount of saline . The E . coli injection was followed by signs of severe shock in eight of 12 rats . Control animals all survived with only minor changes in cardiac output and peripheral hemodynamics . Blood flow in shocked animals was characterized by a reduction of cardiac output, while myocardial and cerebral flows were not reduced . The intact circulation to the brain and to the heart in the shocked rats was at the expense of kidney, spleen, and skin blood flows.

Invasion Metastasis, 1987, 7(2), 83 - 95
Treatment with endotoxins of peritoneal carcinomatosis induced by colon tumor cells in the rat; Lagadec P et al.; Peritoneal carcinomatosis, a common spreading of human colon carcinoma, can be obtained by intraperitoneal injection of colon tumor cells in rats . When BDIX rats are injected with 10(6) syngeneic tumor cells, isolated and cloned from a chemically induced colon carcinoma, they die within 2-3 months with solid peritoneal tumors and hemorrhagic ascites . Repeated intraperitoneal injections of 20 micrograms endotoxins (Escherichia coli W0128:B12) from day 3 after tumor cell challenge inhibited tumor growth . This effect was long-lasting since 7 out of 10 treated rats were still alive and tumor-free 6 months after tumor cell challenge . When the endotoxins were administered from day 15 after the tumor cell challenge, in rats with established tumors visible with the naked eye, the survival times were significantly increased, and 6 out of 30 treated rats were still alive and tumor-free 6 months after tumor cell challenge . The optimum effect was obtained with 5 repeated injections . The different frequencies of injection tested, i.e . 1, 3 or 5 days apart were equally effective . Endotoxins were ineffective when administered intravenously . No side effect was observed.

Nutr Cancer, 1987, 9(2-3), 129 - 42
Dietary fat, plasma lipoproteins, and immune function in middle-aged American men; Berry EM et al.; Dietary fat has been incriminated as a positive risk factor for the development of neoplasia in human populations . We used adipose tissue fatty acid analysis as an index of dietary fat intake to study the association between dietary fat and immune function in a group of 94 free-living American males (avg age 47 years) . Immunocompetence was tested by a battery of T- and B-lymphocyte stimulation tests and also by natural killer (NK) cell activity . Correlations were sought between fatty acid composition, plasma lipids, and immune responsivity . The degree of unsaturation of the diet over a polysaturated-to-saturated fat ratio range of 0.54-1.01 had no predictable effect on the immune function . Stepwise regression analysis showed that the concentrations of plasma triglycerides and cholesterol and its subfractions did not explain any of the variance in the immune tests . Palmitic acid (16:0) was associated with 7% of the variance of the response to C . albicans and E . coli, perhaps through influencing B-cell activity . Stearic acid (18:0) was correlated negatively to concanavalin A responsivity (18% of the variance) and positively to NK activity (20% of the variance) . If impaired in vitro immune function is a marker of increased risk for carcinogenesis, then our data do not support a role for dietary fat influencing in any systematic manner lymphocyte function in vitro, as reflected by proliferative response or NK activity . Further, plasma lipoproteins, in particular cholesterol levels, did not appear to affect any immune function test . It remains to be studied whether dietary fat, lipoproteins, or fat-soluble substances may influence membrane structure and function and prostaglandin formation as alternative pathways in the promotion of neoplasia.

EMBO J, 1987 Jan, 6(1), 43 - 8
Selection of AUG initiation codons differs in plants and animals; Lutcke HA et al.; The influence of the nucleotide at position -3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs . The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide -3 . In each case, translation of mRNAs produced a single protein of Mr = 12,600 . Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position -3, respectively) but no differences in the wheat germ system . Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate . mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ . Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons . Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC . Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.

Cor Vasa, 1987, 29(1), 64 - 9
Ultrastructural evidences of direct endotoxin action on the heart; Bardakhchian EA et al.; In experiments on 13 rabbits, the authors performed heart perfusion according to Langendorf with endotoxin (9 experiments) or with Ringer's solution (4 experiments), and subsequently analysed the myocardial ultrastructure . They found that 30-minute endotoxin perfusion produces contracture changes, intracellular myocytolysis and desquamation of endothelial cells with destruction of the capillary basal membrane . The findings attested to myocardial dysfunction caused by direct endotoxin action on the heart.

Vet Res Commun, 1987, 11(1), 41 - 55
Functional significance of histologic alterations induced by Escherichia coli pig-specific, mouse-negative, heat-stable enterotoxin (STb); Whipp SC et al.; In contrast to cholera enterotoxin and other Escherichia coli enterotoxins, a pig-specific, heat-stable E . coli enterotoxin (STb) causes morphologic lesions (loss of villous epithelial cells and partial villous atrophy) . These lesions reflect a loss of absorptive cells and thus suggest that STb causes impaired absorption as well as inducing net secretion . The present studies assess functional significance of morphologic changes induced by STb . Net fluid movement, mucosal surface area, sucrase activity and the electrical response induced by alanine were measured in swine jejunal loops exposed to E . coli culture filtrates with and without STb . Net fluid secretion (-11.1 +/- 1.1 ml) occurred in some STb loops (secretors) and net absorption (2.7 +/- 0.3 ml) in others (nonsecretors), but net absorption occurred in all control loops (4.9 +/- 0.2 ml) . The mucosal surface area of STb loops was about 20% less than that of controls (P less than 0.01) . Sucrase activity was also lower (about 15%) in STb loops than in control loops (P less than 0.01) . The electrical response induced by alanine in mucosa from nonsecreting STb loops did not differ from that induced in mucosa from control loops . However, the response to alanine in mucosa from secreting STb loops was reduced about 70% from that in mucosa from nonsecreting STb loops or from control loops (P less than 0.05) . It is concluded that reduced sucrase activity is a functional correlate to villous atrophy induced by STb, that STb impairs alanine absorption in some loops (secretors), and that the impaired alanine absorption is independent of the decreased surface area caused by STb . Because the impaired alanine absorption occurred independent of the decreases in surface area, it is suggested that the secretory response to STb is associated with an impairment of active absorption of alanine.

Mol Biol (Mosk), 1987 Jan-Feb, 21(1), 266 - 74
{Effect of the concentration ratio of bi- and monovalent ions on the functional activity of 30S ribosome subunits of Escherichia coli}; Zhuchenko OP et al.; Experiments on poly(U)-dependent binding of Phe-tRNAPhe to 30S subunits revealed the existence of a critical {Mg2+}/{NH4+} ratio in a medium (approximately 0.05-0.1) with respect to the binding capacity of subunits . If the ratio is greater than the critical one, 30S subunits undergo reversible inactivation even at the highest Mg2+ concentrations (up to 20 mM) . The stronger is the deviation from the {Mg2+}/{NH4+} value = 0.05-0.1, the greater are both the rate and extent of such an inactivation . Two sites for tRNA in initially active 30S subunits have been shown to be inactivated in an interdependent way . On the other hand, a progressive decrease of {Mg2+}/{NH4+} ratio in a medium (from the value of 0.05 and lower) does not produce inactivation, but rather results in reduced affinity constants of Phe-tRNAPhe for active sites of 30S subunits.

Mol Biol (Mosk), 1987 Jan-Feb, 21(1), 242 - 9
{Recognition of Escherichia coli promoters from the primary structure of DNA}; Aleksandrov NN et al.; The perceptron algorithm has been applied for E . coli promotors searching . To choose the appropriate promoter signs the statistical analysis was performed . By the modified perceptron method a vector, which exactly divides promoters and non-promotors in the learning sequences and gives nearly the same results as the statistical vector, has been obtained . By using this vector two potential promotors have been found at the phoE-proB chromosome region.

Mol Biol (Mosk), 1987 Jan-Feb, 21(1), 189 - 93
{The role of heat shock proteins in the osmoregulation of Escherichia coli}; Sherman MIu; E . coli has a number of biochemical systems which protect cells from different chemical and physical damages . The aim of this work is to characterize the interaction between two of these: the osmoregulation system and the heat shock system . It is shown that exposure of E . coli to 42 degrees C to induce hsps synthesis, abolish the growth inhibition by high (0.45 M) NaCl concentration . Also, transient pretreatment of cells with high NaCl protect them from heat damage . It is shown that osmotic shock induces the hsps synthesis . The cell growth restoration after the complete inhibition by high (0.6 M) NaCl concentration correlates with the hsps accumulation . Moreover the heat shock treatment reduces the adaptation time.

Microbios, 1987, 49(199), 107 - 13
Degradation of the pyrimidine bases uracil and thymine by Escherichia coli B; Patel BN et al.; Degradation of the pyrimidine bases uracil and thymine by Escherichia coli B was investigated . The known products of the reductive pathway of pyrimidine base catabolism were tested to determine if they could support the growth of E . coli B cells as sole sources of nitrogen or carbon . As might be expected if the reductive pathway was present, it was found that dihydrouracil, N-carbamoyl-beta-alanine, beta-alanine, dihydrothymine and beta-aminoisobutyric acid could sustain the growth of the bacterial cells as sole nitrogen sources by at least a fourteen-fold greater level than that observed if they were included as sole carbon sources . The existence of the reductive pathway of pyrimidine base degradation was confirmed in this micro-organism, since dihydrouracil, N-carbamoyl-beta-alanine and beta-alanine were detected following thin-layer chromatographic separation of the catabolic products of uracil and dihydrouracil.

Mol Gen Genet, 1987 Jan, 206(1), 178 - 80
Characterization of a promoter up mutation in the -35 region of the promoter of the primer for ColE1 replication; Castagnoli L; A point mutation in the -35 region of the promoter of the primer for initiation of DNA replication in the plasmid pMB1 was characterized . This base change causes a promoter up phenotype . The analysis of a second mutant obtained by site-directed mutagenesis allowed the exclusion of a role in the phenotype for the potential intrastrand secondary structure as well as for the methylation state of the DNA in the promoter region . The promoter up phenotype is concluded to be due to a change in the primary structure of the -35 element with the consequent production of a better cluster of hydrogen bond donors and acceptors for the RNA polymerase.

Mol Gen Genet, 1987 Jan, 206(1), 126 - 32
Uncoupling of synthesis and release of cloacin DF13 and its immunity protein by Escherichia coli; Luirink J et al.; The synthesis of the bacteriocin cloacin DF13 and its release into the culture medium were genetically uncoupled by subcloning the gene encoding the bacteriocin release protein (BRP) from pCloDF13 . The gene was cloned under the control of the IPTG-inducible lpp-lac promoter-operator system on the expression vector pINIIIA1, giving pJL1 . A 4 kb DNA fragment of pJL1, containing the tandem lpp-lac promoter, the BRP gene and lacI (BRP cassette), was cloned into the pCloDF13 derivative plasmid pJN67, which encodes cloacin DF13 but not the release protein . Furthermore, the pCloDF13 immunity protein gene was subcloned downstream of the temperature-inducible PL promoter of the expression vector pPLc236, together with the BRP cassette . Growth, induction and excretion experiments with Escherichia coli cells harbouring the constructed plasmids revealed that: the BRP is the only pCloDF13-derived gene product responsible for the observed growth inhibition and apparent lysis of strongly induced cells . This growth inhibition and lysis can be prevented by Mg2+ ions added to the culture medium, and involves induction of phospholipase A activity . The expression of the BRP gene can be regulated by varying the IPTG concentration . A separately controlled and moderate induced BRP synthesis can be used to bring about the release of large amounts of cloacin DF13 under conditions that allow a strong induction of the bacteriocin and which do not result in lysis of cells . Preliminary results indicated that the BRP can stimulate the release of immunity protein in the absence of cloacin or cloacin fragments.

Mol Gen Genet, 1987 Jan, 206(1), 110 - 5
Receptor specificity of the short tail fibres (gp12) of T-even type Escherichia coli phages; Riede I; Short tail fibres of T-even like phages are involved in host recognition . To determine the specificity of the fibres, the region containing gene 12 of phages T2, K3, and K3hx was cloned . The genes 11, 12, wac, and 13, coding for the baseplate outer wedge, short tail fibres, collar wishes, and a head completion component, respectively, were localized on the cloned fragments . Plasmid-encoded gene 12 could be expressed without helper phage . Efficient expression of gene 12 from T2 and K3hx made an extraction of protein 12 possible . Hybrid phages obtained by in vitro complementation, recombination analysis and protein 12 binding to host range mutant bacteria excluded a role of the short tail fibres from T2, K3 or K3hx in the recognition of outer membrane proteins . Binding patterns of protein 12 to different Escherichia coli lipopolysaccharide mutants and inhibition of binding of protein 12 by a monoclonal antibody against the core region of E . coli K12 lipopolysaccharide suggested that heptose residues are necessary for efficient binding . The binding site of the same monoclonal antibody is different from the short tail fibre binding site in an E . coli B strain suggesting different binding specificities of protein 12 . Thus, the ability of different bacterial strains to inactivate phage could be related to differences in the binding specificity of the short tail fibres for the lipopolysaccharides of these bacteria.

J Dairy Sci, 1987 Jan, 70(1), 201 - 8
Endotoxin of Escherichia coli and permeability of the mammary gland of goats; Lengemann FW et al.; Serial collections of milk were used to determine where in the mammary gland endotoxin of Escherichia coli was effective in altering the transfer of selected milk components into blood and blood components into milk . Lactating goats had half the gland infused with 1 microgram of endotoxin and the other half served as a control . Sodium-24 and 42K or {14C} lactose were included with 141Ce in the infusate in some experiments, whereas in others 99mTc-labelled albumin or 24Na and 42K were given intravenously 2 h after the endotoxin infusion . Milk was collected 3 h after endotoxin infusion . Endotoxin increased the loss of 24Na, 42K, and {14C} lactose from the mammary gland and increased the transfer of 24Na and 99mTc-albumin into the gland . The transfer in of 42K was reduced compared with control halves . Movement of stable Na and K was in accord with the movement of the 24Na and 42K . Endotoxin was effective in all parts of the gland but particularly from the mid-portion upward to the alveoli . For the control halves there was evidence that some 24Na and 42K crossed the ductal or cisternal epithelium into blood outside of the alveoli, whereas only 42K provided evidence for transfer from blood to milk in these same regions . There was no demonstrable transfer of lactose and albumin in regions other than the alveoli.

J Biochem (Tokyo), 1987 Jan, 101(1), 207 - 15
Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site; Kato H et al.; Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements . The effects of ligands of dihydrofolate reductase on the reaction of E . coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase . The E . coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim . Methotrexate was studied in detail and proved to bind to an ATP binding site of the E . coli B enzyme with K1 value of 0.1 mM . The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase . These analyses would indicate that the homologous portion of the amino acid sequence of the E . coli B enzyme provides the ATP binding site . This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.

J Biochem (Tokyo), 1987 Jan, 101(1), 123 - 34
Production and isolation of recombinant somatomedin C; Saito Y et al.; High-level production of a growth promoting peptide hormone somatomedin C (insulin-like growth factor I) has been achieved using recombinant DNA techniques in Escherichia coli . We found a new structural protein, designated as LH, to stabilize somatomedin C in vivo, and constructed expression vectors for somatomedin C fusing to LH through a methionine and through a tryptophan, respectively . Each of the fused proteins was produced at approximately 4.5 X 10(5) molecules per single E . coli cell and comprised more than 20% of the total cellular proteins . Somatomedin C was obtained in high yield by limited cyanogen bromide degradation of the methionine-type fused protein, in spite of somatomedin C itself having a Met at the 59th position, followed by renaturation of the resultant reduced peptide . The tryptophan-type fused protein was also converted to the peptide hormone by treating with 3-bromo-2-nitrophenylsulphenyl skatole or N-chlorosuccinimide . The recombinant somatomedin C obtained by these procedures was identical with the native one in amino acid sequence as well as in biological activity of stimulation of DNA synthesis in BALB/c 3T3 cells.

Digestion, 1987, 36(2), 74 - 80
Effect of endotoxin tolerance on drug hepatotoxicity: amelioration of taurolithocholate cholestasis in the perfused rat liver; Utili R et al.; Induction of endotoxin tolerance may cause resistance not only to endotoxin itself but also to the hepatotoxic effects of other membrane-active agents . To further study this effect, we tested whether endotoxin tolerance could ameliorate the adverse effects of taurolithocholate (TLCA) which causes cholestasis by altering liver plasma membrane organization . Isolated perfused rat livers from endotoxin-tolerant rats had a lower basal bile flow than control livers . However, a bolus addition of TLCA at 3 X 10(-5) or 5 X 10(-5) M in the perfusate caused a marked and prolonged decrease of bile flow in controls, but only a transient and significantly less pronounced diminution of bile flow in endotoxin-tolerant livers . Likewise, TLCA caused a significantly lower alteration of hepatocyte membrane permeability, as measured by sucrose permeability studies, in endotoxin-tolerant livers than in controls . Analysis of bile acid composition of bile from endotoxin-tolerant livers demonstrated that they excreted greater amounts of total bile acids, in particular TLCA and taurocholate, than controls . These results demonstrated a protective effect of endotoxin-tolerance against TLCA toxicity which may result from an altered interaction of TLCA with liver membranes and an increased clearance of TLCA.

Circ Shock, 1987, 21(3), 207 - 16
A comparison of Escherichia coli endotoxin single bolus injection with low-dose endotoxin infusion on pulmonary and systemic vascular changes; D'Orio V et al.; The purpose of this study was to compare effects of single bolus endotoxin injection with sustained low-dose endotoxin infusion on systemic and pulmonary hemodynamics in anesthetized dogs . When administered as a bolus (.01 mg/kg), endotoxin induced systemic vascular changes whose evolution could be divided into two consecutive phases . In the early phase, marked hepatic venoconstriction caused a rise in portal pressure followed by abrupt decreases in both cardiac output and blood pressure . Mean pulmonary artery pressure remained unchanged . Because of lowered blood flow, both peripheral and pulmonary resistances increased . The rise in the latter was due to a prominent vasoconstriction of pulmonary arteries . Following a partial spontaneous recovery from shock, the late phase was characterized by a low-output state combined with high systemic vascular resistances . In contrast, when endotoxin was given at a slow infusion rate (250 ng/kg/min) over a 2-hour period of time, cardiovascular effects were basically different from the preceding ones, and they were measurable only after a certain period of time had elapsed from the start of endotoxin insult . First, blood pressure decreased gradually, while cardiac output remained almost unchanged . Therefore, peripheral resistance was decreased . Second, in the pulmonary circulation, the site of vasoconstriction was shifted from arteries to veins . We conclude that there is a fundamental difference in the response of the dog's systemic and pulmonary circulation as a function of endotoxin administration as either a bolus or slow infusion . This difference might be due to sudden elevated portal pressure responsible for an abrupt cardiovascular collapse in dogs subjected to bolus injection.

Can J Microbiol, 1987 Jan, 33(1), 57 - 62
Metabolism of Escherichia coli injured by copper; Domek MJ et al.; Escherichia coli injured by copper in carbonate buffer simulating the drinking water environment showed decreased oxygen utilization . Oxygraph measurements revealed that copper-injured bacteria had a rate of oxygen utilization that was less than 25% of that of control cells . Respirometry experiments measured rates over a longer period of time and showed similar trends . Nuclear magnetic resonance spectroscopy (13C nmr) and gas chromatography were used to identify differences in metabolism between healthy and injured populations of E . coli . The rate of glucose utilization by injured cells under anaerobic conditions was 64% of that of healthy cells . The rates of lactate and ethanol accumulation were 88 and 50% of the control, respectively . The 13C nmr studies of oxygenated cultures revealed differences in the accumulation of acetate and glutamine . Aerobic utilization of glucose and succinate by injured cells were 87 and 21% of the rate of the controls, respectively . Additional studies revealed injured cells had a decreased ability to reduce 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) with a variety of carbohydrate substrates . Injured cells reduced greater quantities of INT than healthy cells when NADH was used as a substrate . A comparison of metabolic end products suggested that injured cells also had considerable differences in carbon flow compared with healthy cells.

Bioorg Khim, 1987 Jan, 13(1), 119 - 21
{The vector containing a signal for specific degradation of chimeric proteins . Synthesis of {Leu5}enkephalin using enteropeptidase}; Dobrynin VN et al.; A plasmid vector (pEK1) coding, in framework of beta-galactosidase gene, for the amino acid sequence (Asp)4Lys which is recognized by bovine enteropeptidase has been constructed . Using this vector and chemically synthesized DNA coding for the {Leu5}-enkephalin, a plasmid (pEK-ENK) has been obtained in which the beta-galactosidase gene is fused, through the enteropeptidase linker, with the gene for {Leu5}enkephalin . The chimeric protein produced by expression of this plasmid has been isolated and then cleaved by the enteropeptidase to give {Leu5}enkephalin with the yield 74%.

Biochem J, 1987 Jan 1, 241(1), 75 - 80
Tripeptidyl peptidase II in haemolysates and liver homogenates of various species; Balow RM et al.; Rabbit antibodies against purified tripeptidyl peptidase II (TPP II) of human erythrocytes have been prepared and used in immunoblot analysis . Antibodies affinity-purified against the denatured 135,000-Mr subunit of the human peptidase were shown to cross-react with purified rat liver TPP II, as well as a polypeptide of Mr 135,000 in haemolysates and liver homogenates from several other species . TPP II activity in haemolysates from monkey, hog, horse and rabbit corresponded to the levels found in human erythrocytes, whereas this activity was absent or low in erythrocytes from calf and hen . On immunoblot analysis of the calf haemolysates, the 135,000-Mr polypeptide could not be detected . In contrast, analysis of liver homogenates from these species revealed both TPP activity and immunoreactive polypeptides . Immunoreactive polypeptides with Mr about 105,000 and 84,000 of variable occurrence were also detected . In addition, extracts of Escherichia coli showed no TPP II activity under the conditions of the standard assay, although polypeptides of Mr 135,000 and 40,000 were revealed on immunoblot analysis of this species.

Vet Q, 1987 Jan, 9(1), 86 - 7
Additional studies on the therapeutic efficacy of sulphadimidine sodium in experimental Escherichia coli infection of broilers; Goren E et al.; In contrast with the recommended dose of 2,000 ppm sulphadimidine sodium in the drinking water for treatment of broilers against colibacillosis, it appeared that under experimental conditions, treatment at 500 ppm (60 mg/kg body weight) gave the best therapeutic effects . At higher concentrations, drinking water consumption and body weight gain were reduced significantly . During treatment high blood plasma-concentrations were measured.

Mol Cell Biol, 1987 Jan, 7(1), 564 - 7
Complementation of a polyamine-deficient Escherichia coli mutant by expression of mouse ornithine decarboxylase; Macrae M et al.; Mouse ornithine decarboxylase (ODCase) cDNA was expressed at a high level in an Escherichia coli mutant deficient in polyamine biosynthesis . The expression of mouse ornithine decarboxylase relieved the dependence of the mutant on an exogenous source of polyamines, presumably by providing putrescine, the product of the enzyme . The effect on the enzymatic activity of deletions that removed carboxy-terminal amino acids of the protein was determined.

Mol Cell Biol, 1987 Jan, 7(1), 541 - 4
Biochemical and biological properties of the human N-ras p21 protein; Trahey M et al.; We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli . No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP . Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43% . Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells . This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.

J Immunoassay, 1987, 8(1), 131 - 43
An enzyme-immunoassay for human interleukin-2; Moriya N et al.; A highly sensitive enzyme-immunoassay (EIA) for human interleukin-2 (IL-2) has been established . The assay is based on a sandwich method that uses two kinds of anti-IL-2 antibodies raised against Escherichia coli-derived recombinant IL-2 (rIL-2) . An affinity-purified-anti-IL-2 goat IgG was used as the first antibody and the Fab' fragment of an affinity-purified-anti-IL-2 rabbit IgG was used as the second antibody after being coupled with horseradish peroxidase (HRP) . As little as 30 pg/ml of IL-2 was detected by the EIA, indicating that this method was about 100 times more sensitive than the bioassay using an IL-2-dependent murine natural killer cell line, NKC3 . There was a good correlation between the EIA and the bioassay (r = 0.998).

J Appl Physiol, 1987 Jan, 62(1), 116 - 21
Neutrophil depletion does not prevent lung edema after endotoxin infusion in goats; Winn R et al.; Neutropenia was produced in goats by injection of either nitrogen mustard, (1.5 mg/kg) or hydroxyurea (200 mg X kg-1 X day-1) . A nitrogen mustard (M + E) group (n = 6), a hydroxyurea (H + E) group (n = 5), and a control (E) group (n = 7) were given 1-h infusions of endotoxin (5 micrograms/kg total dose), then monitored for up to 5 h . Postmortem extravascular lung water (EVLW) was significantly higher in the M + E group (14.2 +/- 4.4 ml/kg) and the E group (11.9 +/- 3.9 ml/kg) when compared with a normal control (6.6 +/- 1.3 ml/kg) group that did not receive endotoxin . EVLW in a group made neutropenic with nitrogen mustard (6.7 +/- 1.3 ml/kg) and the H + E (7.9 +/- 1.5 ml/kg) groups were not statistically different from each other or from normal controls . Circulating neutrophil counts averaged 32 +/- 42 cells/microliter in the M + E group and 180 +/- 210 cells/microliter in the H + E group . Only minimal histological changes were seen in the H + E group, but the E and M + E lungs had severe pulmonary edema . We conclude that neutrophils are not required for increased EVLW and decreased arterial O2 partial pressure after endotoxin infusion, and hydroxyurea prevents at least part of the pulmonary edema after endotoxin by a mechanism that is not neutrophil dependent.

Farmakol Toksikol, 1987 Jan-Feb, 50(1), 57 - 60
{Effect of armin on nonspecific resistance factors of the body and on the primary humoral immune response}; Zabrodskii PF; A dose-dependent increase of parameters of non-specific defence of the organism and depression of primary humoral immune response following a single subcutaneous administration of armin in doses of 0.04-0.16 mg/kg were found during experiments on mice and rats.

