Protein Expr Purif, 1991 Oct-Dec, 2(5-6), 330 - 8 Overexpression and purification of the galactose operon enzymes from Escherichia coli; Vorgias CE et al.; A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented . The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity . The purified gal operon enzymes were biochemically characterized by gel-filtration column chromatography and isoelectric focusing, and the Km values for their substrates were determined. Protein Expr Purif, 1991 Oct-Dec, 2(5-6), 321 - 9 Mutation in the Escherichia coli htpR locus results in stabilization of recombinant expression products that are susceptible to proteolytic degradation; Lindler LE et al.; We compared the expression and degradation of three cloned malarial proteins in a pair of isogeneic strains of Escherichia coli that differed at the htpR locus . The htpR locus encodes an alternate sigma factor necessary for the transcription of heat shock promoters . Plasmodium sequences were cloned from polymerase chain reaction-amplified DNA initiated by oligonucleotide primers that were specific for the gene coding regions to be expressed . The amplified DNA was cloned and expressed in a vector that encodes a strong T7 promoter and translation--initiation signal . The total cell yield of two of the expressed proteins was found to be increased when synthesis occurred in a E . coli htpR mutant . Pulse--chase experiments showed that the increased protein yield correlated with a decrease in the degradation of the protein in the htpR strain . A two- to seven-fold increase in the half-life of the malaria proteins was observed in the E . coli htpR- background as compared to htpR+ . We found no difference in survival of the E . coli K165 htpR mutant and isogeneic parent during thermal induction . Since the synthesis of the heat shock sigma factor did not significantly influence survival of E . coli and htpR expression results in increased degradation of foreign proteins, the E . coli htpR mutant was a valuable host strain for production of foreign proteins. Protein Expr Purif, 1991 Oct-Dec, 2(5-6), 317 - 20 A rapid purification procedure of recombinant integration host factor from Escherichia coli; Vorgias CE et al.; A rapid procedure for the large-scale isolation of recombinant integration host factor (IHF) protein from Escherichia coli is presented . The protein was overproduced in the E . coli K5746 strain, whose construction has already been described . The procedure consists of a mild extraction of protein and fractionation by ammonium sulfate . A single-step affinity chromatography on heparin-Sepharose provided very pure IHF protein . A Mono-S FPLC column was used to highly concentrate the pure IHF for crystallization trials . Attempts to crystallize IHF produced small stable crystals that have a large number of molecules in the asymmetric unit and to date diffract poorly . Further attempts to crystallize IHF under other conditions as well as in a complex with the putative DNA binding site are underway. Protein Expr Purif, 1991 Oct-Dec, 2(5-6), 313 - 6 Purification and properties of recombinant Pneumocystis carinii dihydrofolate reductase; Sirawaraporn W et al.; Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography . The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence . The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7 . The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl . Urea at 2.5 M also increased the enzyme activity twofold . Kinetic analysis of the purified enzyme revealed that the Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat was 70 s-1 . Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P . carinii DHFR and showed little selectivity over the human DHFR . Trimetrexate and piritrexim were much more potent inhibitors of the P . carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR. Zhonghua Jie He He Hu Xi Za Zhi, 1991 Oct, 14(5), 269 - 70, 318 {Effect of activated leukocytes on pulmonary arterial pressure}; Sun RY; Shock model was established by intravenous injection of E Coli endotoxin with a dosage of 5 mg/kg wt in dog . An immediate fall in systemic arterial pressure (SAP) was found after injection, while an increase in pulmonary arterial pressure (PAP) and a markedly leukopenia in circulatory blood were also found . Rats lung was perfused with warm (37 degrees C) krebs solution in constant flow rate . There was an obviously increase in PAP when activated leukocytes had been added to the perfusion solution . The results suggested that activated leukocytes in the lung blood vessels may play an important role in the pathogenesis of pulmonary hypertension. Indian J Pathol Microbiol, 1991 Oct, 34(4), 270 - 5 Predicted secondary structure of glycogen phosphorylase from Escherichia coli as deduced using Chou-Fasman analysis; Venkaiah B et al.; Secondary structure of glycogen phosphorylase from Escherichia coli has been deduced using Chou-Fasman analysis . Out of 809 amino acid residues, 244 residues showed formation of alpha-helix (30%), 218 residues beta-pleated