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 carryin