Eur Biophys J, 1987, 14(3), 169 - 78
Lac repressor-Lac operator complexes . Solution X-ray scattering and electrophoretic studies; Culard F et al.; Complexes between the Lac repressor and a small DNA operator fragment (29 base pairs) were investigated using polyacrylamide gel electrophoresis and solution X-ray scattering . Titration of the DNA fragment with the repressor, followed by gel electrophoresis showed that only two types of complexes are formed with repressor/operator ratios of 0.5 and 2 . Radii of gyration and forward scattered intensities were obtained from Guinier plots for repressor/operator ratios ranging from 0.3 to 2 . They demonstrated that the first complex contains one repressor and two operators, whereas the second one contains four repressors and two operators . Mixing operator and repressor in equimolar concentrations leads to a mixture of both complexes . A possible model for the four repressor/two operator complex is proposed.

Circ Shock, 1987, 21(2), 155 - 68
Leukotriene antagonist FPL 57231 prevents the acute pulmonary effects of Escherichia coli endotoxin in cats; Pacitti N et al.; We studied the effects of a selective leukotriene (LT) antagonist (FPL 57231, 2 mg kg-1 min-1) on the acute cardiopulmonary changes observed in feline endotoxin shock . LTC4 and LTD4 (0.1-3.0 micrograms kg-1) given intravenously had little or no activity on pulmonary arterial pressure (PAP), dynamic lung compliance (Cdyn), and airways resistance (Raw) . They did, however, produce a systemic hypertension, which was significantly attenuated during the FPL 57231 infusion . E . coli endotoxin (2 mg kg-1) administration resulted in decreases in systemic arterial blood pressure and Cdyn, together with increases in both PAP and Raw . During infusion of FPL 57231, all these endotoxin-induced cardiopulmonary changes were attenuated . Radioimmunoassay of blood samples taken from cats given FPL 57231 showed that levels of 6-keto prosta-glandin F1 alpha and thromboxane B2 were not significantly increased by endotoxin, as would normally be expected in cats administered endotoxin . FPL 57231 was also found to antagonise the pulmonary effects of the thromboxanemimetic U46619 and of prostaglandin F2 alpha . These results indicate that it is unlikely that the leukotrienes are involved as important mediators of the acute phase of endotoxin shock in cats.

Am J Vet Res, 1987 Jan, 48(1), 56 - 62
Escherichia coli-induced lung and liver dysfunction in dogs: effects of flunixin meglumine treatment; Hardie EM et al.; Twelve dogs were infused with 10(10) Escherichia coli/kg of body weight through a portal vein catheter over a 1-hour period; 6 dogs were treated with flunixin meglumine (1 mg/kg) 15 minutes after the infusion had begun . Six dogs (controls) were infused with a comparable volume of sterile saline solution over the same period . Over a 4-hour monitoring period, nontreated septicemic dogs developed systemic hypotension, decreased cardiac output, increased portal pressure, increased serum alanine transaminase values, increased extravascular liver water, increased liver glycogen depletion, and decreased arterial oxygen tension compared with control dogs . Accumulations of polymorphonuclear leukocytes and E coli were found in the livers and lungs of septicemic dogs . Flunixin meglumine treatment prevented systemic hypotension and hypoxemia, reversed the early but not the late stages of portal hypertension, and decreased E coli concentrations in the lungs . Other effects of treatment were not noticed.

Acta Anaesthesiol Scand, 1987 Jan, 31(1), 67 - 72
Intermittent and continuous positive-pressure ventilation in the prophylaxis of endotoxin-induced lung insufficiency . A study in pigs; Borg T et al.; The effects of intermittent and continuous positive-pressure ventilation (IPPV and CPPV) instituted prophylactically were evaluated in a porcine model of endotoxin-induced pulmonary and cardiovascular failure . Pigs under ketamine anaesthesia were infused i.v . with E . coli endotoxin over 6 h . Twenty animals, breathing air spontaneously, received endotoxin without treatment . Fifteen animals were treated prophylactically with IPPV (normoventilation with air) . Nine animals received prophylactic treatment with CPPV (positive end-expiratory pressure 0.8 kPa (8 cmH2O) . Endotoxin infusion in spontaneously breathing animals caused profound deterioration of pulmonary gas exchange, a marked rise in pulmonary vascular resistance (PVR) and a moderate increase in extravascular lung water (EVLW) . Cardiac output (Qt) and O2 delivery decreased considerably . Metabolic acidosis indicated oxygen deficit . Eleven of 20 animals died during the observation period . IPPV improved arterial oxygenation during endotoxin infusion, and the increase in EVLW tended to be lower . The alterations in pulmonary haemodynamics, Qt and O2 delivery, were of the same magnitude as in spontaneously breathing animals . Survival was improved . CPPV fully prevented the deterioration in pulmonary gas exchange and the development of pulmonary oedema . There was an accentuated increase in PVR . Qt and O2 delivery decreased markedly and a severe metabolic acidosis developed . All animals treated with CPPV died during the observation period . These results indicate that prophylactic IPPV and CPPV may counteract the development of sepsis-induced lung insufficiency in man . However, it must be emphasized that adequate cardiovascular support is essential in optimizing the treatment.

Vet Pathol, 1987 Jan, 24(1), 71 - 9
Effects of Escherichia coli heat-stable enterotoxin b on small intestinal villi in pigs, rabbits, and lambs; Rose R et al.; Culture supernates from two strains of E . coli were placed into different ligated intestinal sections (loops) of each animal . The two bacterial strains were identical except that one contained a plasmid carrying the heat-stable toxin b (STb) gene, while the other did not . Morphometric techniques were used to assess villous epithelial surface areas and mucosal volumes in both intestinal segments exposed to STb-positive (test) and to STb-negative (control) supernates . In pigs whose intestines were exposed to STb-positive supernatants for 2 hours, both villous epithelial surface area and mucosal volume were significantly smaller in test loops than in control loops (P less than 0.02) . In test loops of pigs incubated for 1 hour, and in test loops of lambs incubated for 2 hours, there was a decrease in villous epithelial surface area which approached the test for significance but did not meet it (0.05 less than P less than 0.10) . Rabbit test loops did not differ from rabbit control loops in either villous epithelial surface area or mucosal volume . Histological examination of the tissues from all three species revealed epithelial changes in porcine and ovine tissues only . In porcine and ovine tissues, epithelium at villous tips was seen to be cuboidal or squamous, or even to be absent . Villi with similarly altered epithelium were seen in control loops, but were seen much more frequently in test loops . These epithelial changes were seen as early as 30 minutes of incubation in pigs . Intestinal tissues from these pigs were examined by transmission electron microscopy, but no difference between test and control tissues was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Med Microbiol Immunol (Berl), 1987, 176(1), 47 - 51
A new ELISA test for HIV antibodies using a bacterially produced viral env gene product; Schneider J et al.; Screening tests for antibodies to the human immunodeficiency virus (HIV), based on the indirect ELISA principle using viral preparations as antigen, yield a substantial number of false-positive and false-negative results . These failures are due to the lack of certain viral polypeptides or contaminating cellular polypeptides in viral preparations . Therefore, the accuracy of the screening tests should be improved by using highly purified, synthetic viral antigens . With establishment of such an ELISA antigen in mind, we examined a bacterially synthesized polypeptide {ENV(80)} that corresponds to 80 conserved amino acids of the HIV gp41 transmembrane glycoprotein . ENV(80) was expressed as a DHFR fusion protein in Escherichia coli . Results obtained by HIV ELISA and immunoprecipitation with 497 serum samples from various groups at risk of AIDS were compared with those obtained with the ENV(80) ELISA . The ENV(80) ELISA was found to be superior to the H9/HTLV-III ELISA with respect to sensitivity and specificity and is almost equivalent in accuracy to immunoprecipitation.

J Steroid Biochem, 1987 Jan, 26(1), 67 - 71
Endotoxin-induced changes in sex steroid hormone levels in male rats; Christeff N et al.; Intravenous administration of Escherichia coli endotoxin (ENDO) was found to induce profound time and dose dependent changes in the serum steroid hormones, oestrone (E1), oestradiol (E2), corticosterone (B), progesterone (P4), 17 alpha-OH progesterone (17 alpha OHP4), and testosterone (T) of intact male rats . These changes were rapid, with a maximal response at 2 h and a return to close to normal values by 4 h . Non-lethal doses (0.01-2 mg/kg) of ENDO induced large increases in oestrogens (3-9-fold), P4 (4-fold) and B (2-3-fold) and decreased serum T (2-fold) . The greatest increase in E2 level was seen with an ENDO dose of 2 mg/kg . Serum E1, E2 and T did not change in response to lethal ENDO doses (4-8 mg/kg); B, P4 and 17 alpha OHP4 levels alone were moderately elevated . Systemic mean arterial pressure was unchanged, except at the highest ENDO dose used . Thus, the hormonal responses are unlikely to be the result of hemodynamic changes . Low doses of ENDO did not produce an increase in serum E1 and E2 in adrenalectomized or orchidectomized rats . These results indicate that oestrogens are largely produced in the testis . The aromatization of the testicular and adrenal androgens can be stimulated by glucocorticoid.

Eur Surg Res, 1987, 19(2), 78 - 85
Protection by aminated glucan in experimental endogenous peritonitis; Almdahl SM et al.; The effect of prophylactic intraperitoneal aminated glucan on the survival rate and formation of adhesions and abscesses was investigated in rats with acute and subacute peritonitis, respectively induced by cecal perforation and ileal ligation . A significantly reduced mortality was found in both forms of peritonitis . Pretreatment by aminated glucan also significantly reduced the number of abscesses and peritoneal adhesions . An about threefold increase in peritoneal macrophages in aminated glucan-treated rats compared to controls was noted . In vitro studies, using 32P-labelled Escherichia coli, demonstrated that peritoneal macrophages from aminated glucan-treated rats had an enhanced ability to engulf and degrade bacteria . Scanning electron microscopy showed that macrophages from aminated glucan-treated animals were highly spread with prominent ruffling and bacteria located intracellularly, as opposed to macrophages from controls which were rounded with bacteria on the cell surface.

Cell Biochem Funct, 1987 Jan, 5(1), 55 - 61
Effect of Escherichia coli lipopolysaccharide on the microviscosity of liver plasma membranes and hepatocyte suspensions and monolayers; Portoles MT et al.; The fluorescence probe 1,6-diphenylhexa-1,3,5-triene (DPH) was used for monitoring structural perturbations induced by lipopolysaccharide (LPS) of Escherichia coli (0111:B4) in plasma membranes of rat liver . Changes in microviscosity were observed in plasma membrane preparations from control rats after treatment with LPS and in plasma membrane preparations from liver perfused with LPS . In both systems fluorescence polarization was measured from which microviscosity was calculated . This parameter increases with LPS treatment . From temperature dependence studies was inferred that LPS interaction with plasma membrane preparations induces an increase of both the polarization term (r0/r-1)-1 and flow activation energy (delta E) . Addition of LPS to hepatocyte suspensions also induces an increase on microviscosity and a delay in the fall of microviscosity induced by a temperature rise in hepatocyte monolayers grown on microcover slides . These data suggest that LPS interaction can be attributed to its binding to membrane hydrophobic regions in a non-specific manner.

Virology, 1987 Jan, 156(1), 122 - 6
Characterization of phage 18, an unstable coliphage; Bess VH et al.; Phage 18, a noninducible coliphage, is quite unstable and therefore difficult to study . Newly developed very gentle lysis and mounting techniques yielded isolated virions for examination by electron microscopy . The phage has a contractile tail with a length of 130 nm and an isometric head with a capsid diameter of 50 nm . Phage 18 is similar in morphology to phage P2 but is heteroimmune to it . DNA extracted from a clear-plaque mutant of phage 18 was subjected to BamHI restriction endonuclease digestion and was found to be easily distinguishable from the published restriction patterns for P2, phage 299, or phage 186 DNA . The genome size was calculated to be 33.5 kb . Using the DNA melting point, phage 18 DNA (G+C) content was determined to be 55.0% and its buoyant density was determined to be 1.715.

Scand J Immunol, 1987 Jan, 25(1), 55 - 60
The protective effect of beta 1-3D-glucan-derivatized plastic beads against Escherichia coli infection in mice; Seljelid R et al.; Pretreatment with beta-1,3-D-glucan-derivatized plastic beads conferred strong protection against Escherichia coli infection in mice . The protective effect showed a dose-response relationship to the amount of beads injected and was dependent on the time point of the injection relative to the infection with E . coli . A similar protection could be obtained in nude mice . Experiments with radioactively labelled bacteria as well as beads indicated a systemic effect of the beads . Macrophages extracted from animals treated with glucan plastic beads appeared highly stimulated . This was also true of cells that did not contain beads and presumably therefore not glucan, which seems to indicate a soluble stimulatory factor.

Mol Cell Biochem, 1987 Jan, 73(1), 61 - 8
Glutathione status and sensitivity to GSH-reacting compounds of Escherichia coli strains deficient in glutathione metabolism and/or catalase activity; Alonso-Moraga A et al.; The intracellular concentrations of total glutathione, GSSG and protein X S-SG, the total excreted glutathione concentration, and the susceptibility towards GSH-reacting compounds were assayed in strains of Escherichia coli deficient in biosynthesis and/or reduction of glutathione . A deficiency in glutathione reductase displaced the glutathione status towards the oxidized forms . This displacement was more clearly appreciated in strains additionally deficient in glutathione biosynthesis . A deficiency in catalase activity also produced an increase in the oxidation of glutathione . The most severe changes were observed in the concentrations of protein-glutathione mixed disulfides and in the amount of glutathione excreted to the medium . Increased sensitivities towards compounds known to interact with cellular GSH were observed in glutathione reductase deficient strains, although these effects were enhanced in strains additionally deficient in GSH biosynthesis.

J Gen Virol, 1987 Jan, 68 ( Pt 1), 135 - 45
Expression of reovirus type 3 (Dearing) sigma 1 and sigma s polypeptides in Escherichia coli; Pelletier J et al.; The reovirus S1 gene codes for two polypeptides: sigma 1 and sigma s . In order to characterize the structure and function of the sigma 1 polypeptide, we have expressed the sigma 1 protein in Escherichia coli . The S1 gene from mammalian reovirus type 3 (Dearing strain) and the variant K strain were subcloned into an expression vector containing the tac (trp-lac) promoter designed to express foreign gene products in E . coli efficiently . The hybrid plasmids, upon induction with isopropyl-beta-D-thiogalactopyranoside, expressed two polypeptides that were detected by {35S}methionine labelling . One of the induced proteins had a relative molecular mass (Mr) of approx . 46,000 and corresponded to sigma 1, as shown by immunoprecipitation with goat anti-reovirus antibody and a monoclonal antibody against sigma 1 . The second induced protein had a Mr of approx . 12,000 and was very similar to sigma s as judged by comparative tryptic peptide map analysis . Protein sigma 1 produced in E . coli was shown to be functional as judged from its ability to bind to mouse L fibroblasts.

Arch Surg, 1987 Jan, 122(1), 105 - 10
Macrophages and translymphatic absorption represent the first line of host defense of the peritoneal cavity; Dunn DL et al.; To quantitate the host defenses of the rat peritoneal cavity, nonviable radiolabeled Escherichia coli were injected intraperitoneally and clearance, leukocyte influx, and phagocytosis were examined . Macrophages (MCs) were present initially and remained relatively constant in number . The polymorphonuclear leukocyte (PMN) response began at one to two hours and was maximal at 24 to 72 hours . A previously unidentified inoculum-dependent PMN response was defined . Clearance and phagocytosis were extremely rapid, and few (less than 3%) free bacteria were present after two hours . Phagocytic activity of MCs and PMNs was identical, but MCs were numerically predominant initially and thus accounted for the majority of early phagocytosis . Thus, MC phagocytosis and clearance represent the primary line of host peritoneal defenses . We hypothesize that the subsequent inoculum-dependent PMN response may have evolved to cope with those larger inocula for which this initial response is inadequate.

Am Rev Respir Dis, 1987 Jan, 135(1), 93 - 9
Role of 5-hydroxytryptamine in endotoxin-induced respiratory failure of pigs; Olson NC; The porcine pulmonary vascular and airway responses to exogenous 5-hydroxytryptamine (5-HT), norepinephrine, prostaglandin F2 alpha (PGF2 alpha), and angiotensin II were evaluated before and after ketanserin, a 5-HT2 receptor antagonist . Ketanserin blocked the 5-HT-induced increases in airway and pulmonary artery pressures, whereas the increases in airway and pulmonary artery pressures caused by norepinephrine, PGF2 alpha, or angiotensin II were not significantly modified by ketanserin, indicating a relatively high degree of specificity for 5-HT2 receptors . The role of endogenous 5-HT in mediating endotoxin-induced respiratory failure was evaluated by treating pigs with ketanserin . Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized 10- to 14-wk-old pigs at 5 micrograms/kg the first h, followed by 2 micrograms/kg/h for 3.5 h . Ketanserin was infused at 300 micrograms/kg before endotoxin plus 67 micrograms/kg/h during endotoxemia . During Phase 1 (i.e., 0 to 2 h), the endotoxin-induced increases in pulmonary vascular resistance and room air alveolar-arterial oxygen difference and the decreased cardiac index and lung dynamic compliance were not significantly modified by ketanserin . However, during Phase 2 (i.e., 2 to 4.5 h) endotoxemia, ketanserin attenuated the endotoxin-induced pulmonary hypertension and the increases in pulmonary vascular resistance, alveolar dead space ventilation, and alveolar-arterial oxygen difference . Ketanserin also attenuated the Phase 2 bronchoconstriction and the decreased cardiac index, but did not modify the endotoxin-induced increase in alveolar-capillary permeability . These results indicate that 5-HT plays little or no role in mediating the early (i.e., less than 2 h) response to endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Am Rev Respir Dis, 1987 Jan, 135(1), 83 - 6
Inhalation of endotoxin stimulates alveolar macrophage production of platelet-activating factor; Rylander R et al.; The production of PAF was studied in alveolar macrophages (AM) and neutrophils recovered by bronchial lavage from guinea pigs exposed to aerosolized bacterial endotoxin (lipopolysaccharide, LPS) . The amount of cell-associated PAF was estimated by measuring serotonin release from rabbit platelets . An increased and dose-related production was found in AM for as long as 2 h after a 40-min exposure . No production was detectable after 4 h . Prolonging the exposure did not prolong the response . When a second exposure was given, no PAF could be detected until the time interval between the 2 exposures was 72 h . The amount of neutrophils in lung lavage fluid was elevated about 100 times at 4 h after the exposure, but only a minor PAF production was found in these cells . In view of the role of LPS-contaminated dusts for the development of human lung disease, particularly airway constriction, the role of PAF needs to be further investigated.

Surg Gynecol Obstet, 1987 Jan, 164(1), 17 - 21
A temporary nontoxic lubricant for a synthetic absorbable suture; Rodeheaver GT et al.; Synthetic absorbable sutures have been coated with lubricants to improve their handling characteristics . A unique surfactant, poloxamer 188, has been used to coat the surface of the polyglycolic acid sutures . This lubricant was chosen because it does not damage the tissues defenses of the host and invite infection . Since poloxamer 188 is readily soluble in aqueous solutions, it is rapidly absorbed in the tissue environment resulting in an uncoated suture that displays increased knot security . The coating of polyglactin 910 is also minimally reactive in tissues and does not damage tissue defense . In contrast with the coated polyglycolic acid sutures, the knot security of the coated polyglactin 910 sutures is not altered by exposure to an aqueous environment or implantation . The increased knot security of the coated polyglycolic acid suture after implantation is considered to be a distinct clinical advantage over that of the coated polyglactin 910 sutures.

Proc Natl Acad Sci U S A, 1987 Jan, 84(2), 412 - 5
Molecular cloning and nucleotide sequence of cDNA for human liver arginase; Haraguchi Y et al.; Arginase (EC 3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals . Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia . To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, we isolated cDNA clones for human liver arginase . Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe . Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted Mr, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment . Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert . RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases . The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively . There are several highly conserved segments among the human, rat, and yeast enzymes.

Proc Natl Acad Sci U S A, 1987 Jan, 84(2), 373 - 7
Membrane association of proline dehydrogenase in Escherichia coli is redox dependent; Wood JM; The PutA protein, product of the Escherichia coli gene putA, has two functions essential for proline utilization and for the regulation of putP and putA expression: as the peripheral membrane flavoprotein, proline dehydrogenase (EC 1.5.99.8), it transfers electrons from proline to the respiratory chain, and, as a repressor, it controls expression of genes putP and putA in response to proline supply . Association of proline dehydrogenase with the membrane was shown to require the simultaneous presence of the soluble enzyme, membranes, and proline . The kinetics of that association, monitored by following proline oxidation in a coupled enzyme assay system, were not altered when the transmembrane proton gradient generated during proline oxidation was dissipated by a proton ionophore . However, D-lactate or NADH could replace proline as a promoter of proline dehydrogenase-membrane association under anaerobic reaction conditions . These data imply that reduction of proline dehydrogenase and/or a membrane constituent promotes enzyme-membrane association . A biochemical mechanism is suggested whereby the concentration of proline dehydrogenase associated with the respiratory chain would be determined by proline supply.

Proc Natl Acad Sci U S A, 1987 Jan, 84(2), 344 - 8
DNA base changes and alkylation following in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea; Richardson KK et al.; Dideoxy chain-termination DNA sequencing was used to determine the specific DNA base changes induced after in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target . The resultant mutation spectra were compared with the levels of O6-alkylguanine and O4-alkylthymidine in genomic DNA immediately after exposure . All (39/39) of the MNU-induced mutations were G X C----A X T transitions . In contrast, 24/33 point mutations isolated following ENU treatment were G X C----A X T transitions, 7/33 were A X T----G X C transitions, 1/33 was a G X C----C X G transversion, and 1/33 was an A X T----C X G transversion . Three large insertions, probably of spontaneous origin, were also isolated . O4-alkylthymidine/O6-alkylguanine ratios were 0.014 for MNU and 0.28 for ENU . These data suggest that the difference in the mutation spectrum of MNU versus ENU may be attributed, in part, to the different ratio of O6-alkylguanine versus O4-alkylthymidine produced in the DNA . Of the G X C----A X T transitions, 82% of the MNU- and 71% of the ENU-induced mutations occurred at the middle guanine of the sequence 5'-GG(A or T)-3'.

Proc Natl Acad Sci U S A, 1987 Jan, 84(2), 334 - 8
Escherichia coli formylmethionine tRNA: mutations in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop; Seong BL et al.; We have generated mutants of Escherichia coli formylmethionine initiator tRNA in which one, two, and all three G X C base pairs in the GGGCCC sequence in the anticodon stem are changed to those found in E . coli elongator methionine tRNA . Overproduction of the mutant tRNAs using M13 recombinants as an expression vector and development of a one-step purification scheme allowed us to purify, characterize, and analyze the function of the mutant tRNAs . After aminoacylation and formylation, the function of mutant formylmethionyl tRNAs was analyzed in an MS2 RNA-directed in vitro protein-synthesizing system, in AUG-dependent ribosomal P site binding, and in initiation factor binding . The mutant tRNAs show progressive loss of activity in initiation, the mutant with all three G X C base pairs substituted being the least active . The mutations affect binding to the ribosomal P site . None of the mutations affects binding to initiation factor 2 . We also show that there is a progressive increase in accessibility of phosphodiester bonds in the anticodon loop of the three mutants to S1 nuclease, such that the cleavage pattern of the mutant with all three G X C base-pair changes resembles that of elongator tRNAs . These results are consistent with the notion that the contiguous G X C base pairs in the anticodon stem of initiator tRNAs impart on the anticodon loop a unique conformation, which may be important in targeting the initiator tRNA to the ribosomal P site during initiation of protein synthesis.

Proc Natl Acad Sci U S A, 1987 Jan, 84(1), 31 - 5
Shared active sites in oligomeric enzymes: model studies with defective mutants of aspartate transcarbamoylase produced by site-directed mutagenesis; Wente SR et al.; Many oligomeric enzymes are functional only in the assembled form, and it is often difficult to determine unambiguously why monomers are inactive . In some cases individual monomers cannot fold into stable correct ("native") conformations without contributions from interchain interactions . For other oligomers, catalysis requires the contributions of amino acid residues at the interface between adjacent polypeptide chains, and monomers are inactive because they cannot form complete active sites . A test for the presence of shared sites was devised that is based on the formation of active hybrid oligomers from appropriate inactive parental mutants produced by site-directed mutagenesis . This approach was applied in a study of the catalytic trimer of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, using three mutants, in which Ser-52 was replaced by His, Lys-84 by Gln, or His-134 by Ala . Hybrid trimers formed from the virtually inactive Ser and Lys mutants were 10(5) more active than the parental proteins, and the specific activities of each hybrid were about 33% that of the wild-type trimer, as expected for the scheme based on shared sites . Hybrids from the His and Lys mutants had comparable specific activities . Moreover, one hybrid with approximately 33% activity had one high-affinity binding site for a bisubstrate analog as compared to about three for wild-type trimer . As a further test, hybrids were also formed from wild-type and double-mutant (Lys-84----Gln and His-134----Ala) trimers . The hybrid containing two chains with the double mutation and one wild-type chain had very little activity, and that composed of one double mutant and two wild-type chains had 32% the specific activity of wild-type trimers . This negative complementation experiment is in quantitative accord with the scheme based on shared sites at or near the interfaces between adjacent chains . The techniques used to demonstrate shared active sites in the catalytic subunits of aspartate transcarbamoylase can be applied generally to various types of oligomers (dimers, tetramers, etc.) to determine whether the participation of amino acid residues from adjoining chains is essential for forming active sites in oligomeric enzymes.

Mutat Res, 1987 Jan, 183(1), 1 - 9
A model for the recA-dependent repair of excision gaps in UV-irradiated Escherichia coli; Smith KC et al.; We have tested and supported the hypothesis that, in UV-irradiated Escherichia coli, recA-dependent nucleotide excision repair only functions in the replicated portion of the chromosome (i.e., where sister duplexes exist) . Using a dnaA(Ts) mutation to align the chromosomes (i.e., all rounds of DNA replication were completed, and new rounds could not be initiated), we studied the genetic control of excision repair (measured as the repair of excision gaps in DNA) in cells with unreplicated chromosomes, and also in cells with partially replicated chromosomes . The excision repair that occurred in cells with unreplicated chromosomes was recA independent, but the excision repair that occurred in cells with partially replicated chromosomes was partially recA dependent . We found no evidence of interchromosomal recombination in recA-dependent excision repair . The majority of this recA-dependent excision repair was recF dependent, and a small portion was recB dependent . The recF and recB genes are suggested to function in excision repair in a manner similar to their function in postreplication repair, i.e., in the replicated portion of a chromosome, the RecF pathway repairs gaps, and the RecB pathway repairs the DNA double-strand breaks that arise at unrepaired gaps.

Mutat Res, 1987 Jan, 176(1), 21 - 8
Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA; Paramio JM et al.; The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied . pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation . After transformation into E . coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed . Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria . Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent) . Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain . In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed . Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery . Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells . Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts . The greatest increase in plasmid recovery was found in the wild-type strain . This likely involved the repair of monoadducts and some fraction of the crosslinks . We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.

J Clin Microbiol, 1987 Jan, 25(1), 146 - 9
Cytotoxic necrotizing factor production by hemolytic strains of Escherichia coli causing extraintestinal infections; Caprioli A et al.; Two hundred and nineteen strains of Escherichia coli from extraintestinal infections and feces of healthy subjects were examined for hemolysin (Hly) and cytotoxic necrotizing factor (CNF) production and for mannose-resistant hemagglutination . Of 105 strains from extraintestinal infections, 42 (40.0%) were positive for production of both Hly and CNF, and 21 (20.0%) were positive for Hly alone; on the contrary, only 1 Hly- and CNF-positive strain and 2 Hly-positive strains were found among 114 strains from normal stools . CNF production was not found to occur among the nonhemolytic strains, confirming the close association existing between these toxic factors . Hemolytic strains positive for CNF showed mannose-resistant hemagglutination less frequently than did Hly-positive, CNF-negative strains (25.6 versus 82.6%), suggesting the existence of two distinct classes among hemolytic strains of E . coli.

J Clin Microbiol, 1987 Jan, 25(1), 106 - 9
Comparative study of synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes to identify enterotoxigenic Escherichia coli; Echeverria P et al.; Escherichia coli isolated from 2,126 children in Thailand and the Philippines was examined for enterotoxin production and for DNA hybridization with synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes . A total of 233 infections with E . coli that were detected by one or more of these assays were identified . Of the infections, 75% (164/233) were identified by all three methods . An additional 18% (43/233) were identified by two of three methods . Isolates from 10% (19/183) of infections with E . coli that hybridized with both the oligonucleotide and cloned enterotoxin gene probes were nontoxigenic, as determined by the Y1 adrenal cell and suckling mouse assays . Although synthetic oligonucleotide probes to detect enterotoxigenic E . coli are more uniform and easier to use than cloned enterotoxin gene probes, the heat-labile toxin oligo probe used in this study did not identify 13% (11/87) of infections with E . coli that produced heat-labile toxin, as identified with the Y1 adrenal cell assay and the cloned enterotoxin gene probe . Synthetic oligonucleotide probes enable laboratories with only minimal equipment to use DNA hybridization assays to identify enterotoxigenic E . coli.

J Bacteriol, 1987 Jan, 169(1), 97 - 101
Effect of growth rate and cell shape on the peptidoglycan composition in Escherichia coli; Driehuis F et al.; The muropeptide composition of peptidoglycan from Escherichia coli W7 cultivated at different growth rates in chemostat cultures was compared by using high-pressure liquid chromatography . At a low growth rate (D = 0.1 h-1), about 40% more covalently bound lipoprotein and at least twofold more diaminopimelyl-diaminopimelic acid cross-bridges were found than at a high growth rate (D = 0.8 h-1) . The total degree of cross-linkage was only slightly increased, and the fraction of trimeric muropeptides and the average length of the glycan chains were not changed significantly . Analysis of the peptidoglycan from a morphological variant strain of W7 revealed that the altered peptidoglycan composition in slowly growing W7 cells was not correlated with the observation that these cells, due to their decreased cell length, were relatively enriched in polar material . In fact, our results suggested that peptidoglycan forming cell poles is chemically identical to that forming lateral wall.

J Bacteriol, 1987 Jan, 169(1), 423 - 6
uvrC gene function has no specific role in repair of N-2-aminofluorene adducts; Bichara M et al.; In Escherichia coli, plasmid DNA modified with N-2-aminofluorene adducts survived equally well in wild-type, uvrA, or uvrB strains . Increased sensitivity was found in uvrC and uvrD strains . Moreover, N-2-aminofluorene-mediated toxicity in the uvrC background was reversed when an additional uvrA mutation was introduced into the strain.

J Bacteriol, 1987 Jan, 169(1), 419 - 22
Intracellular 5',5'-dinucleoside polyphosphate levels remain constant during the Escherichia coli cell cycle; Plateau P et al.; All AppppN and ApppN nucleotides (N = A, C, G, or U) occur in Escherichia coli . Measured cellular concentrations were 2.42 microM AppppA, 0.61 microM AppppC, 0.95 microM AppppG, 1.17 microM AppppU, 0.47 microM ApppA, 0.14 microM ApppC, 0.20 microM ApppG, and 0.12 microM ApppU . These concentrations remained constant during the cell cycle in synchronized exponentially growing cells.

J Bacteriol, 1987 Jan, 169(1), 272 - 7
New regulatory features of the promoters of an Escherichia coli rRNA gene; Lukacsovich T et al.; Recombinant plasmids were constructed by fusing either promoter p1 or p2 or both promoters of the rrnB gene of Escherichia coli to a DNA fragment coding for the N-terminal alpha-peptide of beta-galactosidase . These plasmids contained various lengths of the 5'-leader region of rRNA as the 5'-terminal end of the alpha-peptide messenger . In some cases the entire 5'-terminal rRNA-coding sequence was removed, and alpha-peptide synthesis was governed by rac promoters formed by fusion of rrnBp2 and lac promoters . By measuring the level of alpha peptide, conclusions could be drawn about the activities of the promoters under various physiological conditions . It was found that the rate of transcription starting from promoter p1 or p2 might vary more than 10-fold during the growth cycle, showing a sharp maximum during outgrowth from the stationary phase into exponential growth or during nutritional shift-up . The target sequence of this regulation was localized to the leader region of the rrnB gene.

J Bacteriol, 1987 Jan, 169(1), 26 - 32
Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli; VanBogelen RA et al.; Heat and various inhibitory chemicals were tested in Escherichia coli for the ability to cause accumulation of adenylylated nucleotides and to induce proteins of the heat shock (htpR-controlled), the oxidation stress (oxyR-controlled), and the SOS (lexA-controlled) regulons . Under the conditions used, heat and ethanol initiated solely a heat shock response, hydrogen peroxide and 6-amino-7-chloro-5,8-dioxoquinoline (ACDQ) induced primarily an oxidation stress response and secondarily an SOS response, nalidixic acid and puromycin induced primarily an SOS and secondarily a heat shock response, isoleucine restriction induced a poor heat shock response, and CdCl2 strongly induced all three stress responses . ACDQ, CdCl2, and H2O2 each stimulated the synthesis of approximately 35 proteins by factors of 5- to 50-fold, and the heat shock, oxidation stress, and SOS regulons constituted a minor fraction of the overall cellular response . The pattern of accumulation of adenylylated nucleotides during these treatments was inconsistent with a simple role for these nucleotides as alarmones sufficient for triggering the heat shock response, but was consistent with a role in the oxyR-mediated response.

J Bacteriol, 1987 Jan, 169(1), 210 - 7
Cytochemical localization of lipopolysaccharides during peptidoglycan degradation of Escherichia coli cells; Frehel C et al.; The cytochemical reaction of Thiery {J . P . Thiery, J . Microsc . (Paris) 6:987-1018, 1976} was applied to several Escherichia coli strains having different lipopolysaccharide molecular structures . The granular deposit obtained strongly suggested that part of the R core exposed on the outer membrane was responsible for the staining . As this procedure specifically stains the outer membrane, it was possible to demonstrate that, in E . coli K-12, changes in lipopolysaccharide distribution occurred during autolysis and lysozyme treatment.

J Bacteriol, 1987 Jan, 169(1), 142 - 56
Organization of developing Escherichia coli colonies viewed by scanning electron microscopy; Shapiro JA; Colony growth was initiated by inoculating minimal glucose agar with 1-microliter . spots of a plasmid-free Escherichia coli culture and incubating at 32 degrees C . Inoculations took place over a 3-day period, at the end of which the plates were fixed and dried for scanning electron microscopy . In this way, it was possible to examine the surfaces of colonies ranging in age from 0 to 68 h . Macroscopically, the colonies were organized into different concentric zones, and several morphological features could be seen to develop over this period . These included a shallow depression ring marking the site of inoculation, a deeper indentation ring whose position moved outward as the colony grew, an expanding plateau region between the two rings, a mound outside the indentation ring, and a flat brim extending onto the substrate which was either present or absent at different times . Microscopically, a variety of cell morphologies and cell arrangements were detected . Upon inoculation, the bacteria accumulated at the periphery of the inoculation spot but showed no other kind of order . For the first 7.5 h, all bacteria were rod shaped; at the end of this initial phase, a high degree of alignment was seen in the cells at the colony edge . By 24.5 h, both shorter more ovoid cells and longer filaments had begun to appear, and large multicellular arrays had formed . At later stages of colony development, morphologically distinguishable zones involving cells of different shapes and sizes had formed, and these zones often marked the boundaries of macroscopic features . The edges were particularly interesting and at 68 h displayed very sharp saw-toothed boundaries between concentrically organized groups of bacteria . There were some transient irregularities in the concentric organizations of growing colonies, and one colony had entered upon a distinct developmental pathway.

J Bacteriol, 1987 Jan, 169(1), 110 - 6
In vitro system for molybdopterin biosynthesis; Johnson ME et al.; A high-Mr fraction present in chl+ and chlA1 strains of Escherichia coli synthesizes molybdopterin (MPT) from the low-Mr fraction of several MPT-deficient mutants . Using this in vitro complementation as an assay, we have partially characterized the high-Mr fraction as a protein, termed MPT converting factor, of Mr 45,000, distinguishable from the Mo cofactor carrier protein of similar Mr by its absolute requirement for the low-Mr fraction of a non-chlA1 mutant in the nit-1 reconstitution assay . MPT converting factor was rapidly inactivated in the absence of a reduced sulfhydryl compound . Anaerobic incubation of MPT converting factor with trypsin destroyed its activity . High-performance liquid chromatographic analysis of alkaline KMnO4 oxidation products demonstrated that the factor did not contain any bound pterin . Since mutants lacking MPT converting factor are not auxotrophs for folate or riboflavin, the factor appears to be distinct from known pteridine biosynthetic enzymes in E . coli . We have partially purified and characterized the low-Mr fractions as probable MPT precursors . Several distinct precursors were separable by high-performance liquid chromatography . Like MPT activity, precursor activity was oxygen sensitive . Precursor activity was not correlated with levels of L-threo-neopterin, a major pterin of unknown function in E . coli . Precursor activity was correlated with levels of a new 6-alkylpterin, compound Z, produced by acidic iodine oxidation . Compound Z has the properties expected of an oxidized MPT precursor.

Infect Immun, 1987 Jan, 55(1), 69 - 77
Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa; Knutton S et al.; The adhesion of classic enteropathogenic Escherichia coli (EPEC) strains of human origin to isolated human small intestinal enterocytes and cultured small intestinal mucosa was investigated . An adhesion assay with isolated human enterocytes prepared from duodenal biopsy samples was developed and tested with EPEC strains known to cause diarrhea in healthy adult volunteers . In the assay a mean of 53 and 55% of enterocytes had brush border-adherent E . coli E2348 (O127;H6) and E851 (O142:H6), respectively, whereas the value for a nonpathogenic control strain and a plasmid-cured derivative of strain E2348 was 0% . A collection of 17 EPEC strains was also tested for the ability to colonize cultured human duodenal mucosa . Extensive colonization occurred with 13 strains, including serogroups O55, O86, O111, O114, O119, O127, O128, and O142; and in each case electron microscopic examination of colonized mucosa revealed the characteristic histopathological lesion reported by others in natural and experimental EPEC infections . EPEC strains were seen to adhere intimately to the enterocyte surface, causing localized destruction of microvilli . The plasmid-cured derivative of strain E2348, which colonized cultured mucosa much less efficiently than the parent strain, nevertheless produced an identical lesion, indicating that plasmid-encoded factors are not essential for adhesion and the brush border-damaging property of EPEC.

Infect Immun, 1987 Jan, 55(1), 193 - 7
Presence and expression of aerobactin genes in virulent avian strains of Escherichia coli; Lafont JP et al.; Virulent and nonvirulent isolates of avian Escherichia coli were tested for the presence of aerobactin genes by colony hybridization with a specific gene probe constructed from plasmid pABN1 (A . Bindereif and J . B . Neilands, J . Bacteriol . 153:1111-1113, 1983) . Positive hybridization with the gene probe was highly correlated with virulence, as measured by the 50% lethal dose of the strains for chicks . Evidence for the expression of aerobactin genes in the virulent strains was obtained by demonstrating their susceptibility to cloacin DF13, which binds to the same receptor that binds aerobactin, and their ability to produce aerobactin, as revealed by cross-feeding the E . coli mutant WO987 (aroB fepA iuc iut+), which is unable to synthesize but capable of taking up aerobactin . We suggest that the production of aerobactin is involved in the virulence of avian septicemic E . coli.

J Mol Cell Immunol, 1987, 3(2), 109 - 20
Lymphoma models for B cell activation and tolerance . V . Anti-Ig mediated growth inhibition is reversed by phorbol myristate acetate but does not involve changes in cytosolic free calcium; Scott DW et al.; B cell lymphomas which can be growth inhibited by crosslinking their surface IgM receptors by anti-Ig reagents provide models for normal B cell regulation and tolerance . WEHI-231 and CH31 are two independently derived lines that are exquisitely sensitive to negative signalling by antibodies specific for mu or kappa chains, but are unaffected by antibodies against MHC class 1 or 2 antigens . In order to determine the mechanism of this growth inhibition as a model for tolerance, we have examined the roles played by protein kinase C activation and calcium mobilization/influx during negative signalling in these cells . We found that growth inhibition caused by anti-mu crosslinking was reversed in the presence of either phorbol myristate acetate (PMA) or by lipopolysaccharide (LPS) from E . coli . The effect of PMA on negative signalling was a true reversal since phorbol esters could be added after anti-mu treatment, thus allowing nearly normal cellular progression into the S phase of the cell cycle . In contrast, pretreatment with PMA did not provide protection against the growth inhibition from anti-mu . Indeed, a "desensitization" protocol demonstrated that PMA pretreatment actually decreased reversal by both PMA and LPS of the effects of anti-mu on B lymphoma growth . These studies suggest that both LPS and PMA act via at least one common intermediate, which is assumed to involve activation and translocation of protein kinase C . Analysis of changes in calcium ion concentration after treatment with anti-Ig reagents showed both mobilization from internal stores and influx via calcium channels in WEHI-231, as has been reported for normal B cells . However, these changes did not correlate with negative signalling for the several reasons . Firstly, anti-mu inhibition of the growth of WEHI-231 could be induced in the relative absence of extracellular Ca++ or in quin-2 loaded (buffered) cells . Secondly, pretreatment with high concentrations of PMA ablated calcium mobilization, yet failed to modulate growth inhibition in WEHI-231 cells . Moreover, LPS provided protection from the effects of anti-mu yet did not alter cellular {Cai++} . In addition, PMA posttreatment (under conditions causing a reversal of the effects of anti-mu) can be applied as long as four hours after the initial exposure to anti-mu and the rapid measurable changes in calcium flux . Indeed, such changes in intracellular free calcium occurred in elutriated WEHI-231 lymphoma cells at all phases of the cell cycle, although we have previously identified early G1 as the only critical period in which negative signalling can be delivered.(ABSTRACT TRUNCATED AT 400 WORDS)

Free Radic Res Commun, 1987, 3(6), 389 - 94
Comparative anti-inflammatory activity of different superoxide dismutases and liposomal SOD in ischemia; Jadot G et al.; Comparison of superoxide dismutases from different sources with respect to biological activity in the rat tourniquet poditis model shows that anti-ischemic activity is very variable although all the enzymes have the same specific enzymic activity . Both bovine Cu-SOD and E . coli Mn-SOD have excellent properties whereas yeast Cu-SOD and the homologous rat Cu-SOD show zero activity . The results confirm earlier demonstrations that (1) "All superoxide dismutases are equal but some are more equal than others", (2) at the dose levels used (compatible with possible clinical use) homologous enzyme is inefficient and hence human Cu-SOD may not be effective in humans, (3) liposomal encapsulation of bovine Cu-SOD greatly enhances biological efficacity, provides a slow release mechanism of the enzyme and provides a powerful drug for the treatment of ischemic injury.

Curr Genet, 1987, 12(8), 625 - 7
Neomycin resistance as a dominantly selectable marker for transformation of the zygomycete Absidia glauca; Wostemeyer J et al.; A plasmid (pAmN61) containing the NPT II structural gene (neomycin phosphotransferase) fused to the N-terminal region of a homologous actin gene was used for the transformation of Absidia glauca protoplasts . Neomycin resistant transformants could be selected for on complete medium containing 1.2 mg/ml neomycin sulfate . The physical presence of plasmid DNA in Absidia glauca was demonstrated by retransformation into Escherichia coli and by Southern blot analysis . No integration of plasmid DNA at either one of the two actin loci was observed; Southern blot experiments provide evidence that pAmN61 is autonomously replicated in Absidia glauca.

Diagn Clin Immunol, 1987, 5(4), 201 - 4
Interleukin-1 activity from human cord blood monocytes; Wilmott RW et al.; Cord blood monocyte synthesis of IL-1 was investigated by using a thymocyte proliferation assay . Monocytes from 27 infants ranging in gestation from 31 to 41 weeks (mean 38.9, SE 0.54) with birthweights from 1.20 to 4.31 kg (mean 3.24, SE 0.13) were isolated from cord blood; 2 x 10(5) cells/ml were plated in 15 mm wells and stimulated with 10 micrograms/ml LPS (E . coli) . Control cultures contained medium alone . Supernatants were harvested after 24 hr and tested in a C3H/HeJ mouse thymocyte proliferation assay . The mean response for 27 cord monocyte samples at 24 hr was 14,142 cpm (SE 1,499), not significantly different than that for cells obtained from eight normal adult volunteers (15,137 cpm, SE 3,535) . Vaginally delivered infants with perinatal complications such as amnionitis, fetal distress, or early sepsis had significantly increased unstimulated activity (5,139 vs 1,331 cpm) compared to samples from normal infants, whereas stimulated activity was not significantly different (16,219 vs 12,261 cpm) . Thus, the IL-1 response to lipopolysaccharide is intact in newborn human monocytes and there is evidence of an increased unstimulated activity following neonatal complications.

Curr Genet, 1987, 11(5), 347 - 52
An intron in the 23S rRNA gene of the Chlorella chloroplasts: complete nucleotide sequence of the 23S rRNA gene; Yamada T et al.; A 243 bp intron was found within the 23S rRNA gene of the unicellular green alga Chlorella ellipsoidea . This intron is A+T-rich (63.7%) compared with the 23S rRNA (50.5%) and is located in domain II of the 23S rRNA . In contrast to rRNA introns so far known, this intron is considerably small and does not possess features of group I introns in spite of its possible folded secondary structure; this is a new type rRNA intron . The complete nucleotide sequence of the 23S rRNA gene (2,965 bp) was also compared with that of tobacco chloroplasts and E . coli.

Vet Res Commun, 1987, 11(5), 479 - 91
The efficacy of dexamethasone and flunixin meglumine in treating endotoxin-induced changes in calves; Margolis JH et al.; Eicosanoids have been implicated in the pathophysiology of endotoxic shock . Drugs which alter eicosanoid production such as corticosteroids and non-steroidal anti-inflammatory drugs (NSAID) are beneficial in treating endotoxic shock . Experiments were conducted to investigate the efficacy of dexamethasone, a corticosteroid, and/or flunixin meglumine, a NSAID, in treating endotoxin-induced changes in calves . Fourteen male calves were assigned to one of four treatment groups: group 1, endotoxin-untreated; group 2, endotoxin-flunixin meglumine treated; group 3, endotoxin-dexamethasone-treated; group 4, endotoxin-flunixin meglumine and dexamethasone-treated . Each calf was given three intravenous and intraperitoneal injections of E . coli endotoxin . Hemodynamic, blood gas, blood chemical and eicosanoid level determinations were obtained . Thirty minutes after endotoxin injection, pulmonary artery pressure (PAP) increased and cardiac output (CO) decreased compared with baseline, corresponding to increased thromboxaneB2 levels in groups 1 and 3 . These groups exhibited a decreased mean arterial pressure (MAP) at three and five hours corresponding to increased 6-keto-prostaglandinF1 alpha . The MAP, PAP and CO of group 4 remained near baseline for the entire six hours, except for a late drop in MAP . Lactic acid levels were significantly increased and arterial bicarbonate levels were reduced by six hours in all groups except for group 4 . These results indicate that the combination treatment of flunixin meglumine and dexamethasone prevents many of the metabolic derangements observed during endotoxic shock in calves.

Acta Gastroenterol Latinoam, 1987, 17(1), 25 - 33
{Spontaneous peritonitis in the patient with cirrhosis, our experience}; Terg R et al.; Due to scarce printed information in our country, the incidence regarding spontaneous peritonitis observed during two years in 76 ascites episodes, found in 63 patients with hepatic cirrhosis, is analysed retrospectively . Thirteen patients (17%), showed spontaneous peritonitis and the relationship man-woman was 5 to 1; 70% of the germs found in the ascites fluid was of enteric origin, mainly Escherichia Coli . In three patients the diagnosis was made by both counting the leucocytes and the clinical symptoms, in spite of the negative culture . There were no significant differences in the presence of humoral complications or alterations when patients appeared with sterile ascites and spontaneous peritonitis, but there were differences with the death rate which was 7.9% (5/63), in the former and 38% (5/13), in infected ascites; 80% of the dead patients showed renal deficiency at the end of the evolution and a relationship with the use of aminoglucosides can not be discarded . The search for spontaneous peritonitis in the cirrhotic patient, as a routine, seems to have the same incidence among as, as the one described in the literature.

Circ Shock, 1987, 23(3), 179 - 88
Attenuation of endotoxin-induced increase in glucose metabolism by platelet-activating factor antagonist; Lang CH et al.; Platelet-activating factor (PAF) has recently been proposed as a putative mediator of various pathophysiologic events during endotoxemia . The aim of the present study was to determine the relative importance of PAF in producing the alterations in carbohydrate metabolism following endotoxin . Chronically catheterized conscious rats were treated with SRI 63-441, a specific PAF receptor antagonist, or saline prior to Escherichia coli endotoxin (100 micrograms/100 g body weight, LD10) administration . Hemodynamic and whole-body glucose kinetic changes, the latter assessed by a constant intravenous infusion of {6-3H} glucose, were determined throughout the 4-hr experimental protocol . Endotoxin induced a transient 30-35% reduction in mean arterial blood pressure (MABP) in animals treated with saline . The PAF-antagonist attenuated this hypotensive effect, and MABP was only reduced by 14-18% . Endotoxin increased plasma glucose and lactate levels, as well as the rate of glucose appearance (Ra) in saline-treated rats . The PAF antagonist reduced the hyperglycemia by 60-75% and tended to prevent the hyperlactacidemia . The endotoxin-induced elevation in glucose Ra was also attenuated by 55% . A similar degree of hyperglucagonemia was observed following endotoxin in both groups, and plasma insulin concentrations were not different . However, plasma catecholamine levels were significantly lower (30-70%) in endotoxemic rats treated with the PAF antagonist . These results suggest that the enhanced production of PAF following endotoxin may be responsible, at least in part, for the early hemodynamic changes . The role of PAF as a mediator of endotoxin-induced glucose dyshomeostasis, however, may be secondary to its hemodynamic effects.

J Enzyme Inhib, 1987, 1(4), 259 - 66
A comparative analysis of the synergistic interaction between N1-phenylsulfanilamides and benzylpyrimidines using qsar techniques; Coats EA et al.; Growth inhibition of E . coli cell culture has been determined for a series of 4-substituted-N1-phenylsulfonilamides tested in the presence and absence of synergistic concentrations of trimethoprim . Quantitative structure-activity relationships, established by regression analysis, exhibit an identical dependence of bacterial growth inhibition on sulfonamide pKa irrespective of the presence or absence of trimethoprim . Examination of a small series of benzylpyrimidines in the presence or absence of 4-dimethylamino-N1-phenylsulfanilamide gave similar results . Since the presence of a synergistic agent affords no change in structure-activity relationships, it is concluded that no direct interaction between sulfonamides and benzylpyrimidines occurs and that the synergism observed is solely the result of the kinetic consequences of sequential blockade of the folate biosynthetic pathway.

Biochem Soc Symp, 1987, 54, 93 - 101
Regulation of the enzymes at the branchpoint between the citric acid cycle and the glyoxylate bypass in Escherichia coli; Nimmo HG et al.; During growth of Escherichia coli on acetate, the glyoxylate bypass supplies the precursors needed for biosynthesis . The glyoxylate bypass enzyme isocitrate lyase competes with the citric acid cycle enzyme isocitrate dehydrogenase for the available isocitrate . We have studied the control of metabolic flux at this branchpoint by examining the regulatory properties of the enzymes concerned . Isocitrate dehydrogenase is controlled by reversible phosphorylation catalysed by a bifunctional kinase/phosphatase whose activities are regulated by isocitrate, biosynthetic precursors and adenine nucleotides . The flux through isocitrate lyase is mainly controlled by the intracellular concentration of isocitrate . The phosphorylation system responds to the availability of energy and precursors and maintains isocitrate at a concentration high enough to sustain the flux through the glyoxylate bypass necessary for biosynthesis.

Biochem Soc Symp, 1987, 54, 67 - 81
2-Oxo acid dehydrogenase multi-enzyme complexes: in the beginning and halfway there; Perham RN et al.; The lipoate acyltransferase components of the pyruvate and 2-oxoglutarate dehydrogenase complexes are highly segmented proteins, forming the structural and mechanistic cores of the complexes . Various functional domains can be isolated by controlled proteolysis, the cleavage sites occurring in conformationally flexible segments of polypeptide chain with unusual sequences . The complexes exhibit novel properties of active-site coupling, which stem from their unusual quaternary structure and the polypeptide chain flexibility that enables protein domains to move with respect to the three contributing active sites during catalysis . In vitro mutagenesis, high resolution n.m.r . spectroscopy and the methods of protein engineering are providing important insights into the mechanics of these processes, with more general implications for the design principles of macromolecular assemblies.

Biochem Soc Symp, 1987, 54, 183 - 95
Compensatory regulation in metabolic pathways--responses to increases and decreases in citrate synthase levels; Walsh K et al.; The level of citrate synthase was varied in Escherichia coli by recombinant DNA methods to elucidate regulatory interactions between the individual steps of the citric acid cycle . The effects of overproduction and underproduction of citrate synthase were assessed by measuring metabolite levels, rates of carbon flow, the phosphorylation state of isocitrate dehydrogenase, and the growth rate of the culture . This analysis revealed that the levels of citrate synthase and isocitrate dehydrogenase activity are co-ordinated for efficient growth on acetate . When citrate synthase was overproduced the isocitrate dehydrogenase reaction became rate limiting and prevented large increases in the flux through the citric acid cycle . Furthermore, changes in the level of citrate synthase were found to modulate the phosphorylation state of isocitrate dehydrogenase which regulates the distribution of carbon flow between the citric acid cycle and the glycoxylate shunt . These adjustments allowed the organism to maintain a relatively constant metabolic state despite changes in the level of a central metabolic enzyme . The interplay between citrate synthase and isocitrate dehydrogenase illustrates how living systems can compensate for variations in their internal environment.

Biochem Soc Symp, 1987, 54, 17 - 31
Control of flux through the citric acid cycle and the glyoxylate bypass in Escherichia coli; Holms WH; The glyoxylate bypass and citric acid cycle operate concurrently in Escherichia coli when acetate is the sole source of carbon and energy to sustain aerobic growth . The overall carbon balance allows fluxes through the central metabolic pathways (CMPs) to be computed on the assumption that these metabolic pathways are known . Acetate is fluxed via the CMPs to the precursors required for synthesis of new biomass and also to generate the reducing power and ATP required to convert these precursors to biomass . Under these circumstances, a junction is created at isocitrate where isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) compete for their common substrate . In general, flux through ICL generates the precursors used for biosynthesis while the larger part of the flux (95%) through ICDH is dedicated to the supply of reducing power and ATP . The system sustains a large intracellular pool of isocitrate to accommodate the rather low affinity of ICL for this substrate . Excessive flux of isocitrate through ICDH is prevented by regulation of ICDH activity: reversible inactivation of ICDH is achieved by a bifunctional kinase/phosphatase, as the phosphorylated form of ICDH has no activity . The kinase/phosphatase responds to two classes of effectors--intermediates of the CMPs generated by flux through ICL and the lower energy forms of ATP and NADPH (ADP, AMP and NADP+) generated when these intermediates are used for biosynthesis . The effect is to adjust flux through ICDH so that the rate of supply of NADPH and ATP is equal to the demands of biosynthesis . Biosynthetic fluxes are limited by the rate of supply of precursors which depends on flux through ICL . Growth rate is most likely limited by the primary flux of acetate to acetyl-CoA or flux through ICL . In the steady state, the flux through ICDH is regulated to be twice the throughput of ICL . The evolution of this complex pattern of control may have depended on alternatives to the citric acid for energy generation.

Biochem Soc Symp, 1987, 54, 103 - 11
The subunits of succinyl-coenzyme A synthetase--function and assembly; Bridger WA et al.; Succinyl-CoA synthetase is made up of two kinds of subunits, designated alpha and beta . The enzyme from Escherichia coli is an alpha 2 beta 2 tetramer (mol . mass . 142 kDa), whereas the mammalian mitochondrial species is an alpha beta dimer . By means of active enzyme centrifugation, we have shown that the active form of the bacterial enzyme is the tetramer even at very low assay concentrations, while the pig heart enzyme is a non-associating dimer over a wide concentration range . The E . coli enzyme shows distinct half-of-the-sites reactivity with respect to the phosphorylation of a histidine residue in the alpha-subunit that represents a step in catalysis . Many lines of evidence (hybrid enzyme formation, oxygen exchange kinetics, 31P-n.m.r . studies) suggest that co-operative interactions between alternatingly functional active sites on the two halves of the E . coli enzyme contribute to its catalytic efficacy . In further refining this model for catalysis, we have shown that the monothiophosphorylated E . coli enzyme does not catalyse exchange of 18O from the beta, gamma-bridge to the beta-non-bridge position of ATP, indicating that the enzyme does not undergo even transient bis-phosphorylation . As a first step in studying the in vivo synthesis and assembly of the enzyme in the mammalian mitochondrial matrix, we have cloned and sequenced a 900 bp cDNA fragment that encodes most of the alpha subunit of rat liver succinyl-CoA synthetase . The derived amino acid sequence shows an impressive degree of homology to that of the alpha subunit of the enzyme from E . coli . We have shown that the alpha subunit in rat liver is a discrete nuclear gene product, complete with cleavable signal sequence to specify mitochondrial targetting.

Biochem Soc Symp, 1987, 53, 103 - 14
Structure and function of candidate vaccine antigens in Plasmodium falciparum; Anders RF et al.; Analysis of Plasmodium falciparum antigens expressed in Escherichia coli has identified several different proteins as potential vaccine components . Fragments of one of these antigens, the ring-infected erythrocyte surface antigen (RESA) have been used to protect Aotus monkeys against overwhelming infection with P . falciparum . In this vaccine, trial protection correlated with antibody responses to two of three repetitive sequences in RESA . RESA is released from merozoites and becomes associated with the erythrocyte membrane at the time of merozoite invasion . The 3' repeat structure of RESA encodes a polyacidic sequence that has homology with the N-terminal sequence (cytoplasmic domain) of band 3, the erythrocyte anion transporter . These homologous sequences both include a recognition sequence for a protein tyrosine kinase . It is postulated that RESA disrupts the normal intermolecular interactions of the cytoplasmic domain of band 3 and that phosphorylation of RESA by an erythrocyte membrane kinase in some way regulates this function of RESA.

J Cell Sci Suppl, 1987, 7, 1 - 13
Enzyme systems initiating replication at the origin of the Escherichia coli chromosome; Kornberg A; More than ten proteins are known to participate in replication of plasmids bearing the unique origin of the Escherichia coli chromosome (oriC) . Initiation of replication of oriC plasmids has been resolved into five separable stages . An initial complex formation (Stage I) requires an oriC plasmid, dnaA protein and HU protein . In the presence of ATP at a temperature of greater than 28 degrees C, a dnaB-C protein complex interacts to form a prepriming complex (Stage II) . This is followed by extensive unwinding of the template that depends on the further addition of gyrase and single-strand binding protein (SSB) (Stage III) . Hydrolysis of an rNTP by dnaB protein (a helicase action) and of ATP by gyrase (a swivelling action) drives the extreme unwinding of the template . This unwound template-protein complex is the substrate for priming by primase (Stage IV) and elongation by DNA polymerase III holoenzyme (Stage V) . Priming of all DNA chains is done by primase; RNA polymerase functions in template activation rather than priming . DNA polymerase III holoenzyme, composed of at least seven subunits, synthesizes the DNA chains . The alpha subunit is the polymerase, the epsilon subunit is the 3'----5' exonuclease; alpha + epsilon is the proofreading activity . Following the synthesis of new DNA chains, DNA polymerase I and ribonuclease H remove the RNA primers, polymerase I fills the gaps, and ligase seals the daughter strands (Stage VI) . Replication produces plasmids identical in structure and sequence to the initial template.

Curr Genet, 1987, 12(8), 599 - 603
DNA sequence and functional analysis of an ARS-element from the zygomycete Absidia glauca; Burmester A et al.; A DNA fragment of mitochondrial origin from the mucoraceous fungus Absidia glauca promoting autonomous plasmid replication in Saccharomyces cerevisiae was sequenced and functionally characterised . We could show that the original mitochondrial insert cloned in Yip5 contains two regions with ARS activity which mutually inhibit each other . All plasmid derivatives replicating in yeast are rapidly lost during growth under non-selective conditions . In addition to one ARS consensus sequence with only one base substitution, the mitochondrial insert contains 18 related sequences with two base pair exchanges . With one exception all consensus sequences are preceded by sequence motifs strongly resembling ARS boxes.

Braz J Med Biol Res, 1987, 20(6), 873 - 5
Chloramphenicol pretreatment enhances resistance to UV, X-rays and H2O2 damage in Escherichia coli but not of infecting UV-damaged phages; Fernandes PM et al.; Chloramphenicol pretreatment of Escherichia coli increases resistance to UV, X-rays and H2O2 damage . Increased survival of UV-irradiated, infecting lambda phages is not observed . The hypothesis that enhanced survival of chloramphenicol-pretreated cells is due to the induction of plasmid "repairons" is questioned.

Braz J Med Biol Res, 1987, 20(6), 869 - 71
Effect of hydrogen peroxide on Escherichia coli radC; Felzenszwalb I et al.; High sensitivity to ionizing radiation is observed in Escherichia coli radC mutants . This is not seen for H2O2-treated cells but when the polA mutation is also present, cells are more sensitive than in the presence of the recA mutation . An increase in inactivation was observed for strains tested when cells are grown in minimal medium and starvation-induced resistance is observed in H2O2-treated cells.

Cold Spring Harb Symp Quant Biol, 1987, 52, 631 - 8
Implications for enzymic catalysis from free-energy reaction coordinate profiles; Fierke CA et al.; The constraints on the internal ground and transition states of enzyme-bound intermediates are mandated by the overall free-energy change in the direction of flux (Chin 1983; Stackhouse et al . 1985; Raines 1986) and the barrier for combination with reagent . Within these confines there are many solutions to transit the reaction coordinate . The path taken may reflect the function of the enzyme in question; particularly, whether in addition to catalysis there is a need to optimize reaction accuracy (Cramer and Freist 1987), to perform mechanochemical coupling (Jencks 1980), or to create metabolic control (Newsholme and Crabtree 1981; Koshland 1984).

Oncogene, 1987, 1(4), 387 - 93
The human c-myc exon 1 product: preparation of antisera and analysis of its expression; Ferre F et al.; We investigated the coding capacity of the previously reported open reading frame (ORF) of the human c-myc exon 1 . By in vitro translation assay, we found that exon 1 ORF was translated into a 20-kd protein (p20 protein) . In order to obtain antisera raised against the p20 protein, we constructed a plasmid vector which expressed most of the exon 1 ORF as a 25-kd (P25) protein in Escherichia coli . Polyclonal antisera raised against this P25 protein specifically precipitated a chimeric protein which contained exon 1-related amino acid sequences . We used these antisera to test for the existence of an exon 1 product in human cells . In the human cell lines tested, these antisera have failed so far to detect any exon 1-related proteins . However, exon 1-related proteins were detected with the anti-p20 antisera in quail embryonic cells (QEC) transfected by human c-myc recombinants constructed to express such proteins, but were expressed at low levels compared with the human c-myc protein also expressed in the transfected QEC . Our results suggest that secondary structure of the mRNA could be responsible for the low expression of the exon 1 product in QEC.

Braz J Med Biol Res, 1987, 20(5), 599 - 601
Antipyretic activity of an aqueous extract of Zizyphus joazeiro Mart . (Rhamnaceae); Nunes PH et al.; Bark infusions of Zizyphus joazeiro Mart . (Rhamnaceae) have been employed in Northeastern Brazil as a remedy for fever . This study investigated the antipyretic activity of an aqueous extract of the plant in rabbits rendered febrile by intravenous injection of E . coli endotoxin . Fever responses were significantly decreased (P less than 0.05) by the oral administration of a bark infusion of Z . joazeiro Mart . These results lend support to the popular use of infusions of this plant in folk medicine as a remedy for fever and suggest that the characterization of the principle(s) responsible for such activity deserves further investigation.

Acta Microbiol Hung, 1987, 34(3-4), 233 - 40
The effects of cannabinoids and cannabispiro compounds on Escherichia coli adhesion to tissue culture cells and on leukocyte functions in vitro; Molnar J et al.; delta 9-Tetrahydrocannabinol, cannabidiol, cannabidiolic acid, tetrahydrocannabidiolic acid, cannabispirol, acetylcannabispirol, cannabispirone, and cannabispirenone in a low concentration did not affect the adhesion of Escherichia coli on cultured HEp-2 cells . Cannabinoids at 10(-6) M increased the chemiluminescence of human polymorphonuclear leukocytes, while the cannabispiro compounds failed to enhance the oxidative burst of leukocytes . In lymphocyte and granulocyte function tests (E- and EA-rosette formation, blast transformation of T-lymphocytes in the presence of phytohaemagglutinin and concanavalin-A, ADCC and phagocytosis) all compounds displayed immunosuppressive effect at 1.5 X 10(-5) M . Tetrahydrocannabidiolic acid exerted the weakest immunosuppression on human leukocyte functions.

Acta Microbiol Hung, 1987, 34(3-4), 225 - 32
Age dependent effect of toxic and detoxified endotoxins on the natural anti-DNA antibody level in rats; Elekes E et al.; Toxic endotoxin (LPS) and its irradiated detoxified form (RD-LPS) induced only small changes in the DNA level in the sera of 4, 12 and 18 months old rats . Changes in the anti-DNA titres were age-dependent . In 4 months old rats one or two injections of the LPS preparations failed to increase the circulating anti-DNA level . In 12 and 18 months old rats both toxic and detoxified LPS preparations raised the anti-DNA titres . The second LPS injection evoked a smaller response in 18 months old rats than the first one.

Curr Genet, 1987, 12(7), 547 - 54
The isolation of specific genes from the basidiomycete Schizophyllum commune; Froeliger EH et al.; We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune . Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation . The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ura1, encoding orotidine-5'-phosphate decarboxylase; OMPdecase), tryptophan (trp1, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1) . In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S . commune mutant . Total DNA from transformants was restricted, religated, and used to transform E . coli . Ampicillin resistant plasmids were recovered from E . coli and tested for their ability to transform the corresponding mutant of S . commune . Plasmids complementing the ade2, ade5, pab1, trp1, and ura1 mutations were recovered.

Proteins, 1987, 2(3), 210 - 24
Prediction of the tertiary structure of the alpha-subunit of tryptophan synthase; Hurle MR et al.; The tertiary structure of the alpha-subunit of tryptophan synthase was proposed using a combination of experimental data and computational methods . The vacuum-ultraviolet circular dichroism spectrum was used to assign the protein to the alpha/beta-class of supersecondary structures . The two-domain structure of the alpha-subunit (Miles et al.: Biochemistry 21:2586, 1982; Beasty and Matthews: Biochemistry 24:3547, 1985) eliminated consideration of a barrel structure and focused attention on a beta-sheet structure . An algorithm (Cohen et al.: Biochemistry 22:4894, 1983) was used to generate a secondary structure prediction that was consistent with the sequence data of the alpha-subunit from five species . Three potential secondary structures were then packed into tertiary structures using other algorithms . The assumption of nearest neighbors from second-site revertant data eliminated 97% of the possible tertiary structures; consideration of conserved hydrophobic packing regions on the beta-sheet eliminated all but one structure . The native structure is predicted to have a parallel beta-sheet flanked on both sides by alpha-helices, and is consistent with the available data on chemical cross-linking, chemical modification, and limited proteolysis . In addition, an active site region containing appropriate residues could be identified as well as an interface for beta 2-subunit association . The ability of experimental data to facilitate the prediction of protein structure is discussed.

Proteins, 1987, 2(2), 81 - 9
Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells; Schneider WP et al.; We have produced a series of hybrid IgG1.IgG2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differences between immunoglobulin isotypes . Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse gamma 1 and gamma 2a gene segments . Crossovers occurred throughout these segments although the frequency was highest in regions of high nucleotide sequence homology . Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H2L2 tetramers and properly glycosylated . In addition, all 11 immunoglobulins have identical antigen combining sites specific for the fluorescent hapten epsilon-dansyl-L-lysine . Protein A binding was used as a probe of the structural integrity of the Fc portion of these variant antibodies . Differences in protein A binding between IgG1 and IgG2a appear to be due to amino acid differences at positions 252 (Thr----Met) and 254 (Thr----Ser) of the heavy chain (EU numbering).

Proteins, 1987, 2(1), 54 - 63
Characterization of a slow folding reaction for the alpha subunit of tryptophan synthase; Hurle MR et al.; The equilibria and kinetics of urea-induced unfolding and refolding of the alpha subunit of tryptophan synthase of E . coli have been examined for their dependences on viscosity, pH, and temperature in order to investigate the properties of one of the rate-limiting steps, domain association . A viscosity enhancer, 0.58 M sucrose, was found to slow unfolding and accelerate refolding . This apparently anomalous result was shown to be due to the stabilizing effect of sucrose on the folding reaction . After accounting for this stabilization effect by using linear free-energy plots, the unfolding and refolding kinetics were found to have a viscosity dependence . A decrease in pH was found to stabilize the domain association reaction by increasing the refolding rate and decreasing the unfolding rate . This effect was accounted for by protonation of a single residue with a pK value of 8.8 in the native state and 7.1 in the intermediate, in which the two domains are not yet associated . The activation energy of unfolding is 4.8 kcal/mol, close to the diffusion limit . The negative activation entropy of unfolding, -47 cal/deg-mol, which controls this reaction, may result from ordering of solvent about the newly exposed domain interface of the transition state . These results may provide information on the types of noncovalent interactions involved in domain association and improve the ability to interpret the folding of mutants with single amino-acid substitutions at the interface.

Gene, 1987, 61(3), 231 - 41
A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library; Richardson MA et al.; A new shuttle cosmid vector, pKC505, was constructed for the cloning of Streptomyces DNA . This vector, which can be conjugally transferred between different streptomycetes, was used to construct a genomic library from a spiramycin-producing S . ambofaciens strain . By transformation of the spiramycin-sensitive S . griseofuscus with the library, three phenotypically different spiramycin-resistance genes were isolated . S . ambofaciens DNA in these clones was colinear with the chromosome, and the cloned DNA was stable in E . coli, S . griseofuscus and S . fradiae . These cosmids could be isolated easily from S . griseofuscus, an improvement over the previous shuttle cosmid vector, pKC462a {Stanzak et al., Bio/Technology 4 (1986) 229-232}, which was somewhat difficult to isolate from S . lividans.

Microbiol Immunol, 1987, 31(11), 1071 - 83
Nucleotide sequence of the pnd gene in plasmid R483 and role of the pnd gene product in plasmolysis; Ono K et al.; The pnd gene of R plasmid R483, like the srnB gene of the F plasmid, increases the degradation of stable RNA in Escherichia coli . The nucleotide sequence of the pnd locus was determined and compared with that of the srnB locus . The genes have open reading frames that are 54% homologous, and both have an upstream inverted repeat sequence . The pnd gene expression seems to decrease the osmotic barrier of the cytoplasmic membrane, since no plasmolytic vacuoles were formed in the cells carrying the gene when the cells were exposed to hypertonic sucrose solution . This result suggests that RNase I in the periplasm passes through the altered membrane to degrade stable RNA in the cytoplasm.

Gene, 1987, 61(1), 63 - 74
High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection; Takeshita S et al.; Three types of alpha-complementation plasmid vectors were constructed which contain a chloramphenicol- or kanamycin-resistance (CmR or KmR) gene and polylinker cloning sites within the coding region of lacZ' . These vectors are essentially based on high- or low-copy-number replicons . The low-copy-number vectors, 3.61 kb in size, confer CmR and contain the pSC101 replicon and pUC8-/pUC9-type polylinker . On the other hand, the high-copy-number vectors, 2.21 to 2.68 kb in size, confer either CmR or KmR, and contain the pBR322 replicon and pUC18-/pUC19-type or other modified polylinkers . All cloning sites except HindIII and SmaI sites in the KmR vectors are unique in each plasmid . Since almost all frequently used plasmid vectors confer ampicillin resistance, these vectors may be useful to simplify the subcloning/DNA joining experiments due to unnecessity of radioisotope labelling, size fractionation and purification of foreign DNA segments.

Gene, 1987, 61(1), 51 - 62
Expression of the alpha and beta tubulin genes of the African trypanosome in Escherichia coli; Wu J et al.; The African trypanosome, Trypanosoma brucei, contains multiple genes for both alpha- and beta-tubulins, which code for similar if not identical proteins . Studies of the structure and function of trypanosome microtubules have been limited due to the difficulties in obtaining sufficient amounts of purified tubulin . To produce large amounts of purified tubulin for studies of structure and function and to begin developing a system for producing systematic alterations of tubulin structure we have cloned and expressed the alpha- and beta-tubulin genes of T . brucei in Escherichia coli to produce the unfused proteins . Controlled high-level expression of both alpha- and beta-tubulin was achieved using a plasmid vector, pOTS, in which expression is controlled by phage lambda promoter/operator and a temperature-sensitive lambda repressor . The tubulins produced are insoluble, as has been found for many other proteins expressed to high levels in E . coli; they are readily purified to near homogeneity by chromatography on DEAE-cellulose in 7 M urea . N-terminal analysis of the purified proteins indicates that they are initiated correctly and that the N-formyl group is removed from the initiating methionine . This factor will probably prove important in the reconstitution of biologically active tubulin.

Gene, 1987, 60(2-3), 303 - 6
pUC12-W1: a plasmid vector for the study of cloned DNA conformation; Wang Y et al.; pUC12-W1 is a new cloning vector for the study of torsion-induced structural transitions of insert DNA . It was derived from pUC12 by deleting three A + T-rich sequences which can undergo structural transitions when torsionally stressed . Transitions at these sites have low energy of activation and undefined structures . They complicate studies on transitions of DNA inserts by diverting torsional force and causing the vector to be undefined in helical and energetic terms . The new vector pUC12-W1, from which these segments have been deleted, will facilitate studies of torsion-induced structural transitions of insert DNA.

Gene, 1987, 60(2-3), 197 - 204
Carbohydrate-binding protein 35: molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli; Jia S et al.; Affinity-purified antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a lambda gt 11 expression library derived from mRNA of 3T3 fibroblasts . This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing beta-galactosidase and CBP35 sequences . Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35 . Such a limited proteolysis followed by affinity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity . This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against beta-galactosidase . These results indicate that we have identified a cDNA clone for CBP35 that yields a recombinant polypeptide with lectin activity produced in Escherichia coli . Using this cDNA clone as a probe, Northern-blot analysis showed an increased expression of the CBP35 gene when quiescent 3T3 cells were activated by the addition of serum growth factors.

Res Exp Med (Berl), 1987, 187(6), 451 - 9
Role of fluid replacement, increased oxygen availability by perfluorochemicals and enhanced RES function in the treatment of mesenteric occlusion shock; Hamar J et al.; Experiments were carried out on 183 rats to study the effect of a complex therapy for treatment of mesenteric shock . The superior mesenteric artery (SMA) was temporarily ligated for 90 min under ether anesthesia; this was followed by an analgesic treatment . After release of the ligated artery, fluid therapy was instituted by administering the equivalent of 7.5% of body weight of one of three different solutions: ringer lactate (RL), hydroxyethyl starch (HES) in RL, and perfluorochemicals (PFC, 4 g/kg b.wt . in RL with HES), the latter with the aim to improve the oxygen transport to the tissue . The same fluid therapy was carried out on rats pretreated with E . coli endotoxin . Endotoxin pretreatment was chosen to compensate the negative effect of PFC on the reticuloendothelial system (RES) as shown in previous studies . Survival time and survival rate were recorded as well as hematocrit values at different times before and after treatment . Experimental groups were: Controls: (1) SMA occlusion without release; (2) 90-min occlusion without therapy . Treated animals: (3) RL therapy; (4) therapy with HES in RL; (5) therapy with PFC in RL and HES; (6), (7), and (8) identical therapies as Groups 3, 4, and 5, respectively, but with endotoxin pretreatment . Survival time increased to the same extent if HES or PFC were added to RL . There was a further increase both in survival times and rates with endotoxin pretreatment (Groups 6, 7, and 8 vs . Groups 3, 4, and 5) . The highest survival time and rate were obtained in Group 8, which received PFC therapy with endotoxin pretreatment . There was a slight negative correlation between survival time and hematocrit values if all groups were considered together.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1987, 60(1), 1 - 11
Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12; Oliver G et al.; We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, that contains the structural genes for both the large and small subunits of the Escherichia coli K-12 glutamate synthase (GOGAT) enzyme, as well as the 5'- and 3'-flanking and intercistronic DNA regions . The Mrs of the two subunits, as deduced from the nucleotide (nt) sequence, were estimated as 166,208 and 52,246 . Partial amino acid sequence of the GOGAT enzyme revealed that the large subunit starts with a cysteine residue that is probably generated by a proteolytic cleavage . Northern blotting experiments revealed a transcript of approximately 7300 nt, that at least contains the cistrons for both subunits . A transcriptional start point and a functional promoter were identified in the 5' DNA flanking region of the large subunit gene . The messenger RNA nontranslated leader region has 120 nt and shares identity with the leader regions of E . coli ribosomal operons, in particular around the so-called boxA sequence implicated in antitermination . Other possible regulatory sequences are described.

Med Cutan Ibero Lat Am, 1987, 15(5), 419 - 24
{Behavior of delayed-type hypersensitivity in parapsoriasis}; Paschoal LH et al.; The authors present a study of behaviour of the late hypersensitivity in fourteen patients carrying parapsoriasis with age varying from 36 to 64 years being nine men and five women with time of the illness development from one to eleven years . There were thirteen white sick people and a yellow one . The parapsoriasis diagnosis was confirmed in all of them clinica and histopathologica . The patients were not getting any therapeutic with corticosteroids or immunosuppressive drugs at the time of study . They were studied immunological through methods in vivo . 1 . Methods in vivo . a) Intradermoreactions with the infectious antigens: PPD, trichophytin, levedurin, E . coli . It was used as a control group of fourty normal fellows . b) Sensibility to DNCB . The results obtained allow the following thesis: 1 . Low table of positivity to the intradermal reactions to the late reading only one of the antigens tested on the patients (E . coli) related to the others . According to these elements we cannot get at a conclusion for depression of the inespecific cellular immunity in our sick patients . 2 . Significant statistics difference on the sensibility test to DNCB on the sick patients related to the controls, a fact which shows a certain degree of the depression of the inespecific cellular immunity . 3 . The sick patients with parapsoriasis before taking some treatment should go through the study of a late hypersensitivity.

Methods Enzymol, 1987, 154, 329 - 50
Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template; Zoller MJ et al.; The important features of the protocol described here are as follows: First, the procedure consists of a few simple steps and results in a reasonably high frequency of mutagenesis . Second, using two primers, there is no need to isolate covalently closed double-stranded molecules as in our previous method . Third, the use of vectors derived from single-stranded phage facilitates template preparation, mutagenesis efficiency, screening, and DNA sequencing . Fourth, the same basic steps can be directly applied when using the single-stranded pUC derivatives.

J Membr Biol, 1987, 100(1), 21 - 9
Mechanism of sugar transport through the sugar-specific LamB channel of Escherichia coli outer membrane; Benz R et al.; Lipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel of Escherichia coli outer membrane . Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior . The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase . Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars . The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel . These carbonyl groups may also be involved in the sugar binding via hydrogen bonds . The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M . Luckey and H . Nikaido (Proc . Natl . Acad . Sci . USA 77:165-171 (1980a)) and the stability constants for the sugar binding given in this study.

Gene, 1987, 57(1), 143 - 8
Localization of the T4 phage ribonucleotide reductase B1 subunit gene and the nucleotide sequence of its upstream and 5' coding regions; Chu FK et al.; The nucleotide (nt) sequence in a 757-bp {corrected} segment downstream from the intron-containing T4 phage thymidylate synthase gene (td) has been determined . This region was found to contain two open reading frames (ORFs) . The first ORF(ORF2) {corrected} 261 bp {corrected} in length, is 24 {corrected} nt downstream from the td gene . The second ORF(ORF3) {corrected}) is 200 bp long at 558 {corrected} nt from the td gene and extends to the end of the Eco RI fragment . The amino acid (aa) sequence (66 aa residues) deduced from the second truncated ORF shows 59% homology to the sequence of the N-terminal portion of the ribonucleotide reductase large subunit of either Escherichia coli (B1 subunit) or mouse (M1 subunit) . This tentatively identifies the truncated gene to be the 5' end of the T4 phage ribonucleotide reductase subunit B1 (nrdA) gene and pinpoints its exact location on the T4 phage genomic map . Southern hybridization analysis suggests good sequence homology among the nrdA genes of various T-even phages.

Circ Shock, 1987, 23(3), 205 - 13
Experimental endotoxemia increases plasma von Willebrand factor antigen concentrations in dogs with and without free-radical scavenger therapy; Novotny MJ et al.; Pentobarbital-anesthetized beagles were infused with physiologic saline (control dogs) or E . coli endotoxin (1.5 mg/kg i.v.) for 1 min . The plasma von Willebrand factor antigen (vWf:Ag) concentration, a potential index of endothelial damage, was monitored before and for 4 h after endotoxin challenge . The plasma vWf:Ag concentration increased only slightly in the control dogs (n = 6); whereas, in dogs infused with endotoxin (n = 20), the vWf:Ag concentration increased progressively to 2.1 times prechallenge values in 4 h . In addition to endotoxin, some of these dogs were treated with free-radical scavengers; allopurinol (n = 3), allopurinol plus superoxide dismutase and catalase (n = 6), or deferoxamine (n = 5) . The free-radical scavengers did not prevent endotoxin-induced increases in vWf:Ag concentration.

Arch Microbiol, 1987, 149(1), 36 - 42
Cloning and sequence of the mdh structural gene of Escherichia coli coding for malate dehydrogenase; Vogel RF et al.; The malate dehydrogenase gene of Escherichia coli, which is susceptible to catabolite and anaerobic repression, has been cloned using plasmic pLC32-38 of Clarke and Carbon (1976) . The nucleotide sequence was determined of a 2.47 kbp fragment, containing the mdh structural gene . All information necessary for expression of the mdh structural gene was mapped within a 1.3 kbp SphI-BstEII fragment . Compared with the untransformed wild type, transformations with pUC19 vector, containing this fragment, gave up to 40-fold more malate dehydrogenase activity in both E . coli wild type and mdh mutant recipients . Catabolite repression was not affected in the transformants . A possible CRP binding site in the promotor region of the mdh gene provides evidence for a co-regulation with fumA gene, the structural gene of fumarase, which is also subject to catabolite repression . The structures for transcription initiation and termination were similar to those previously described for E . coli . Amino acid sequence homologies between pro- and eucaryotic malate dehydrogenases are discussed.

Nucleic Acids Symp Ser, 1987, (18), 69 - 72
Facile preparation of purine and pyrimidine 2-deoxy-beta-D-ribonucleosides by biotransformation on encapsulated cells; Holy A et al.; A preparative-scale synthesis of pyrimidine or purine 2'-deoxy-beta-D-ribonucleosides from a heterocyclic base and 2'-deoxyuridine proceeds by biotransformation reaction in the presence of thymine-dependent E . coli mutant cells encapsulated in alginate gel . The products are isolated by octadecyl-silica or ion-exchange chromatography.

Nucleic Acids Symp Ser, 1987, (18), 131 - 5
Carbocyclic analogues of dTTP and UTP: properties in polymerase enzyme-catalyzed reactions; Sagi J et al.; Racemic carbocyclic analogues of dTTP {(+/-)-C-dTTP} and its ribo counterpart, 5-methyl-UTP {(+/-)-C-m5UTP} were synthesized and examined, in comparison with dTTP and UTP (and m5UTP), as potential substrates of E . coli DNA and RNA polymerases, respectively . Unexpectedly, only a very low (terminal) incorporation of C-dTMP into DNAs of different structure was observed, C-dTTP did not serve as a substrate for chain elongation by the Klenow DNA polymerase . Inhibition of DNA replication was, however, observed in the presence of (+/-)-C-dTTP . The UTP analogue, (+/-)-C-m5UTP proved neither a substrate nor an inhibitor of the RNA polymerase enzyme.

Nucleic Acids Symp Ser, 1987, (18), 121 - 4
Some aspects of DNA polymerase functioning; Krayevsky AA et al.; The group of DNA polymerases was studied using some new nucleoside 5'-triphosphate analogs with termination substrate properties . Among DNA polymerases tested the least specific appeared reverse transcriptases of retroviruses and the most specific were DNA polymerases alpha type from high eucaryotes including mammalians.

Neoplasma, 1987, 34(5), 615 - 25
Postirradiation utility of insulin in radiotherapy; Chaudhari CA et al.; The radiobiological effect of insulin was studied under laboratory conditions to find its utility in radiotherapy . Balb/c mice receiving injections of insulin after irradiation exhibited rapid recovery from radiation effect . This was evident from the data on their life span, organ weights and spleen colony assay studies, carried out under conditions of whole body and partial body irradiation . This trend was absent in mice injected with insulin before irradiation . The results of experiments on E . coli B/r and HA cells irradiated in the presence of insulin under oxic conditions suggest radioprotective effect of insulin . The E . coli B/r cells irradiated in the presence of insulin under hypoxia, however, showed a moderate radiosensitizing effect of insulin.

Gut, 1987, 28 Suppl, 175 - 80
Pathogenesis of the mucosal hyperplasia in self-filling blind loops of rat jejunum: a morphometric study in germ free animals; Menge H et al.; Bacterial overgrowth and high intraluminal concentrations of deconjugated bile acids are thought to be responsible for mucosal hyperplasia in self-filling blind loops of rat jejunum . To investigate this hypothesis further we have assessed the three dimensional architecture of these loops created in germ free animals without or with di- or mono-association of different bacterial species . It was found that mucosal hyperplasia develops in the absence of any bacterial contamination and that bacterial association does not lead to a more pronounced mucosal proliferation . This implies that other mechanisms provoke this morphological phenomenon . Increased bulk contents in these loops or immunological events are probably the most likely explanation.

Gene, 1987, 58(1), 77 - 86
Modification of mRNA secondary structure and alteration of the expression of human interferon alpha 1 in Escherichia coli; Lee N et al.; A plasmid (pNL015) was constructed to contain a human interferon alpha 1 (IFN-alpha 1) gene under the transcriptional control of the Escherichia coli lipoprotein promoter . The E . coli cells harboring this plasmid produce 2.8 x 10(4) units/ml of IFN . Secondary structure analysis of the transcripts produced by pNL015 showed that the coding region could base pair with the Shine-Dalgarno (SD) region with a delta G = -3.9kcal/mol . A new plasmid pNL008 was constructed by modifying pNL015 with an 11-bp deletion and a 2-bp insertion in the coding region, so that the SD region is not involved in the secondary structure . E . coli cells harboring pNL008 produce ten times more IFN activity than cells harboring pNL015 . A series of experiments were carried out to show that the specific activities of IFN, differential rates of IFN transcription, protein degradation or mRNA degradation could not account for the difference observed in expression . A rigorous test on this model of translational inhibition was conducted by the construction of pNL017 with a single bp substitution which did not change the amino acid sequence of the IFN (synonymous codon substitution) but which increased the calculated energy of interaction with the SD sequence to delta G = -10.8 kcal/mol . The E . coli cells harboring pNL017 produced no detectable IFN activity.

Gene, 1987, 57(2-3), 267 - 72
A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli; Hoffman CS et al.; A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described . To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform . Centrifugation of this mixture separates the DNA from cellular debris . E . coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA . This procedure also releases chromosomal DNA . Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.

Circ Shock, 1987, 23(4), 231 - 40
Endotoxin-induced hemodynamic and prostaglandin changes in ponies: effects of flunixin meglumine, dexamethasone, and prednisolone; Templeton CB et al.; Shock was induced in four groups of anesthetized ponies with an intravenous injection of Escherichia coli endotoxin {125 micrograms/kg} . Five minutes after endotoxin injection, the ponies were given no treatment (group A), flunixin meglumine (FM:1.1 mg/kg) (group B), dexamethasone (2 mg/kg) (group C), or prednisolone (10 mg/kg) (group D) . Additionally, FM was given every 3 hours, and the steroids were given at 3, 9, and 24 hours following endotoxin . Hemodynamic measurements were made during the 4-hour anesthetic period . Blood samples were collected for the analysis of prostaglandins, blood chemicals, and enzymes until death . Microspheres labeled with one of four radionuclides were used to determine regional blood flow at 0, 0.1, 1, and 2 hours after endotoxin was given . Plasma levels of both thromboxane and prostaglandin I2 increased from less than 1 ng/ml to between 3 and 5 ng/ml following the injection of endotoxin . The elevated thromboxane corresponded with high pulmonary arterial pressure {between 35 and 55 mm Hg} and low mean systemic arterial pressure (between 40 and 65 mm Hg) during the first 5-10 minutes following endotoxin . Increased concentrations of prostaglandin I2 were temporally related to systemic arterial hypotension, which occurred 1-2 hours following endotoxin in all groups except group B . The rise of prostaglandin I2 and hypotension were not observed in the flunixin meglumine-treated ponies . Dexamethasone was less effective, and prednisolone was ineffective in preventing the synthesis of prostaglandin I2 and the accompanying hemodynamic changes that occurred during the first 2 hours following endotoxin . This is probably due to the fact that steroids require a longer period of time before prostaglandin synthesis is reduced . Although not statistically significant, increased survival trends were observed in ponies treated with flunixin meglumine.

Cancer Metastasis Rev, 1987, 6(3), 261 - 81
Drug resistance and DNA repair; Fox M et al.; DNA repair confers resistance to anticancer drugs which kill cells by reacting with DNA . A review of our current information on the topic will be presented here . Our understanding of the molecular biology of repair of 0(6)-alkylguanine adducts in DNA has advanced as a result of the molecular cloning of the E . coli ada gene but the precise role of this lesion in the cytotoxic effects of alkylating agents in mammalian cells is not completely understood . Less progress has been made in understanding the enzymology and molecular biology of DNA cross-link repair even though such lesions are important for the cytotoxic effects of the widely used bifunctional alkylating agents and platinum compounds . It is evident that drug sensitive or resistant phenotypes are as highly complex as are the effects of DNA damage on cell metabolism and various aspects of these effects are discussed . Few clear correlations have been made between quantitative differences in DNA repair capacity and cellular sensitivity but assays which were developed to measure fidelity and intragenomic heterogeneity in DNA repair are beginning to be applied . Such studies may reveal subtle differences between sensitive and resistant cell lines . The molecular cloning of human DNA repair genes by transfection into drug sensitive rodent cells has been attempted . Some success has been achieved in this area but the functions of the cloned genes have yet to be identified.

J Reprod Fertil Suppl, 1987, 35, 317 - 25
Dynamics of the acute uterine response to infection, endotoxin infusion and physical manipulation of the reproductive tract in the mare; Williamson P et al.; The uterine responses after the infusion of saline (PBS), a bacterial suspension, or lipopolysaccharide derived from Escherichia coli, and after stimulation of the reproductive tract were compared . All infusions provoked a response involving both serum proteins and leucocytes . Protein levels peaked within a few hours of infusion, whereas leucocyte concentration peaked later at around 6 h . Bacterial recovery from the uterus followed a similar pattern, with recovery falling dramatically by 12 h . In mares known to be susceptible to infection large numbers of bacteria were again recovered after 24 h . No differences were apparent between resistant and susceptible mares in protein or leucocyte concentrations . Stimulation of the cervix and uterus resulted in a protein and neutrophil response . In contrast, vaginal stimulation failed to provoke the uterine defences.

IARC Sci Publ, 1987, (84), 41 - 3
Repair of synthetic oligonucleotides containing O6-methylguanine, O6-ethylguanine and O4-methylthymine, by O6-alkylguanine-DNA alkyltransferase; Graves RJ et al.; 32P-Labelled self-complementary oligonucleotides containing O6-methylguanine, O6-ethylguanine, and O4-methylthymine have been synthesized and used as substrates for the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AAT) . The reaction was second-order with rate constants of 2.6 X 10(7) M-1 sec-1 for O6-methylguanine, 2.6 X 10(4) M-1 sec-1 for O6-ethylguanine and 2.5 X 10(3) M-1 sec-1 for O4-methylthymine . These oligomers should allow sensitive and specific assay of the mammalian enzyme.

IARC Sci Publ, 1987, (84), 37 - 40
O-Alkyl deoxythymidines are recognized by DNA polymerase I as deoxythymidine or deoxycytidine; Singer B et al.; The O2- and O4-methyldeoxythymidine triphosphates (O-alkyl dTTP) can be used to substitute for dTTP in Escherichia coli DNA polymerase I (Pol I)-catalysed synthesis of poly{deoxyadenosine-deoxythymidine} (dA-dT) . When incorporated into the polynucleotide, no detectable perturbation of structure occurred with even 20% O-methyldeoxythymidine in place of dT . However, on replication of such polymers with Pol I, significant amounts of deoxyguanosine triphosphate (dGTP) were incorporated, as well as high levels of deoxyadenosine triphosphate (dATP), indicating tautomer-like behaviour . Higher homologues, such as O4-ethyl (e4) dTTP or O4-isopropyl (ip4) dTTP, could also replace dTTP, but with lower efficiency . Nevertheless, their presence, like O4-methyl (m4) dT substitutions, caused transitions as well as inhibiting enzyme digestion with a variety of 3' nucleases, particularly to the 3'----5' exonuclease activity (proofreading) of polymerases . Further proof of mutagenicity comes from site-directed experiments placing m4dT or e4dT in place of dT at position 587 in am3 of phi X174, in which all revertants sequenced had A----G transitions . This implies that, since m4dT and e4dT are poorly repaired in eukaryotes, it is likely that they will remain in the DNA and lead to effects on enzyme activity, as well as mutations which contribute to the carcinogenicity of N-nitroso compounds.

Gene, 1987, 56(2-3), 309 - 12
New and versatile cloning vectors with kanamycin-resistance marker; Pridmore RD; Described here is a pair of small multi-copy kanamycin-resistance plasmids, containing the pUC lacZ alpha-complementation peptide and the pUC18 and pUC19 multiple cloning site . These plasmids and their derivatives allow simple and rapid transfer of inserts from one replicon to another without the necessity of purifying the insert from vector.

Gene, 1987, 56(2-3), 185 - 98
The complete nucleotide sequence of the ilvGMEDA cluster of Escherichia coli K-12; Cox JL et al.; The ilvGMEDA gene cluster of Escherichia coli K-12 has been the focus of intensive genetic and biochemical analysis for the past 30 years . Genetic regulation of the ilvGMEDA cluster involves attenuation, internal promoters, internal Rho-dependent termination sites, a site of polarity in the ilvG pseudogene of the wild-type organism, and autoregulation by the ilvA gene product, the biosynthetic L-threonine deaminase . We have now completed the nucleotide sequence of the 6600-bp cluster and have analyzed it, along with the ilvYC, ilvBN, and ilvIH genes, for codon frequencies and possible evolutionary relationships . The isoleucine content of each of the gene products of the ilvGMEDA cluster is quite similar (less than a two-fold variation), thus excluding one possible interpretation of the isoleucine-specific downstream amplification phenomenon . There is no evidence for retrograde evolution in the cluster since no significant homologies are detectable among genes that catalyze sequential reactions of the pathway . A highly significant homology does exist, however, between the threonine deaminases of yeast mitochondria and E . coli . The sequence at the boundary of the ilvA and ilvD genes is TAATAATG, so that the second TAA stop codon of ilvD overlaps the ATG initiation codon of ilvA.

Gene, 1987, 56(1), 153 - 60
Reovirus major capsid protein expressed in Escherichia coli; Giantini M et al.; A DNA copy of the open reading frame of the S4 gene of reovirus type 3 was cloned into a temperature-regulated bacterial expression vector . Induction at 42 degrees C resulted in the synthesis of a polypeptide that comigrated with virion capsid protein sigma 3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reacted with sigma 3-specific antisera . The protein was expressed in bacteria as insoluble aggregates that accumulated in polar inclusion bodies . Aggregated product also resulted when the expression system was manipulated to induce bacterial sigma 3 (b sigma 3) synthesis at temperatures below 42 degrees C . Various methods used to solubilize b sigma 3 did not yield the monomeric protein . The results indicate that sigma 3, the major surface component of reovirions, is expressed in transfected Escherichia coli as an aggregated, disulfide cross-linked protein.

Gene, 1987, 56(1), 125 - 35
Vectors for selective expression of cloned DNAs by T7 RNA polymerase; Rosenberg AH et al.; Plasmid vectors are described that allow cloning of target DNAs at sites where they will be minimally transcribed by Escherichia coli RNA polymerase but selectively and actively transcribed by T7 RNA polymerase, in vitro or in E . coli cells . Transcription is controlled by the strong phi 10 promoter for T7 RNA polymerase, and in some cases by the T phi transcription terminator . The RNA produced can have as few as two foreign nucleotides ahead of the target sequence or can be cut by RNase III at the end of the target sequence . Target mRNAs can be translated from their own start signals or can be placed under control of start signals for the major capsid protein of T7, with the target coding sequence fused at the start codon or after the 2nd, 11th or 260th codon for the T7 protein . The controlling elements are contained on small DNA fragments that can easily be removed and used to create new expression vectors.

Environ Mol Mutagen, 1987, 10(3), 311 - 5
Mutagenicity testing of ethylene oxide in Escherichia coli strains with different repair capacities; Kolman A et al.; Mutagenicity of ethylene oxide in Escherichia coli B strains with different repair capacities was studied . Deficiencies in excision repair (uvrA, polA) led to a considerable increase in mutation frequency compared with that in the wild-type strain and in strains deficient in error-prone repair (recA, lexA).

Circ Shock, 1987, 23(2), 93 - 106
Volume expansion, dobutamine and noradrenaline for treatment of right ventricular dysfunction in porcine septic shock: a combined invasive and radionuclide study; Schneider AJ et al.; The purposes of the present study were 1) to explore if volume loading in combination with dobutamine or noradrenaline would be superior to volume loading alone in the treatment of right ventricular (RV) dysfunction in septic shock complicated by acute pulmonary hypertension and 2) to determine whether noradrenaline would be superior to dobutamine because of its vasoconstrictor effects on the peripheral circulation, resulting in increased RV coronary perfusion pressure . Experiments were performed on 21 anesthetized, ventilated pigs . Gated blood pool studies and hemodynamic measurements were performed simultaneously . All animals were given 3-4 X 10(8)/kg live E . coli bacteria, resulting in an abrupt increase in pulmonary arterial pressure and a decrease in arterial pressure, cardiac output, and RV ejection fraction . Right ventricular end-systolic volume was unchanged; RV end-diastolic volume and RV coronary perfusion pressure fell . After randomization, the control group (I, n = 5) was subjected to volume loading, and treatment groups (each n = 8) received volume loading in combination with dobutamine (group II, 5-10 micrograms/kg/min) or noradrenaline (group III, 0.25-0.50 micrograms/kg/min) . In contrast to volume loading alone, dobutamine and noradrenaline increased cardiac output and RV ejection fraction, but only noradrenaline restored mean arterial pressure . Noradrenaline improved RV contractility, as judged from the RV end-systolic pressure-volume relationship, probably because it increased the RV perfusion . Thus, noradrenaline in combination with volume loading may be the treatment of choice to improve RV performance in porcine septic shock associated with pulmonary hypertension.

Circ Shock, 1987, 23(2), 131 - 41
Glucose kinetics and pyruvate dehydrogenase activity in septic rats treated with dichloroacetate; Lang CH et al.; Decreased pyruvate dehydrogenase (PDH) activity in skeletal muscle has been observed during sepsis and may contribute to the altered glucose kinetics seen in this condition . The purpose of the present study was to determine if dichloroacetate (DCA), a known stimulator of PDH activity, could reverse the sepsis-induced increase in glucose metabolism . Hypermetabolic sepsis was produced in chronically catheterized rats by repeated subcutaneous injections of live Escherichia coli . Whole body glucose kinetics, assessed by a constant iv infusion of {6-3H and U-14C}-glucose, were determined in fasted septic and nonseptic rats before and for 4 hr after an injection of DCA (30 mg/100 g BW, iv) . Sepsis produced hyperthermia (+1.6 degrees C) and increased the rates of glucose appearance (Ra; 95%), recycling (318%), metabolic clearance (MCR; 114%), and elevated plasma lactate levels (295%) compared to nonseptic controls . After injection of DCA into septic rats, glucose levels gradually fell, and the sepsis-induced hyperlactacidemia was completely reversed . Treatment of septic rats with DCA reversed the elevated glucose Ra; recycling, although reduced, was still elevated by 50% compared to control animals . DCA did not alter the hyperglucagonemia seen in septic animals, but it did reduce the plasma insulin levels by 60% . Hepatic and muscle PDH activities were not different in saline-treated septic and nonseptic animals . DCA elevated PDH activity in muscle from septic rats, but the increase was smaller than that seen in control animals . This may explain the smaller decline in glucose recycling and plasma lactate in septic animals . These results are consistent with DCA reducing the elevated glucose Ra in sepsis by partial activation of PDH, which reduces the elevated precursor (lactate) supply for gluconeogenesis . However, alterations in PDH activity did not appear to contribute to the underlying increase in glucose Ra and recycling observed in sepsis.

Biosensors, 1987-88, 3(1), 27 - 43
L-lactate enzyme electrode obtained with immobilized respiratory chain from Escherichia coli and oxygen probe for specific determination of L-lactate in yogurt, wine and blood; Adamowicz E et al.; An enzyme electrode for L-lactate measurements in various biological media was prepared with an immobilized bacterial respiratory chain fixed to a Clark electrode . The enzymatic film, which was easy to prepare, contained bacteria immobilized in gelatin, tanned with glutaraldehyde . This electrode was sensitive to 0.1 mM L-lactate and could be utilized to 10 mM . The apparent K50 was 5 mM . Less than 8% of the respiration rate with L-lactate was measured with D-lactate and succinate . The competitive inhibitors D-lactate and pyruvate had a K50 of 50 mM . They could be quantitatively measured by inhibition in a range between 5 and 50 mM . It was also possible to discriminate between L-lactate and various metabolites of the respiratory chain: L-malate, succinate, 3-glycero-phosphate or NAD(P)H . Growing E . coli on 1% D-L-lactate as the sole carbon source in minimal medium induced L-lactate respiration tenfold . All other respiratory activities remained below 10% of the activity with L-lactate . A computerized instrument allowed successive measurements every 3 min for more than 10 h with the same enzymatic film . Most of the measured samples required dilution but no clarification or purification . This enzyme electrode may have many applications in basic research (metabolism, enzymology) and applied research (blood, yogurt, juices, wine).

Acta Biochim Pol, 1987, 34(2), 183 - 93
Are Escherichia coli dam- as compared to dam+ hypermutable by base analogs?
Janion C, Bebenek K, Plewako S.
It was shown that some base analogs, like n2Pur and n2oh6Ade (but not n2om6Ade or oh4Cyd) which strongly inhibit growth of dam- cells, mutagenize and preselect dam- populations . As a result dam- mut(-)--devoid of mismatch repair, dam+ revertants, or dam(-)--insensitive to a base analog are exclusively obtained after mutagenesis . The composition of these mutants depends on the base analog applied . By using reconstruction experiments, the frequency of these mutations induced by n2Pur was calculated.

Gene, 1987, 55(2-3), 295 - 301
Expression of protein IIIa of human adenovirus type 2 in Escherichia coli; Cuillel M et al.; We have constructed a plasmid encoding the protein IIIa gene of human adenovirus type 2 . The gene was expressed under the control of the hybrid tac (trp-lac) promoter; the protein was synthesized at levels up to 5% of newly synthesized protein after IPTG induction . The protein IIIa produced in Escherichia coli has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 67 kDa, and was revealed with anti-adenovirus serum in Western blotting . The protein IIIa produced in bacteria was phosphorylated in the presence of {gamma-32P}ATP.

Exp Lung Res, 1987, 13(2), 97 - 112
Comparative humoral responses to Escherichia coli and sheep red blood cell antigens introduced via the respiratory tract; Scheuchenzuber WJ et al.; The numbers of specific antibody-forming cells (AFC) in mediastinal lymph nodes (MLN) and spleen were determined in Balb/cByJ mice following inhalation or intratracheal (IT) injection of acetone-dried E . coli or its lipopolysaccharide (LPS) . These responses were compared with those obtained using sheep red blood cells (SRBC), an antigen previously used in studies of pulmonary immunity . Inhalation of aerosolized E . coli for as little as 2 min produced significant numbers of AFC in both MLN and spleen, while equivalent administrations of SRBC produced few AFC at either site . Similarly, IT instillations of E . coli resulted in recovery of AFC from MLN and spleen, while IT SRBC produced AFC in MLN but few splenic AFC . IT installations of radiolabeled SRBC and E . coli were used to examine antigen dissemination, and no differences were found in the amounts of radiolabel recovered from various tissues following instillation of either antigen . Experiments using endotoxin resistant mice, and using administrations of LPS in combination with SRBC, were unable to demonstrate alterations in AFC production due to effects of LPS . It was found, however, that inhaled or IT injected E . coli or LPS produced greater pulmonary inflammation than did similar administrations of SRBC, and this may be at least partly responsible for the enhanced induction of systemic immunization.

Chem Biol Interact, 1987, 63(2), 185 - 94
DNA damage by 5-nitro-2-furylacrylic acid, a nitrofuran derivative; Chatterjee SN et al.; 5-Nitro-2-furylacrylic acid (5-NFA) caused dose dependent inhibition of growth of Escherichia coli K-12 strain AB 2480 (uvr-, rec-), the 37% (D37) and 10% (D10) survival doses being 1.0 microgram/ml.h and 1.75 micrograms/ml.h, respectively . Although much higher doses of drug were required to achieve comparable inhibition of growth of E . coli strain 1157 (repair proficient), significant filamentation of these cells was produced by treatment with 1.0 microgram/ml 5-NFA for 4 hr . Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that 5-NFA treatment of E . coli strain AB 2480 produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the initial part of the reaction obeyed a first order relation . 5-NFA also produced dose-dependent increase of prophage induction in E . coli strain GY 5027: envA, uvrB, ampA1, strA (lambda) . The implications of the action of 5-NFA on DNA in relation to the induction of 'SOS' functions and carcinogenesis were discussed.

Arkh Patol, 1987, 49(6), 54 - 8
{Septicemia against a background of immunodeficiency}; Avtsyn AP et al.; Macroscopic diagnosis of septicemia at autopsy is extremely difficult to make in cases where relevant diagnostic cues, notably changes in the spleen characteristic for sepsis are lacking . Histologic, bacterioscopic, immunomorphologic, fluorescence electron-microscopic studies of autopsy material are described which, in combination, enabled the authors to establish pathologic diagnosis of septicemia, identify its etiology, and characterize the immune status.

Nahrung, 1987, 31(5-6), 465 - 7
Treatment of gastrointestinal infections in infants by oral administration of colostral antibodies; Lodinova-Zadnikova R et al.; Passive immunization used for treatment of gastrointestinal infections represents a safe and effective method in premature and full-term newborns, avoiding the use of oral antibiotics.

Med Microbiol Immunol (Berl), 1987, 176(5), 241 - 4
Enterotoxigenic Escherichia coli O153:H45 from an outbreak of diarrhoea in Spain; Escribano A et al.; Enterotoxigenic Escherichia coli (ETEC) strains belonging to a characteristic serobiotype, O153:H45 ST+, were isolated from an outbreak of neonatal diarrhoea in Valencia, Spain . The restriction of the geographical location of this clone to South America and Spain is discussed.

J Cell Sci Suppl, 1987, 6, 303 - 21
Plasmid genes affecting DNA repair and mutation; Strike P et al.; Many bacterial plasmids have the effect of increasing the ultraviolet (u.v.) resistance of host cells that harbour them, apparently by an error-prone repair mechanism that leads to a high level of mutation amongst the survivors . These plasmid systems are apparently analogues of the Escherichia coli umuD/C operon, which is absolutely required in this organism for mutation induced by u.v . light and by many chemical mutagens . This article reviews the extensive and sometimes conflicting literature relating to this phenomenon, and describes the further characterization of one such plasmid system, the imp (I group mutation and protection) operon of the I1 group plasmid TP110 . It is demonstrated that each of the protection mutation systems well characterized to date shows a similar genetic arrangement, and that significant homology can be detected at the amino acid level between the proteins encoded by these different systems.

J Cell Sci Suppl, 1987, 6, 289 - 301
Toxicity, mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide; Linn S et al.; Two modes of killing of Escherichia coli by hydrogen peroxide can be distinguished . Mode-one killing is maximal at 1-2 mM; at higher concentrations the killing rate is approximately half-maximal and is independent of H2O2 concentration but first order with respect to exposure time . Mutagenesis and induction of a phage lambda lysogen are similarly affected by H2O2 concentration, with reduced levels of response above 1-2 mM-H2O2 . Mutagenesis is not affected by inactivation of umuC . Mode-one killing requires active metabolism during the H2O2 challenge and it results in sfiA-independent filamentation of both cells that survive and those that are killed by the challenge . This mode of killing is enhanced in xth, polA, recA and recB strains; however, it is unaffected by mutations in the nth, uvrA, uvrB, uvrC, uvrD, rep, gyrA, htpR and rel loci . Mode-one killing is normal in strains totally lacking catalase activity (katE, katG), glutathione reductase (gor) or glutathione synthetase (gshB), but enhanced in a strain lacking NADH dehydrogenase (ndh) . Mode-one killing is accelerated by the presence of CN- or by an unidentified function that is induced by anoxic growth and is under the control of the fnr locus . A strain carrying both xth and recA mutations and certain polA mutants appear to undergo spontaneous mode-one killing only under aerobic conditions . Taken together, these observations imply that mode-one killing results from DNA damage that normally occurs at a low, non-lethal level during aerobic growth . Models for the resistance to mode-one killing at dose above 1-2 mM-H2O2 will be discussed . Mode-two killing occurs at high concentrations of H2O2 and longer times . It does not require active metabolism, and cells that are killed do not filament, although survivors demonstrate a dose-dependent growth lag followed by a period of filamentation . Mode-two killing is accompanied by enhanced mutagenesis, but strains with DNA repair defects were not observed to be especially sensitive to this mode of killing.

Gene, 1987, 54(2-3), 255 - 9
A simple procedure for large-scale purification of plasmid DNA; Gomez-Marquez J et al.; We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures . It is a modification of the boiling method of Holmes and Quigley {Anal . Biochem . 114 (1981) 193-197} and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA . This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.

Gene, 1987, 54(2-3), 185 - 95
Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12; Claverie-Martin F et al.; The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space . The gene is physically located between the recC and recB loci on the E . coli chromosome . The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined . S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon . The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA . Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters . A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence . The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.

Eur Urol, 1987, 13(4), 219 - 23
Microvascular changes in the early stage of reflux pyelonephritis . An experimental study in the pig kidney; Androulakakis PA et al.; Renal microvascular changes were studied in the pig kidney during the initial stage of reflux pyelonephritis . In 10 piglets vesicoureteric reflux was surgically induced . The animals were then infected with a strain of Escherichia coli and sacrificed 2 weeks following the initiation of infection . Following injection of the kidneys with Microfil, microvascular changes were investigated by a combined stereomicroscopic, microradiographic and histological technique . Renal microcirculation was found to be impaired in the areas involved in the acute inflammatory process, owing to the compression of glomeruli, small peritubular capillaries and vasa rectae by the ensuing interstitial oedema . These findings indicate that acute focal ischaemia caused by microvascular obstruction could be an important factor in the mechanism of renal damage in reflux pyelonephritis.

Vet Med Nauki, 1987, 24(4), 3 - 6
{Lyophilization of live CA-80 vaccine against coli enteritis in pigs}; Ninov NV et al.; A live vaccine against coli enteritis in pigs, CA-80, with a protection medium of 5 per cent hemodex, was successfully freeze-dried in ampoules or vials at the rate of 5 to 10 vaccinal doses each, the titer ranging from 10(9) to 10(10) live bacteria per cu . cm, of a 12-month shelf-life period . The vaccine was found to be innocuous for pigs, and was intended for active immunoprophylaxis on both infected and menaced farms and complexes, applying it to sows in the last third of the gestation period.

J Basic Microbiol, 1987, 27(3), 131 - 7
Regulation of L-carnitine metabolism in Escherichia coli; Jung K et al.; The metabolization of L-carnitine was studied using whole cells of Escherichia coli 044 K74 . It showed features of an epigenetical control . L-carnitine and crotonobetaine were able to induce the carnitine-reducing system . Oxygen and nitrate as electron acceptors, gamma-butyrobetaine as final product of carnitine transformation in E . coli as well as glucose repress the carnitine metabolization . Other betaines and structurally related compounds did not show any effect neither as inductors nor as repressors.

Folia Microbiol (Praha), 1987, 32(3), 194 - 9
Inhibition of mutation induction and unchanged mutational specificity in Escherichia coli K12 overproducing the RecA protein; Koukalova B et al.; Induced mutagenesis was studied in Escherichia coli K12 cells in relation to the level of RecA-protein (P-RecA) . In experiments strains AB2497, AB2497(pBR322) and AB2497(pX02) were used . The multicopy plasmid pX02 is a recombinant of pBR322 and recA+ gene of E . coli K12 . Cells carrying this plasmid overproduce the P-RecA constitutively . Mutagenesis was induced by the decay of incorporated 6-3H-thymidine . Mutations of the argE3 (ochre) to Arg+ prototrophy were followed . Besides the frequency of mutations, mutagenic specificity was determined . In cells AB2497(pX02) which overproduce the P-RecA the yield of Arg+ revertants was markedly reduced compared with that in strains AB2497 or AB2497(pBR322), whereas the mutagenic specificity was not changed . In all the strains studied the predominant type of mutation produced was the base substitution in the A:T base pair.

Gene, 1987, 55(1), 67 - 74
Positive selection vectors based on xylose utilization suppression; Stevis PE et al.; High levels of xylose isomerase activity in wild-type Escherichia coli strains results in a Xyl- phenotype . This phenomenon was exploited for the development of a versatile positive selection system . The xylA promoter was deleted with the exonuclease BAL 31 and the resulting structural gene was inserted into the SmaI site of pUC9, yielding the prototype vector, pLX100 . In this construct xylA expression is placed under the transcriptional control of the lac promoter . Transformation of any wild-type E . coli strain with pLX100 results in high levels of xylose isomerase and a Xyl- phenotype . Decreasing the activity below a critical level (approx . 100 u) restores the Xyl+ phenotype . pLX100 contains contiguous restriction sites for HindIII, PstI, BamHI and XhoI, suitable for positive selection cloning experiments . E . coli transformants containing pLX100 cannot grow in minimal medium with xylose unless a DNA fragment is inserted into any one of the unique restriction sites . This makes the plasmid an ideal positive-selection cloning vector.

Dev Comp Immunol, 1987 Spring, 11(2), 385 - 94
Influence of cell sources, stimulating agents, and incubation conditions on release of interleukin-1 from chicken macrophages; Klasing KC et al.; Experiments were conducted to determine the cell source, stimulating agents, and incubation conditions that maximize interleukin-1 (IL-1) release by chicken macrophages/monocytes . Thymocyte co-mitogen proliferation was used to assay IL-1 activity of conditioned or partially purified supernatants . Monolayers of a transformed chicken macrophage cell line, HD11, released greater amounts of IL-1 than adherent cells isolated from peripheral blood, peritoneal cavity, or spleen . E . coli endotoxin and heat-killed S . aureus induced greater release of IL-1 by HD11 and splenic macrophages than latex or a super induction protocol with mezerien . Blocking macrophage eicosanoid synthesis with indomethacin did not influence IL-1 release from HD11 macrophages . Removing low molecular weight compounds from conditioned supernatants by dialysis did not influence IL-1 activity . IL-1 release was increased by incubating macrophages at 42 C compared to 39 C . Thymocyte co-mitogenic activity of IL-1 was increased by incubating thymocytes at 42 C compared to 39 C . Species cross reactivity between chicken and mammalian IL-1 was also investigated . Chicken IL-1 had slight co-stimulation activity on murine thymocytes, but murine and human IL-1 were without activity on chicken thymocytes.

Circ Shock, 1987, 22(3), 231 - 40
Influence of indomethacin on the endotoxin-induced cardiodepressant effect of serum and steroid hormone changes in male rats; Nunez EA et al.; Escherichia coli endotoxin has been shown to induce a cardiodepressant effect (CDE) and changes in steroid hormone concentrations in the serum of male rats, especially increases in estrogens and a decrease in testosterone . Pretreatment of rats with indomethacin (INDO) abolished these responses to endotoxin . Direct addition of INDO to primary cultures of rat heart cells blocked the cardiodepressant response of these cells to endotoxin-treated rat serum . These data, together with previous results, suggest that relationships between estrogens and CDE are possible, while these two parameters have different time courses and dose dependencies; in any case, the prostanoid system is likely involved since INDO is able to suppress both of them.

AIDS Res Hum Retroviruses, 1987 Spring, 3(1), 95 - 105
Analysis of human serum antibodies to human immunodeficiency virus (HIV) using recombinant ENV and GAG antigens; Kenealy W et al.; Recombinant proteins representing gag and env amino acid sequences of the Human Immunodeficiency Virus (HIV) (HTLV-IIIb) were produced in Escherichia coli and used to analyze sera for the presence of antibodies to HIV . ENV-9 is a protein representing the carboxy terminus of gp120 and part of gp41 which is highly immunoreactive . GAG-1 represents 83% and GAG-55 100% of the amino acids of the gag open reading frame . The purified proteins allow sensitive detection by enzyme linked immunosorbent assay (ELISA) of antibodies directed against either env or gag of HIV . We have determined the reactivity of sera from several HIV exposed individuals, either form high risk populations or with clinically defined conditions, in the ENV-9, GAG-55, and GAG-1 assays and found that two major seropositive groups are observed . The quantitative analysis of sera with env and gag antigens by ELISA showed AIDS patients had very low gag reactivity while retaining high env reactivity . Results obtained with authentic p24 viral protein in both ELISA and radioimmunoassay correlated to those from the GAG-55 ELISA . This correlation and the analysis of sera with both the ENV and GAG ELISAs indicate that the antibodies reactive to gag are specifically affected relative to env reactivity and that different levels of antibodies to separate viral components in these sera may correlate with disease state.

Vet Med Nauki, 1987, 24(1), 15 - 8
{Adsorption of antigens K88 and K99 on aluminum hydroxide gel}; Kapurdova M et al.; Studied was the effect of the pH value of the medium, of the concentration of the antigen and the adsorbent on the adsorption of K 88 and K 99 on aluminium hydroxide-gel . Use was made of strains O 45:K 88 and O 14:K 99 . The optimal parameters of pH and the concentration of aluminium oxide as well as of aluminium hydroxide-gel as an adjuvant, substantiating the maximum adsorption capacity of the latter with regard to the two Escherichia coli antigens used . Established was the maximum amount of the antigens which could be adsorbed by as much as 1 cm3 of aluminium hydroxide-gel . According to the authors such amount could serve to indicate the way how to produce effective preparations to be deposited with the two-antigen adjuvant.

Prog Clin Biol Res, 1987, 236A, 115 - 9
Granulocyte elastase and white cell counts in septic pigs; Siebeck M et al.; Our data are consistent with the assumption that in early septicemia polymorphonuclear granulocytes are sequestered and release lysosomal elastase locally into the respiratory tract and into the circulation.

Gene, 1987, 54(1), 57 - 64
Static bend of DNA helix at the activator recognition site of the ompF promoter in Escherichia coli; Mizuno T; Expression of the ompF gene coding for a major outer membrane protein of Escherichia coli is regulated by osmolarity of the medium . The ompF expression is controlled by a transcriptional activation mechanism which requires the ompR gene product acting on a region located upstream from the canonical -35 and -10 regions of the ompF gene . Evidence is presented that the upstream region of the ompF promoter contains a static bend of DNA having two sets of oligo (dA X dT) tracts with periodical spacing . It was demonstrated that the bending region overlaps with the recognition site for the activator protein, OmpR.

Gene, 1987, 53(2-3), 287 - 92
Sequence determinants in the lamB gene of Escherichia coli influencing the binding and pore selectivity of maltoporin; Heine HG et al.; Maltoporin (LamB protein) is a malto-oligosaccharide-selective pore protein in the outer membrane of Escherichia coli . The genetic basis of binding and transport specificity was investigated through cloning, mapping and sequencing lamB genes from seven independent mutants with various changes in maltodextrin binding affinities; these mutants were unchanged in binding phage lambda . Single amino acid substitutions specifically resulting in maltodextrin affinity changes were as follows: Arg8----His in two independent mutants resulted in much reduced affinity for all ligands and a smaller pore no longer selective for maltodextrins . A Trp74----Arg substitution resulted in a lower affinity for starch, a slight increase in maltose affinity but no striking pore changes . An Arg82----Ser resulted in lowered maltodextrin affinity, but increased affinity for sucrose in both binding and pore function . A Tyr118----Phe resulted in a higher affinity for both starch and maltose, a slightly larger pore and increased transport of maltohexaose by the pores . Asp121----Gly in two independent isolates resulted in a higher affinity for large dextrins and a marginally larger pore . These results suggest that the maltodextrin-selective functions reside in the N-terminal sequence of maltoporin and are separate from the phage lambda binding domains.

Gene, 1987, 53(2-3), 275 - 81
Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promoters within the insert: effect on DNA yield and cloned insert frequency; Gibson TJ et al.; Transcription terminators have been included in a phage-lambda-replicon-based cosmid vector, Lorist2, to insulate vector genes against transcriptional interference from cloned insert DNA . DNA yields of recombinant clones containing Escherichia coli genomic DNA inserts are more even for Lorist2 than with its progenitor LoristB . However, the terminators provide only a partial reduction in the over-representation of r X DNA-containing clones generally observed in cosmid libraries of Caenorhabditis elegans DNA, suggesting that causes other than transcriptional readthrough into the vector contribute to this problem.

Gene, 1987, 53(2-3), 227 - 34
Cloning of the 3'-phosphoadenylyl sulfate reductase and sulfite reductase genes from Escherichia coli K-12; Li C et al.; The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation . pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants . Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5) . The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter . The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene . Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant . The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E . coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase . Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively.

Gene, 1987, 53(2-3), 219 - 26
Expression vector promoting the synthesis and export of the human growth-hormone-releasing factor in Escherichia coli; Anba J et al.; We have studied the synthesis, processing and export of human growth-hormone-releasing factor (hGRF) in Escherichia coli transformed with a plasmid constructed for the expression of hGRF as a hybrid protein . A DNA fragment containing the entire sequence of phosphate-binding protein gene (phoS) is fused to a modified hGRF-coding sequence (phoS-mhGRF) . The hybrid protein, PhoS-mhGRF, was recovered in the supernatant fluid after spheroplasting treatment indicating correct export to the periplasmic space . Pulse-chase experiments demonstrated that the hybrid protein was similarly processed as the PhoS precursor.

Gene, 1987, 52(2-3), 267 - 77
Sequence, structure and promoter characterization of the human thymidine kinase gene; Flemington E et al.; The 12.9-kb human thymidine kinase gene (tk) has been sequenced in its entirety along with flanking regions . Consistent with the previously sequenced chicken tk sequence, the human tk is composed of seven exons . The intron sizes differ substantially, and are responsible for the four-fold greater size of the human relative to the chicken gene . Within the introns are found 13 Alu family repeated sequences and a polypyrimidine stretch . A functional promoter region has been located by fusing sequences from the 5' end of the tk gene to the chloramphenicl acetyl transferase (CAT) gene and assaying CAT activity following transfection into mouse L cells . Several putative transcription signals have been identified in the 5' end including 'TATAA' and 'CCAAT' sequences and 'G-C' elements, two of which are arranged in a 27-bp inverted repeat . There is also a 12-bp repeat, containing an inverted 'CCAAT' element . This repeat shows strong homology to a repeat in the chicken tk promoter as well as the 5' regions of other cell-cycle regulated genes, suggesting that it may be part of the promoter or a regulatory signal . The 5' flanking sequence is G + C-rich and has a high concentration of CpG dinucleotides.

Dev Biol Stand, 1987, 67, 51 - 7
Protein C, isolation and potential use in prevention of thrombosis; Esmon CT et al.; Protein C and protein S serve as natural anticoagulants . Deficiencies of these proteins are often associated with recurrent deep vein thrombosis and coumarin induced skin necrosis . These two proteins function by selectively inactivating factors Va and VIIIa, two of the "cofactors" of blood coagulation . Hence, inhibition of coagulation by this pathway complements the better known inhibition mediated by the antithrombin III-heparin system . These observations suggest that protein C and/or activated protein C may prove useful in controlling thrombosis and/or DIC . We have developed a Ca2+ dependent monoclonal antibody which allows the rapid isolation of human protein C . This rapid isolation has allowed us to demonstrate that activated protein C can protect baboons from the lethal effects of E . coli/endotoxin and that protein C supplementation can minimize fibrinogen consumption following tissue factor infusion into dogs.

Dev Biol Stand, 1987, 67, 177 - 83
Isolation and characterization of genes for blood proteins; Tu GF et al.; The expression vector lambda gt11Amp3 has been used to construct a cDNA library from rat liver polyadenylated RNA . Clones expressing antigenic determinants for rat albumin, transferrin, transthyretin, apolipoprotein E and apolipoprotein AII have been identified . Albumin clones containing cDNA inserts ranging from 0.9 kb to 1.9 kb were further identified by restriction mapping and nucleic acid sequencing . The largest insert contained the entire coding sequence for albumin . Characterization of the expressed proteins by acrylamide gel electrophoresis followed by immunological detection indicated that the proteins were produced as hybrids linked to the bacterial beta-galactosidase . A cDNA library for human liver polyadenylated RNA has also been constructed . Clones expressing antigenic determinants for human serum albumin, transferrin and apolipoproteins AI, AII, AIV and E have been isolated and their identity established by nucleotide sequencing and restriction mapping . Both rat and human serum protein cDNA clones are currently being used to study the tissue specific expression of serum proteins and for the isolation and characterization of the corresponding genes.

Drug Nutr Interact, 1987, 5(2), 103 - 11
The effect of copper deficiency on the immune response in mice; Blakley BR et al.; Weanling female Swiss mice were fed copper-deficient or copper-replete diets for 28 days . Mice fed the copper-deficient diet exhibited typical signs copper deficiency, which included reduced weight gains, anemia, and low liver copper concentrations . The effect of copper deficiency on antibody production, in particular, T-lymphocyte dependent and independent antibody responses, lymphocyte blastogenesis, and sensitivity to endotoxin were evaluated . Antibody production against sheep red blood cells, a T-lymphocyte dependent response, was suppressed in copper-deficient mice (P less than .0001) . In contrast, antibody production against dinitrophenyl-ficoll, a T-lymphocyte independent response was not altered by copper deficiency (P = 0.90) . Lymphocyte blastogenesis studies demonstrated that copper deficiency did not alter T-lymphocyte blastogenesis induced by concanavalin A (P = 0.27) or B-lymphocyte blastogenesis induced by Escherichia coli lipopolysaccharide (P = 0.40) . These results indicate that the immunosuppressive effects are not due to an impairment of lymphocyte blastogenesis, an intermediate step involved in the generation of an immune response, but rather are a manifestation of impaired T-lymphocyte function associated with antibody production . Increased susceptibility to endotoxin, involving nonspecific defense mechanisms, was also observed in copper-deficient mice . Mortality associated with the endotoxin was 68% in the copper-deficient mice as compared to 35% in the copper-replete mice (P = 0.0026) . Impaired T-lymphocyte dependent antibody production and enhanced susceptibility to endotoxin were observed in copper-deficient mice exhibiting classical manifestations of copper deficiency.

Circ Shock, 1987, 22(1), 65 - 72
Changes in central hemodynamics during experimental septic shock in conscious rats; Palsson J et al.; This study describes the hemodynamic, metabolic, and respiratory effects of a 4-h continuous intravenous infusion of live Escherichia coli bacteria (10(9)/h) in conscious, unrestrained rats . The early response to bacterial infusion was moderate hypotension and a marked and sustained increase in heart rate and respiratory rate . During later stages of bacteria infusion a marked decrease in stroke volume and cardiac output was observed, while total peripheral resistance increased . Arterial blood gas measurements showed an early primary respiratory alkalosis, while later stages of bacteria infusion were accompanied by progressive development of metabolic acidosis . This small-sized animal shock model may be useful for further studies, particularly since conscious rats were used to avoid the influence of anesthesia upon the development of septic shock.

Bull World Health Organ, 1987, 65(2), 207 - 15
Potential sources of enterotoxigenic Escherichia coli in homes of children with diarrhoea in Thailand; Echeverria P et al.; PIP: A year-long study was performed to identify potential sources of enterotoxigenic Escherichia coli (ETEC) within the homes of children with diarrhea in Bangkok . ETEC was identified in 8% (10 of 130) of the inhabitants of 42 homes with children with ETEC diarrhea and 6% (8 of 137) of their neighbors, but in only 2% (49 of 3077) of those individuals living in 866 homes not associated with children with ETEC diarrhea . While 46% (13 of 28) of the children under age 2 infected with ETEC were identified on home visits as having had a recent history of diarrhea, only 13% (5 of 39) of those over age 2 presented such a history . ETEC was isolated from 14% of the mothers' hands, 13% of the children's hands, and 7% of jars containing bath water that was used for washing the children after defecation . Drinking water was identified as a probable source of infection in 1 of 42 cases . Further studies are needed to determine whether ETEC from water stored in the home can spread and cause secondary infections . author's modified

Acta Paediatr Scand Suppl, 1987, 331, 5 - 8
Production of authentic recombinant somatropin; Fryklund L; Further developments in recombinant DNA technology, exploiting a natural mechanism for export using a signal peptide in E . coli, have enabled the production of recombinant somatropin, with full biological activity and extremely high purity . The secretion of the protein into the periplasmic space has simplified purification procedures, providing a unique and simple process and a pure and active product.

Acta Paediatr Scand Suppl, 1987, 331, 28 - 34
Clinical trial with authentic recombinant somatropin in Sweden and Finland; Albertsson-Wikland K; A total of 47 prepubertal children with hGH deficiency were treated for up to 6 months with recombinant somatropin . All the children markedly increased their growth rate; 21 of them were naive (not previously treated with hGH), and increased their growth rate from 4.2 +/- 0.2 cm/year to 13.9 +/- 0.9 cm/year (calculated from growth data after 6 months' treatment, n = 11) . Of the 47 children, 26 had been previously treated for 2 +/- 0.3 years (range 0.3-8.3 years) with pituitary hGH . After a period of 0.9 +/- 0.03 years (range 5-15 months) without any hGH therapy, their growth rate increased from 2.9 cm/year to 11.1 cm/year on recombinant somatropin therapy (calculated from growth data after 6 months' treatment, n = 10) . One child reacted with temporary local erythema at the injection site . Anti-hGH antibodies, with a binding capacity of 0.02 mg/litre, were detected in 1 of the 16 children after 6 months of therapy . No adverse effect on her growth rate was seen . No changes in levels of antibodies to Escherichia coli proteins were detected . No other allergic manifestations or systemic side-effects were demonstrable.

Acta Biochim Pol, 1987, 34(1), 35 - 44
Cloning of cysB mutant alleles of S . typhimurium; Jagura-Burdzy G et al.; Two cysB mutant alleles of S . typhimurium have been cloned onto pBR vectors . The product of the constitutive cysBc 1352 allele present on the plasmid was found to fulfill regulatory functions: as an activator of the cysteine regulon and as an autorepressor . CysB70 auxotrophic mutation impairs both regulatory functions cysB protein . Transfer of the clones cysBc 1352 allele from E . coli to S . typhimurium and from S . typhimurium to E . coli and biochemical analysis of transformants suggest involvement of a restriction-modification system in the constitutive expression of the cysteine regulon.

Vox Sang, 1987, 52(3), 203 - 5
Human immunodeficiency virus antibody screening in blood donors from India, Nigeria and Thailand; Kuhnl P et al.; Screening for human immunodeficiency virus (HIV) (LAV/HTLV-III) antibodies in 3 blood donor populations from India (n = 1,000), Nigeria (n = 500) and Thailand (n = 650; sampling in 1982) with a sensitive enzyme immunoassay (EIA; Abbott) yielded seropositivity rates of 0.5, 2.2 and 1.7%, respectively . Two EIAs with control antigens prepared from uninfected cell cultures ('ELAVIA', VIRGO'), a recombinant Escherichia coli DNA EIA ('ENV/CORE'), Western blot, an immunofluorescence assay and a radio-immunoprecipitation assay confirmed none of the EIA-reactive specimens as truly positive . The lack of specificity of the screening test was also attributable to monochromatic evaluation of the test trays at 492 nm only, and to reactivities against determinants of H9 cells used to grow HIV (HLA antibodies).

J Basic Microbiol, 1987, 27(1), 63 - 7
RNA polymerase binding sites on a plasmid R6K derivative with increased copy number; Nesvera J et al.; The specific binding of Escherichia coli RNA polymerase molecules to the DNA of plasmid pNH602, a deletion derivative of R6K having an increased copy number, was detected by electron microscopy . Seven strong RNApol binding sites were found on pNH602 DNA linearized with BamHI or EcoRI restriction endonuclease . All of these specific sites occur in genetically defined regions of the pNH602 molecule . Two of them correspond with the recently reported transcription initiation sites within a region essential for plasmid R6K replication.

Int Arch Allergy Appl Immunol, 1987, 83(3), 238 - 46
Ectophospholipase A2 activity of the rabbit peritoneal neutrophil; Kennedy SP et al.; Intact rabbit neutrophils were found to express phospholipase A2 activity against {14C}oleate-labeled autoclaved Escherichia coli . Cells obtained 12-14 h rather than 4 h after intraperitoneal injection of glycogen had approximately threefold higher activity . The higher activity was due neither to contaminating mono-nuclear cells nor to the degranulation associated with the prolonged inflammatory response in the peritoneum . Of the phospholipase A2 activity of the intact 12-hour cells, approximately one half remained cell-associated, but the other half was released from the neutrophils during incubation . The cell-associated activity was not due to phagocytosis of substrate and subsequent release of {14C}fatty acid . The cell-associated activity was inhibited by the membrane-impermeant diazonium salt of sulfanilic acid . The results indicate that the cell-associated activity of intact neutrophils is due to an ectophospholipase A2.

Gene, 1987, 51(2-3), 179 - 86
Two promoters from the Streptomyces plasmid pIJ101 and their expression in Escherichia coli; Buttner MJ et al.; An RNA polymerase-binding restriction fragment from the small, high-copy-number Streptomyces plasmid pIJ101 has been shown to have promoter activity in vivo using a promoter-probe vector . The nucleotide sequence of the promoter (the pIJ101B promoter) and the approximate position of the transcription start point as identified by in vitro run-off transcription are presented . Both the pIJ101B promoter and the previously characterised pIJ101A promoter were found to promote transcription in Escherichia coli . The transcription start point in E . coli for the pIJ101A promoter has been determined using high-resolution S1 mapping . Initiation occurs at the same point or within 1 or 2 nucleotides of the transcription start point previously identified in Streptomyces lividans, indicating that the same transcriptional signals are recognised in both genera . The data support the idea that one type of RNA polymerase holoenzyme in Streptomyces recognises a class of promoters similar to the major consensus promoters of E . coli, and that the manner of promoter recognition is similar in both genera.

Gene, 1987, 51(2-3), 149 - 61
Possible new genes as revealed by molecular analysis of a 5-kb Escherichia coli chromosomal region 5' to the rpsU-dnaG-rpoD macromolecular-synthesis operon; Nesin M et al.; The complete nucleotide sequence of 5-kb DNA fragment immediately 5' to the rpsU-dnaG-rpoD macromolecular-synthesis operon in Escherichia coli has been determined . It encodes for six open reading frames . Transcriptional and translational analysis have shown that three of them (orfx, orfz1 and orfz2) are expressed in exponentially growing E . coli cells . The orfx, directly 5' to the rpsU gene but transcribed in the opposite direction, may be part of the rpsU-dnaG-rpoD macromolecular-synthesis operon.

Circ Shock, 1987, 22(2), 173 - 83
Pulmonary vascular response to live Escherichia coli: influences of different antiplatelet substances; Svartholm E et al.; The aim of this study was to investigate whether pretreatment with drugs that interfere with platelet functions in different ways could modify the pulmonary vascular response in a porcine septic shock model . Septic shock was induced by i.v . infusion of live Escherichia coli bacteria . Bacteriemic animals were divided into five groups: untreated or pretreated with a thromboxane-A2 synthetase inhibitor (UK 38 485), a serotonin-receptor antagonist (ketanserin), a combination of these two drugs, or a platelet antiaggregating drug (dipyridamole) . E . coli induced significant pulmonary hemodynamic and respiratory changes . The pulmonary responses to E . coli infusion were attenuated after pretreatment with UK 38 485 but unaffected by prior administration of ketanserin or dipyridamole . The combined pretreatment did not attenuate the pulmonary hypertension or other pulmonary responses to E . coli more than UK 38 485 alone . Dipyridamole did not alter the pulmonary circulation after bacterial infusion . It was concluded that thromboxane-A2 is an important, but not the only, mediator of the pulmonary vascular response in septic-shocked pigs and that factors such as serotonin and platelet aggregability seem to be of minor, if any, importance for the hemodynamic response.

Circ Shock, 1987, 22(2), 141 - 54
Effects of vasodilators prostaglandin E1 and methylprednisolone on pulmonary hypertension and right ventricular performance during volume loading in porcine septic shock: a combined invasive and radionuclide study; Schneider AJ et al.; In 16 anesthetized pigs the cardiovascular effects of prostaglandin E1 and methylprednisolone (MPS) during E . coli sepsis were studied . Gated blood pool scans and hemodynamic studies were simultaneously performed . A control group, group I (n = 4), received volume loading alone; groups II, III, and IV received (each n = 4) volume loading after intravenous administration of MPS, prostaglandin E1, and both MPS and prostaglandin E1, respectively . Groups were formed by randomization, such that the effects of prostaglandin E1 (0.1 microgram/kg/min) and MPS (30 mg/kg) could be analyzed separately and in combination . Eight animals treated with prostaglandin E1 were compared with eight animals not receiving prostaglandin E1 . The same method was applied to the MPS group . E . coli infusion resulted in an abrupt increase in pulmonary arterial pressure while systemic blood pressure gradually fell . Cardiac output decreased . Gated blood pool studies showed an increase in right ventricular end-diastolic volume and a decrease in right ventricular ejection fraction . Consequently, right-to-left ventricular end-diastolic volume ratio increased . Pulmonary arterial pressure was lowered in the treatment groups compared to control group . During volume loading right ventricular ejection fraction improved in the prostaglandin E1 group but remained low in the MPS group . Compared to control group, cardiac output did not change and mean systemic arterial pressure significantly decreased in the prostaglandin E1 group . Treatment with prostaglandin E1, MPS, or both drugs and volume loading did not reveal any difference between the four groups with respect to cardiac output, right and left ventricular volumes, and left ventricular ejection fraction . The present study indicates that in a porcine model of E . coli septic shock with acute pulmonary hypertension, prostaglandin E1 and MPS treatment decrease pulmonary vascular resistance but also systemic vascular resistance . Prior to and during volume loading right ventricular ejection fraction increased in the prostaglandin E1 group . However, neither prostaglandin E1 nor MPS improved right ventricular performance and forward flow during volume loading.

Circ Shock, 1987, 22(2), 127 - 39
Humoral factors in primate endotoxin shock; May M et al.; Many biologically active substances are released from the cell in endotoxic shock and thought to contribute to morbidity and mortality . Whether or not these substances are independently toxic or require endotoxin as a catalyst is uncertain; 20 mg/kg E . coli endotoxin was infused into seven rhesus (R) macaca monkeys (toxin donor) . Seven R macaques received an equivalent volume of normal saline (control donor) . Animals were monitored for 12 h and lactated Ringer's was infused to maintain a minimum MABP of 40 mmHg . Donor animals were exsanguinated; plasma was extracted and a Limulus lysate assay (LAL) was utilized to determine the approximate amount of endotoxin present in toxin donor plasma . This volume of endotoxin (0.125 mg/kg) was added to control donor plasma . Plasma was infused into seven toxin receivers and six control receivers . Receiver animals were monitored for 24 h and killed after 3 d . Lungs were evaluated histologically for evidence of acute injury . Five of the seven toxin receivers and no control receivers died within 3 d (P less than .005) . Significantly more hemorrhage occurred in toxin receivers (P less than .01) . Our data suggest a humoral factor present in the plasma of toxin donor capable of promoting tissue injury and death of recipient animals independent of endotoxin.

Mikrobiologiia, 1987 Jan-Feb, 56(1), 58 - 63
{Energy aspects of the growth of Escherichia coli synchronized by starvation}; Tkachenko AG et al.; The quantitative determination of adenyl nucleotides based on the separation of their dansyl derivatives by thin layer chromatography has made it possible to study the dynamics of changes in the pool of ATP, ADP and AMP in Escherichia coli K-12 during its synchronous growth after glucose starvation . The energy parameters (the adenylate pool, energy charge, teh ATP/ADP ratio, the rates of oxygen uptake and ATP generation, the economic coefficients of oxygen and ATP utilization) were compared with changes in the growth characteristics (the rate of growth and biomass concentration) . This comparison allowed the authors to draw the conclusion about the uncoupled constructive and energy metabolism and about the possible regulatory role of energy parameters in the synchronised culture growth.

Mikrobiologiia, 1987 Jan-Feb, 56(1), 150 - 1
{Effect of R-plasmids on the type of growth of Escherichia coli}; Krolichenko TP et al.; The effect of plasmids belonging to four incompatibility groups on the lag phase duration and the growth rate of E . coli was studied . The lag phase was much longer in the presence of plasmids belonging to the IncP and IncI groups in E . coli cells . No correlation in growth rate changes was found between plasmid strains and those which did not contain plasmids.

Braz J Med Biol Res, 1987, 20(6), 877 - 81
Antioxidant capacity of cyclo-oxygenase and lipoxygenase inhibitors; Felzenszwalb I et al.; The present study analyzes the possible scavenger capacity of several anti-inflammatory drugs on growth of Escherichia coli K12, BW9 109, a strain hypersensitive to H2O2, in medium containing H2O2 . Although all cyclo-oxygenase and/or lipoxygenase inhibitors protected the cells against H2O2, no correlation was found between their relative protective abilities and reported anti-inflammatory potencies.

Ciba Found Symp, 1987, 131, 109 - 23
Structure-function relationship of tumour necrosis factor and its mechanism of action; Fiers W et al.; We have cloned the cDNAs of both human and mouse TNF and expressed them to high efficiency in Escherichia coli . Many transformed cell lines are sensitive to the cytotoxic action of TNF, especially in the presence of gamma-interferon, whereas normal cells either are unaffected or respond mitogenically . A number of human-mouse chimeric TNF genes have been constructed and expressed . All show biological activity but none of the chimeric proteins is neutralized by monoclonal antibodies to TNF . TNF has potent antitumour activity in nude mice carrying human xenografts or in mice bearing syngeneic tumours . In some systems direct effects can be demonstrated (in combination with species-specific gamma-interferon) but in others TNF acts indirectly . Combination of TNF with cytostatic drugs can also be effective in curing in vivo . The major limitation of the use of TNF is its toxicity . On many cell types TNF has an action similar to interleukin 1 (IL-1) . At least some of the secondary, intracellular events may be identical for the two effectors . A possible mechanism of action of TNF is the release and metabolism of polyunsaturated fatty acids, which would explain the synthesis of prostaglandins and leukotrienes by many cell types after TNF treatment . The activation of the phospholipase can be blocked by corticoids . Some protease inhibitors protect cells from TNF-induced cytotoxicity but the target of these inhibitors has not been identified . Several genes are switched on by TNF (and by IL-1), including the gene for the 26 kDa protein recently identified as B cell stimulation factor 2 . Events preceding death in rats include hypothermia, hypotension, acidosis and hypoglycaemia . All these effects can be largely eliminated by indomethacin pretreatment, with a resulting improvement in survival . As indomethacin does not inhibit the cytotoxic action of TNF on malignant cells it may form the basis for improved treatment protocols.

Zentralbl Mikrobiol, 1987, 142(7), 541 - 7
Stimulatory effect of phosphate on colicin V synthesis/secretion by strains of Escherichia coli; Rowbury RJ et al.; Larger inhibitory zones were produced by ColV+ strains on sensitive indicators if the strains were grown on nutrient agar and phosphate rather than nutrient agar . This was due to an effect of phosphate on colicin V synthesis/secretion and not to an effect on the response of the indicator . Phosphate produced an effect at 25 degrees C as well as at 37 degrees C (although zones were larger at 37 degrees C) . It seems likely that phosphate stimulation of colicin V synthesis/secretion results from its effect in reducing divalent cation levels in NA because adding EDTA to NA also increased colicin V synthesis/secretion whereas the addition of magnesium or calcium salts had the opposite effect . It is possible that the extent of colicin V synthesis/secretion may depend on the strength of the LPS-LPS bonds in the outer membrane . Phosphate also stimulated synthesis/secretion of colicins E4, G and N but not of the other colicins or microcins tested . Studies with a wild Col+ E . coli isolate suggest that production of some colicins may not be detected on nutrient agar and that addition of phosphate may reveal the synthesis of new colicins.

Gene, 1987, 61(2), 155 - 64
A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei; Penttila M et al.; An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed . Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T . reesei . The transformation frequency can be up to 600 transformants per microgram of transforming DNA . The efficiency of co-transformation with unselected DNA was high (approx . 80%) . The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T . reesei genome . In addition, the Escherichia coli beta-galactosidase was expressed in T . reesei in enzymatically active form from the A . nidulans gpd promoter.

Folia Microbiol (Praha), 1987, 32(6), 531 - 4
Occurrence of alkali-labile sites in pulse labelled DNA of Escherichia coli after the action of DNA-damaging chemicals; Slezarikova V et al.; UV irradiated E . coli cells contain alkali labile sites in their DNA daughter chains . It is shown here that the occurrence of alkali labile sites in the newly synthesized DNA is a more common phenomenon . It occurred after treatment with two different DNA damaging chemicals, but not after treatment with nalidixic acid.

Acta Biochim Biophys Hung, 1987, 22(1), 85 - 97
Effect of the pKM101 plasmid on the repair of single-strand breaks in DNA induced by ionizing irradiation in Escherichia coli; Francia I et al.; The effect of pKM101 plasmid on repair of single-strand breaks in DNA induced by 60Co-gamma irradiation in E . coli K12 AB1157 (wild type) and in its recA- and recB- mutant cells was studied by alkaline sucrose gradient sedimentation method . For quantitative analysis of sedimentation profiles we calculated the S1/2 values described by Veatch and Okada . The S1/2 values of unirradiated cells were 21.10, and after 200 Gray irradiation 11.35, due to the original incidence of single-strand breaks . The presence of pKM101 did not influence these values in either cases . This means that pKM101 had no effect on the rise of single-strand breaks in DNA . During a post-irradiation incubation period at 37 degrees C for 60 min the S1/2 value of the wild type strain increased from 11.35 to 19.22, that of the recB- from 11.50 to 15.23, while the S1/2 value of the recA- mutant did not change owing to the lack of repair of single-strand breaks . pKM101 plasmid markedly increased the S1/2 value in wild type strain and in recB- mutant, while it had no effect on S1/2 in recA- cells, during this post-irradiation incubation period . Thus the effect of pKM101 on the repair of single-strand breaks in DNA proved to be dependent on recA+ genotype . Nalidixic acid at 100 micrograms/ml concentration inhibited the repair of single-strand breaks in both wild type and recB- mutant cells harbouring pKM101 plasmid.

Eur J Nucl Med, 1987, 13(7), 353 - 7
99mTc-HM-PAO for leukocyte labeling--experimental comparison with 111In oxine in dogs; McAfee JG et al.; 99mTc-hexamethylpropylene amine oxime d,l diastereoisomer (HM-PAO), developed as a diffusible brain imaging agent, labels leukocyte suspensions in saline with an efficiency of 80% using 1-200 micrograms quantities . In dogs, the recovery and survival of reinjected cells in the bloodstream resemble those of 111In-oxine labeled cells at least for several hours . Images in control animals at 18 h show the spleen, liver, marrow, and bladder, minimal pulmonary activity and some gastrointestinal activity . Induced E . coli abscesses and joint inflammatory lesions in dogs are shown on 18 h images . This complex appears promising as an agent for abscess detection in humans . However, strict quality control of this agent is necessary, and it must be used immediately after the 99mTc complex is formed for labeling cells.

Nucleic Acids Symp Ser, 1987, (18), 137 - 40
Substrate specificity of uridine and purine nucleoside phosphorylases of the whole cells of Escherichia coli; Zintchenko AI et al.; Substrate specificity of uridine and purine nucleoside phosphorylases of the whole cells of Escherichia coli BM-11 has been studied . Both enzymes reveal similar requirements to the structure and stereochemistry of uracil nucleosides and of the pentofuranose-1-phosphates, respectively, viz, a) modifications at C-3' decreased the substrate activity to a greater extent as compared with the same modifications at C-2'; b) substitution of a methyl group for one of the 5'-CH2 protons does not lead to essential alterations of the substrate activity of such analogs vs . the natural substrates - uridine and ribofuranose-1-phosphate, respectively . PNP exhibits a very broad specificity for the purine acceptor.

Miner Electrolyte Metab, 1987, 13(6), 405 - 8
Immune responses to dialyzer contents by dialysis patients; Sherlock JE et al.; Serum antibodies to the contents of cuprophan hollow-fiber dialyzers were demonstrated by immunodiffusion in 36 of 68 hemodialysis patients . The antibodies were not absorbed by ethylene oxide or washed cuprophan hollow fibers . No cross-reactivity with Escherichia coli lipopolysaccharide and pneumococcal polysaccharides was noted . The antibodies were IgM and probably IgG . Washed cuprophan hollow fibers did not absorb IgE detected by radioallergosorbent test (RAST) . Lymphocyte transformation to the contents of both cuprophan hollow fiber and polyacrylonitrile plate dialyzers was demonstrated . Materials added in manufacture of dialyzers are immunogenic . The long-term biologic effects are unknown.

Microbiol Immunol, 1987, 31(9), 851 - 8
Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells; Minami J et al.; We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells . An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells . It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities . However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation . The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay . The number of adhered bacteria thus obtained was in good agreement with a viable cell count . The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope . The present method also makes it easy to detect adherent strains of E . coli in large numbers of specimens.

Histochemistry, 1987, 87(4), 293 - 300
Immunohistochemical detection of human epidermal growth factor in submandibular glands and their tumors using a polyclonal antiserum and a monoclonal antibody; Tsukitani K et al.; We applied immunohistochemical procedures to detect hEGF in salivary glands and pleomorphic adenomas of salivary-gland origin using a polyclonal hEGF antiserum and a monoclonal antibody against hEGF synthesized by applying the synthetic gene technique using Escherichia coli . In normal salivary glands, hEGF was mainly localized in the ductal system (i.e., intercalated, striated, and excretory ducts) . The staining intensity and intracellular localization exhibited some variation depending on the fixative used . When a polyclonal hEGF antiserum was used for immunostaining, slight background staining was observed in sections prepared using the fixatives tested . Therefore, precise localization of hEGF was obtainable only in formalin-fixed sections using the monoclonal antibody against hEGF . In pleomorphic adenomas, positive hEGF staining was seen on the luminal side of tumors and in cells of ductal origin; no reactivity was present on the outer side of tumors or in cells of myoepithelial origin . Occasionally, long, spindle-shaped tumor cells and chondroidally changed tumor cells also exhibited positive staining for hEGF.

Acta Haematol, 1987, 78(2-3), 85 - 9
X-ray crystallographic and functional studies of human haemoglobin mutants produced in Escherichia coli; Luisi BF et al.; Human beta-globin was produced in Escherichia coli as a cleavable fusion protein using the expression vector pLcII {Nagai and Thogersen, Nature 301, p . 810, 1984} . The fusion protein CIIFX beta-globin was purified under denaturing conditions to homogeneity and the authentic beta-globin was liberated by blood coagulation factor Xa . beta-Globin was then folded and reconstituted with haem and alpha-subunits to form fully functional alpha 2 beta 2-tetramers {Nagai et al., Proc . natn . Acad . Sci . USA 82, p . 7252, 1985} . This has enabled us to produce mutants with amino acid substitutions in the beta-subunit at will and in sufficient quantities to study their oxygen-binding properties and three-dimensional structures . We have crystallised three mutants, Hb Nympheas {Cys-93 beta----Ser}, Hb Daphne {Cys-93 beta----Ser, His-143 beta----Arg} and Hb Sandra {Cys-93 beta----Ser, Asp-94 beta----Glu}, and have solved their structures to high resolution by x-ray crystallography.

Gene, 1987, 56(1), 79 - 86
Cloning of Streptomyces griseus and Streptomyces lividans genes for glycerol dissimilation; Biro S et al.; Streptomyces lividans gyl DNA (for glycerol utilisation) was cloned by complementation of a Streptomyces coelicolor gyl mutant . Restriction mapping showed that the cloned DNA was highly homologous (perhaps 99%) to S . coelicolor gyl DNA . Using phage-mediated mutational cloning, an internal fragment of the S . coelicolor gyl operon was used to generate a gyl mutant of S . lividans, which subsequently served as recipient in the cloning of gyl DNA from S . griseus . A 7.5-kb SstI-generated fragment of S . griseus DNA was obtained which, as judged by analysis of restriction sites, was only perhaps 87% homologous with the S . coelicolor gyl operon . The cloned S . griseus DNA appears to contain intact gylA and gylB genes and probably also an upstream gene related to the putative gyl regulatory '0.9-kb' gene of S . coelicolor . Cloning of the fragment on a high-copy-number vector in S . lividans did not lead to high levels of the enzymes encoded by gylA and gylB . The S . griseus gylA and gylB genes were not detectably expressed in Escherichia coli glp mutants.

Neoplasma, 1987, 34(4), 379 - 87
Chromotest estimation of SOS functions as a screening method for antitumor platinum complexes; Drobnik J et al.; The chromotest which measures the induction of SOS functions in an Escherichia coli strain bearing the lacZ gene under the sfiA gene control was evaluated as a screening method for platinum complexes with antitumor activity . Four types of complexes were used: (a) those with two nitrogen ligands in cis position and chlorine or carboxylic acid as anionic ligand; (b) four nitrogen atoms surrounding platinum; (c) complexes involving sulphur and amino group of methionine; (d) complexes of tetravalent platinum . The trans-diaminodichloro complex was used as negative control . Groups (b) and (c) were inactive in the chromotest which correlated well with their absence of antitumor activity . Antitumor activity and a positive chromotest also correlated in group (d) . In group (a), which includes complexes with well established antitumor activity, the chromotest separated complexes with fast and slow rate of hydrolysis . The results fitted well in the mutation induction test.

J Mol Evol, 1987, 25(2), 151 - 8
Regulatory mutations that allow the growth of Escherichia coli on butanol as carbon source; Clark DP et al.; Starting with adhC mutants of Escherichia coli in which alcohol dehydrogenase (ADH) and acetaldehyde CoA dehydrogenase (ACDH) are expressed constitutively at high levels, we selected mutants with still higher levels of both enzymes . Selection for growth on ethanol in the presence of inhibitors of ADH gave several mutants that had from 2- to 10-fold increases in the levels of both enzymes . These mutations were found to map far from the adhC locus at around 90 min . Such adhR mutants were unable to grow on acetate or ethanol in certain media unless supplemented with extra manganese . This growth disability was suppressed by secondary mutations, one of which, aceX, increased sensitivity to several toxic metals and may perhaps derepress Mn transport . When the adhR mutation expressing the highest ADH and ACDH levels was present together with fadR and atoC mutations (allowing efficient catabolism of acetoacetyl-CoA) and with an aceX mutation, the resulting strains became capable of using n-butanol as sole carbon and energy source . The use of butanol by E . coli illustrates the artificial evolution of a new catabolic pathway, in this case by the selection of four successive regulatory mutations (fadR, adhC, atoC, and adhR) together with the poorly defined aceX mutation . Each stage in the acquisition of this novel pathway confers the ability to use a new growth substrate: decanoic acid (fadR), ethanol (adhC), butyric acid (atoC), and butanol (adhR, when present with aceX).

J Cell Sci Suppl, 1987, 6, 83 - 96
Functional expression of the Escherichia coli alkyltransferase gene in mammalian cells; Margison GP et al.; Alkylating agents can produce a variety of biological effects in mammalian cells and organisms including toxicity, mutagenicity and malignant transformation . These agents react with oxygen and nitrogen atoms in DNA resulting in 12 products some of which are known to be eliminated from DNA by repair systems . One method of assessing the relative importance of a specific product in any of the biological effects of DNA alkylation would be to convert a cell line that is deficient in a particular repair function into a repair-proficient cell line and to determine whether this influences the magnitude of the effect . The cloning and expression in mammalian cells of the Escherichia coli DNA repair gene coding for the O6-alkylguanine-alkylphosphotriester dual alkyltransferase will be described . The E . coli gene product acts on damage produced in host cell DNA by treatment with methylnitrosourea, and reduces the toxicity and mutagenicity of this agent . The effects on the toxicity of a variety of other mono and bifunctional alkylating agents have also been assessed.

Folia Biol (Praha), 1987, 33(3), 145 - 53
Subcloning of prochymosin cDNA for Plac controlled expression; Sedlacek J et al.; Two overlapping segments of prochymosin cDNA clones (Liebscher et al., 1985) were used to construct plasmids that expressed an activable zymogen product and thus verified the integrity of the reverse transcripts . The pUC9 vector was used for the expression, under the control of the lac promoter . The expression product (a fused protein consisting of the N-terminus of beta-galactosidase, a polylinker-coded peptide and prochymosin from its 5th amino acid) displayed, upon activation by the usual procedure, the properties of calf chymosin . The active product was identified by milk-clotting tests, "caseinography" and protein electrophoresis of immunoprecipitates . The "boxing" of prochymosin cDNA in the constructed plasmids makes them a versatile source of this cDNA for other expression constructs.

Gerontology, 1987, 33(2), 99 - 108
Age and protein malnutrition: effects on the febrile response; Bradley SF et al.; Elderly hospitalized patients frequently do not mount a vigorous febrile response . This diminished response could be due to the combined effects of age and protein malnutrition, a common problem in the elderly . We studied the effect of mild protein deprivation on the febrile response to interleukin-1 (IL-1) and endotoxin in young, middle-aged, and elderly Fischer rats . In each age group, rats received either a standard (23% casein) or low protein (8% casein) diet for 5 weeks (young rats) or 8 weeks (middle-aged and elderly rats) . Each rat received an intraperitoneal injection of IL-1, 10 micrograms/kg endotoxin, or 100 micrograms/kg endotoxin and temperatures were measured over 6 h by implanted biotelemetry devices . Protein deprivation decreased the febrile response to IL-1 in all age groups and to endotoxin only in the young and middle-aged rats . Increasing age along did not decrease the febrile response to either pyrogen . Increasing age and protein deprivation did not have an additive effect in decreasing the febrile response to IL-1 or endotoxin.

Gene, 1987, 51(2-3), 269 - 74
Characterization of human ferritin H chain synthetized in Escherichia coli; Levi S et al.; We have inserted the coding region of the cDNA for human ferritin H chain into the expression vector pEMBLex2 . The plasmid obtained is able to direct the synthesis of the ferritin H chain in Escherichia coli up to a concentration of 15% of total soluble proteins . All expressed subunits are found correctly assembled in the complete ferritin molecule, which can be easily purified . We have shown that the ferritin synthesized in E . coli has an Mr, electrophoretic mobility, and thermal stability similar to natural human isoferritins and is recognized by monoclonal antibodies specific for the H, but not by those for the L human ferritin chains.

Gene, 1987, 51(2-3), 255 - 67
Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli; Stark MJ; A series of new expression vectors (the pTTQ series) has been constructed for the regulated expression of genes in Escherichia coli . Based on the pUC plasmids, the pTTQ vectors contain a polylinker/lacZ alpha region flanked by the strong hybrid trp-lac (tac) promoter and the rrnB transcription terminator . Foreign genes can be inserted into the polylinker region of this expression cassette, to give either transcriptional or translational fusions within the lacZ alpha coding region . In most commonly used strains of E . coli, multiple copies of the lac operator titrate out the lac repressor . This phenomenon leads to significant expression from tac or lac promoters present on multicopy plasmids, even in the absence of inducers such as IPTG . To ensure maximal repression of the tac promoter on the pTTQ vectors in any host strain, the lacIQ allele of the lac repressor gene was added to the vectors . This makes them particularly useful for cloning genes when expression at high level is desired but is detrimental to cell growth.

Circ Shock, 1987, 21(4), 261 - 70
Renal microthrombosis following endotoxin infusion may be mediated by lipoxygenase products; Schaub RG et al.; Renal microvascular thrombosis following endotoxin infusion was assessed by measuring accumulation of 125I-labeled fibrinogen and transmission electron microscopy . Endotoxin shock was induced in unanesthetized Sprague-Dawley rats using 14 mg/kg Escherichia coli endotoxin (Difco) infused intravenously over a 5-hour period . Fifteen minutes after infusion of endotoxin was started, intravenous treatment was initiated . Two-thirds of the treatment was given over a period of 20 minutes followed by the remaining dose over the next 2 hours . Five rats received methyl prednisolone sodium succinate (Solu-Medrol) (U-9,088) (39.5 mg/kg) . Six rats received a Solu-Medrol analog (methyl prednisolone 21(N-Methyltaurosuberate), sodium salt) (U-67,590A) (61.08 mg/kg) . Six rats received a 5-lipoxygenase inhibitor (1-naphthalenol,2,3,diethyl-4-methoxy-acetate) (U-66,855) (20 mg/kg) . Six rats received a prostacyclin analog (pentanoic acid, 5- less than hexahydro-5-hydroxy-6-(3-hydroxy-1-octenyl)-3a-methyl-2(1H)- pentalenylidene greater than -calcium salt, hydrate) (U-61,431F) (0.04 mg/kg) . Six rats received a thromboxane synthase inhibitor (2-benzofurancarboxylic acid, 5-(3-pyridinylmethyl), sodium salt monohydrate) (U-63,557A) (18 mg/kg) . Eight endotoxin-infused rats received only normal saline . Six control rats received no endotoxin infusion, only saline . Composition and location of thrombi were assessed by transmission electron microscopy . Glomerular thrombosis was quantitated by determination of whole blood equivalents of 125I fibrin/g of tissue . Electron microscopy and radioactive quantitation demonstrated microthrombosis in the nontreated endotoxin group . The thrombi contained fibrin, platelets, erythrocytes, and leukocytes . The extent of thrombosis was significantly elevated compared to saline-treated controls . Three treated groups (U-9,088, U-67,590A, and U-66,855) all had significantly less thrombosis than the nontreated endotoxin rats . Two treated groups (U-63,557A and U-61,431F) developed glomerular thrombosis similar to untreated endotoxin rats . The results suggest that endotoxin stimulation of procoagulant activity and microthrombosis may be mediated by production of arachidonic acid metabolites via the lipoxygenase pathway.

Circ Shock, 1987, 21(3), 185 - 95
Transient pulmonary platelet sequestration during endotoxemia in dogs; Gutmann FD et al.; We evaluated the time-course of regional platelet sequestration, following a bolus dose of endotoxin in anesthetized dogs . Autologous indium 111 labeled platelets, representing less than 1% of the circulating platelets, were injected 35-60 min prior to administering endotoxin intravenously to dogs . A gamma camera was used to monitor the distribution of these platelets within the thorax and abdomen . Alterations in the circulating blood platelet count paralleled the changes in blood radioactivity, enabling us to use external imaging to evaluate platelet kinetics . Marked hypotension and thrombocytopenia occurred within 6 min after administering endotoxin . The platelet pool in the lungs peaked at 9 min and was temporally related to the decrease in circulating platelet count, hypotension and increase in liver size . Translocation of platelets from the lungs to the circulating platelet pool occurred during the subsequent hour with sequestration occurring in the liver and possibly other organs . During this phase there was a recovery in platelet count to 35% of baseline levels but without significant recovery in mean arterial pressure . Based on these results we propose that endotoxin-induced thrombocytopenia results from pulmonary and hepatic sequestration of platelets, but that sequestration of platelets in the lungs is only transient . The mechanism and significance of subsequent translocation of platelets from the lungs to other sites, particularly the liver and the circulating platelet pool, remain to be investigated.

Genetics, 1987 Jan, 115(1), 25 - 31
Metabolic flux and fitness; Dykhuizen DE et al.; Studies of Escherichia coli under competition for lactose in chemostat cultures have been used to determine the selective effects of variation in the level of the beta-galactoside permease and the beta-galactosidase enzyme . The results determine the adaptive topography of these gene products relative to growth in limiting lactose and enable predictions concerning the selective effects of genetic variants found in natural populations . In the terms of metabolic control theory, the beta-galactosidase enzyme at wild-type-induced levels has a small control coefficient with respect to fitness (C = 0.018), and hence genetic variants resulting in minor changes in enzyme activity have disproportionately small effects on fitness . However, the apparent control coefficient of the beta-galactoside permease at wild-type-induced levels is large (C = 0.551), and hence even minor changes in activity affect fitness . Therefore, we predict that genetic polymorphisms in the lacZ gene are subject to less effective selection in natural populations than are those in the lacY gene . The beta-galactoside permease is also less efficient than might be expected, and possible forces resulting in selection for an intermediate optimum level of permease activity are considered . The selective forces that maintain the lactose operon in a regulated state in natural populations are also discussed.

Appl Environ Microbiol, 1987 Jan, 53(1), 204 - 7
Expression of cloned monkey metallothionein in Escherichia coli; Murooka Y et al.; Expression vectors were constructed in which a cDNA specifying the monkey kidney metallothionein-II (MT-II) was linked directly to the lambda PR promoter . Enhanced expression of MT-II in Escherichia coli was observed when two initiation signals were tandemly linked to the MT-II gene and the lambda cI+ host cells were induced by nalidixic acid.

Proc Soc Exp Biol Med, 1987 Jan, 184(1), 7 - 13
Presence of acute phase changes in zinc, iron, and copper metabolism in turkey embryos; Klasing KC et al.; Acute phase changes in trace mineral metabolism were examined in turkey embryos . An endotoxin injection resulted in increased concentrations of serum copper and liver zinc and decreased concentrations of serum zinc in embryos incubated either in ovo or ex ovo . Changes in zinc and copper metabolism occurred when endotoxin either was injected intramuscularly, into the amnionic fluid, or administered onto the chorioallantoic membrane . Unlike poults, embryos did not respond to an inflammatory challenge with decreased serum iron concentrations . Acute phase changes in embryo serum zinc and copper as well as liver zinc concentrations were similar to those in poults . Increased liver zinc concentrations were associated with increased zinc in metallothionein (MT) . An injection of a crude interleukin 1 preparation into embryos resulted in similar increases in hepatic zinc and MT concentrations as an endotoxin injection, suggesting a role for this cytokine in mediating the acute phase changes in embryonic zinc metabolism.

Proc Natl Acad Sci U S A, 1987 Jan, 84(1), 156 - 60
Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer; Price J et al.; We describe a cell-lineage marking system applicable to the vertebrate nervous system . The basis of the technique is gene transfer using the retroviral vector system . We used Escherichia coli beta-galactosidase as a marker gene and demonstrate a high level of expression of this marker from the viral long terminal repeat promoter, with simultaneous expression of the Tn5 neo gene from the simian virus 40 early promoter . This expression has allowed us to detect individual infected cells histochemically . We applied this marking technique to the study of lineage relationships in the developing vertebrate nervous system, both in vivo and in culture . In the rat retina, we injected virus in vivo and histochemically identified clones of marked neural cells . In addition, we used this virus to infect cultures of rat cerebral cortex and have analyzed the clonal relationships of morphologically different neural cell types . The host range of the marking system extends to avian as well as mammalian species . Thus, this system should have broad applicability as a means of gene transfer and expression in the nervous system.

Mutat Res, 1987 Jan, 183(1), 11 - 20
Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities; Costa de Oliveira R et al.; The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities . The SOS response was measured using an sfiA::lacZ operon fusion . The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain . In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response . The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response . We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions . In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations . The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal" . However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production . Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.

J Clin Microbiol, 1987 Jan, 25(1), 76 - 80
Production, characterization, and species specificity of five monoclonal antibodies to Mycobacterium tuberculosis; Kadival GV et al.; The production and characterization of five monoclonal antibodies to Mycobacterium tuberculosis are described . Specificity of the monoclonal antibodies was tested against other mycobacterial species by an enzyme-linked immunosorbent assay and immunoblots . HGT 3a, an immunoglobulin M (IgM) antibody, recognizes a molecule of 38,000 molecular weight present only in the tuberculosis complex of M . tuberculosis and Mycobacterium bovis BCG . HGT 6, an IgG1 antibody, recognized two molecules with molecular weights of 43,000 and 45,000 and showed limited cross-reactivity . Three other antibodies, HGT 1, HGT 2, and HGT 4, all belonging to the IgG1 type, recognized multiple bands and showed broad reactivity among all mycobacterial antigens tested, Escherichia coli and Nocardia asteroides.

J Bacteriol, 1987 Jan, 169(1), 19 - 25
Genetic and morphological characterization of ftsB and nrdB mutants of Escherichia coli; Taschner PE et al.; The ftsB gene of Escherichia coli is believed to be involved in cell division . In this report, we show that plasmids containing the nrdB gene could complement the ftsB mutation, suggesting that ftsB is an allele of nrdB . We compared changes in the cell shape of isogenic nrdA, nrdB, ftsB, and pbpB strains at permissive and restrictive temperatures . Although in rich medium all strains produced filaments at the restrictive temperature, in minimal medium only a 50 to 100% increase in mean cell mass occurred in the nrdA, nrdB, and ftsB strains . The typical pbpB cell division mutant also formed long filaments at low growth rates . Visualization of nucleoid structure by fluorescence microscopy demonstrated that nucleoid segregation was affected by nrdA, nrdB, and ftsB mutations at the restrictive temperature . Measurements of beta-galactosidase activity in lambda p(sfiA::lac) lysogenic nrdA, nrdB, and ftsB mutants in rich medium at the restrictive temperature showed that filamentation in the nrdA mutant was caused by sfiA (sulA) induction, while filamentation in nrdB and ftsB mutants was sfiA independent, suggesting an SOS-independent inhibition of cell division.

J Basic Microbiol, 1987, 27(3), 139 - 46
Effect of nourseothricin (streptothricin) on the outer membrane of sensitive and resistant Escherichia coli strains; Seltmann G et al.; Nourseothricin (streptothricin) causes disturbances (perforations) in the outer membrane of sensitive E . coli strains allowing lysozyme and deoxycholate, but not the periplasmic alkaline phosphatase to penetrate . EDTA slightly increases, but Mg++ ions slightly decrease this effect . The cell walls of three from four nourseothricin-resistant strains do not become permeable under these conditions, but remain sensitive against TRIS/EDTA . Nourseothricin is supposed to pass the outer membrane of sensitive bacteria via some kind of "self-promoting" pathway . This way can (but need not) be blocked in resistant strains.

Gene, 1987, 55(1), 29 - 40
Contingent replication assay (CRA) procedure for rapid isolation of enhancers; Vasavada HA et al.; A rapid procedure for the isolation of functional enhancer sequences consists of the construction of a shotgun DNA library in SV40-based plasmid shuttle vectors which depend on an enhancer for replication, the replication in monkey (CVI) cells of those vectors into which an enhancer sequence was inserted, the selective cleavage of unreplicated vectors by DpnI and the recovery of the replicated vectors by transfection into Escherichia coli . We describe conditions for the fusion of protoplasts to CVI cells, under which conditions the probability of only one type of plasmid entering a cell is increased and thus complementation and rescue of enhancer-less plasmids are decreased . The effectiveness of the procedure is demonstrated by the recovery of enhancers from bovine papillomavirus and Moloney murine sarcoma virus.

Ann Rech Vet, 1987, 18(2), 121 - 5
Molecular cloning of the bovine viral diarrhea virus genomic RNA; Renard A et al.; The genomic RNA from Bovine Viral Diarrhea Virus (BVD) was cloned in E . coli . The complete sequence has been obtained and the genomic organization has been deduced . Some of the BVD specific cDNA have been expressed in bacteria, eucaryotic cells and in vitro . We suggest that BVDV belongs to a new family different from Togaviridae and Flaviviridae.

Prog Clin Biol Res, 1987, 236A, 401 - 18
Perturbation of transmembrane signaling mechanisms in acute and chronic endotoxemia; Spitzer JA et al.; Our results reviewed here may be summarized as follows: 1 . Continuous endotoxemia significantly interferes with Ca2+-dependent information flow in the liver . 2 . The subcellular sites where these molecular lesions can be localized include: a.) the plasma membrane-there are effects at the level of alpha 1-adrenergic and vasopressin binding, and also in the coupling of receptor activation to inositol lipid metabolism in terms of PIP2 degradation and resynthesis b.) the endoplasmic reticulum in terms of Ca2+ release and PI synthesis . Another one of the sequelae of Ca2+-associated receptor activation, namely, cytosolic ionized Ca2+ concentration is also affected . 3 . Finally, in addition to seeing the impact of acute or continuous endotoxemia at the level of receptor activation and signal generation, we can also document alterations in the expression of physiologic function subserved by these Ca2+- and inositol lipid-associated signaling processes--i.e . in glycogen phosphorylase activity-being consistent with the above described changes . In conclusion, we state that a causal link is shown between receptor binding, agonist-induced phosphoinositide hydrolysis, intracellular Ca2+ mobilization and activation of phosphorylase a in the liver, suggesting that these alterations may underlie some of the metabolic consequences of chronic sepsis.

Gene, 1987, 53(2-3), 145 - 52
RNA polymerase makes important contacts upstream from base pair -49 at the Escherichia coli galactose operon P1 promoter; Busby S et al.; A G:C to T:A transversion at bp position -19 in the gal operon promoter region relieves the dependence of galP1 promoter activity on the cAMP-CRP complex . Deletion analysis shows that expression from the promoter is decreased on replacement of the sequence between 49 and 54 bp upstream from the P1 start point . Moreover, protection experiments show that RNA polymerase interacts with this region in open complexes at P1 . We propose that this contact is necessary for optimal P1 activity; point mutations in the gal promoter region can alter DNA flexibility and hence the strength of this contact; CRP factor activates P1 transcription by favouring formation of this contact; and the gal repressor blocks P1 activity by binding to this zone.

Gene, 1987, 52(2-3), 245 - 56
A low-copy-number vector utilizing beta-galactosidase for the analysis of gene control elements; Koop AH et al.; A low-copy-number vector, pFZY1, with the multiple restriction site linker of M13mp18 inserted upstream from a promoterless beta-galactosidase (beta Gal)-coding lacZ gene has been constructed to provide a convenient and accurate system to analyze regulatory elements in vivo . The plasmid contains the oriF replication origin without the par locus and is present in the cell in one to two copies per genome . It is retained in the host by the presence of ampicillin, and each inserted promoter yielded consistent values of beta Gal activity under all the conditions tested . A series of tetracycline resistance (TcR) promoter fragments and lac promoter fragments have been compared in pFZY1 and the high-copy-number pKO-vector series . The transcriptional activity measured for different fragments containing the same TcR promoter varied within a six-fold range among the several constructs tested . Regulation of the wild-type lac promoter and mutants in pFZY1 was similar to that observed for lac promoters in the chromosome while their regulation in pKO-1mp18 was significantly affected by the high copy number, as expected.

Gene, 1987, 52(2-3), 225 - 33
A family of yeast expression vectors containing the phage f1 intergenic region; Vernet T et al.; The construction and characterization of a family of yeast expression vectors is described . They have the following features: plasmid replication and selection (ApR) in Escherichia coli, packaging of single-stranded (ss) DNA upon infection of E . coli with a filamentous helper phage, replication in Saccharomyces cerevisiae based on the 2 mu plasmid origin of replication (ori), selection in yeast by complementation of LEU2 (pVT-L series, size 6.3 kb) or URA3 gene (pVT-U series, size 6.9 kb) and seven unique restriction sites for cloning within an 'expression cassette' which includes the promoter and 3' sequence of the ADH1 gene . The multiple cloning site as well as the ori and intergenic region of the phage f1 have been cloned in two orientations for convenient gene cloning and ssDNA strand selection . As a result any of these eight vectors can be chosen for cloning, expressing genes in yeast, sequencing and mutagenesis without the need for recloning into specialized vectors.

Gene, 1987, 52(2-3), 215 - 23
Production of chimeric protein coded by the fused viral H-ras and human N-ras genes in Escherichia coli; Matsui T et al.; A new method for the production of a chimeric protein of two related genes has been developed . The nucleotide sequences of the region from the N terminus to the 86th amino acid (aa) residue of human N-ras and of the Harvey sarcoma virus (Ha-MuSV) H-ras are 80% homologous . We isolated the DNA fragment encoding the N-terminal portion up to the 70th aa residue from plasmid pH-1 which encodes the total genome of Ha-MuSV, and the DNA fragment encoding the C-terminal portion from the 40th aa to the C terminus from plasmid p6a1 which includes the human N-ras cDNA but lacks the N-terminal portion . After partial digestion of both fragments with phage lambda exonuclease, which creates 3'-protruding ends, a hybrid was formed between 73% homologous single-stranded DNA portions at the 3' ends of both fragments . The hybrid was recloned on pBR322 after repairing with Escherichia coli DNA polymerase I and DNA ligase . The chimeric v-H/N-ras gene composed of the N-terminal portion of v-H-ras gene and the remaining region of N-ras gene was inserted into an expression vector containing two tandem trp promoters and a terminator, and expressed in E . coli . The chimeric protein was found to accumulate to approx . 10% of total cellular proteins.

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