sheet (27%) and 192 residues (24%) showed formation of reverse beta turn, distributed all over the sequence . There are total 27 alpha-helix and 31 beta-pleated sheets distributed all over the molecule . A structure consisting of three consecutive strands of beta-pleated sheets and two joining alpha-helix is predicted for the stretch of the primary sequence from residues 325 to 372, thus showing the presence of a Rossman fold super secondary structure . There is a tyrosine at position 350 in the super secondary structure, in the area to contain a reverse beta turn . Several amino acids pairs are present in the sequence having Rossman fold super secondary structure. Pigment Cell Res, 1991 Oct, 4(4), 186 - 92 A recombinant vaccinia virus infects Xenopus melanophores; Potenza MN et al.; A recombinant vaccinia virus was employed to demonstrate infection of cultured Xenopus laevis melanophores . The recombinant virus contains one copy each of the Escherichia coli lac Z and human growth hormone genes under the transcriptional control of two separate viral promoters . Western blot analysis and in situ staining revealed the dependency of beta-galactosidase production in infected Xenopus cells on time and multiplicity of infection (MOI) . Western blot analysis was used to demonstrate the production of a 65 kD vaccinia late protein and its variation over time and with MOI . When virus preparations from infected Xenopus cells were attempted, no amplification of virus was observed and only a minute portion of the original innoculum was recovered . We therefore propose an abortive infection of Xenopus pigment cells by vaccinia virus: The amphibian cells allow for the synthesis of viral proteins, but not for the efficient replication of competent virus . The findings have implications not only for our understanding of the virus/host interaction, but also for the efficient expression of exogenously introduced genes in cultured Xenopus melanophores. Microb Pathog, 1991 Oct, 11(4), 297 - 304 Presence of cfaD-homologous sequences and expression of coli surface antigen 4 on enterotoxigenic Escherichia coli; relevance for diagnostic procedures; Sommerfelt H et al.; We examined the ability of a colonization factor antigen I (CFA/I) polynucleotide probe to identify coli-surface antigen 4 producing (CS4+) strains of enterotoxigenic Escherichia coli (ETEC) . At low stringency (LS) the probe hybridized to colony lysates of strains previously shown to produce CS4 or CFA/I fimbriae . Only DNA from CFA/I+ strains maintained a stable probe-target hybrid under high stringency (HS) conditions . On examination of several clones from three previous CS4 producers, identified as positive in LS and negative in HS colony hybridization, spontaneous loss of nucleotide sequences homologous to a gene encoding a positive CFA/I regulator, CfaD, was found to be associated with lacking expression of CS4 . Our findings indicate that, on stored or subcultured isolates of ETEC, identification of CS4 strains may benefit from applying gene probe technology. Microb Pathog, 1991 Oct, 11(4), 259 - 68 Identification of carbohydrate structures as receptors for localised adherent enteropathogenic Escherichia coli; Jagannatha HM et al.; Enteropathogenic Escherichia coli strains of diffused adherent (DA) and localised adherent (LA) phenotypes were tested for their ability to bind to glycolipids . DA strains did not bind to the glycolipids tested, while LA strains bound to asialo GM1, asialo GM2, globoside and lacto-N-neotetraose in decreasing order of avidity . The minimum common sequence among the four glycolipids could be delineated as GalNac beta 1-4 Gal as the binding epitope with GalNac beta 1-3 Gal and GlcNac beta 1-3 Gal serving as relatively weaker binders . The binding was not inhibited by a variety of free oligosaccharides or by the neoglycoproteins tested . Adhesion-negative mutants of an enteropathogenic LA strain showed a markedly reduced binding to asialo GM1 indicating that the recognition of GalNac beta 1-4 Gal was correlated with the ability to adhere to HeLa cells . Thus recognition and binding to glycolipids could play an important role in colonisation through adherence to intestinal surfaces. Indian J Biochem Biophys, 1991 Oct-Dec, 28(5-6), 541 - 5 Metabolic analysis of galactose toxicity in Escherichia coli with 2-deoxygalactose as the probe; Raut N et al.; Biochemical basis of galactose toxicity has been studied in gal T mutants (CGSC 4974) using 2-deoxygalactose, a non-metabolizable analogue of galactose, as the probe . It is found that biochemical features of toxicity in wild type cells either with 2-deoxygalactose or with 2-deoxyglucose are very similar to the picture obtained with gal T mutants and the observed bacteriostasis is probably due to futile phosphorylation and not due to any specific inhibitory effect of phosphorylated galactose. Indian J Biochem Biophys, 1991 Oct-Dec, 28(5-6), 374 - 80 Molecular interactions between ribosomal proteins--a study of S4-S9 interaction; Prakash V; The ribosomal proteins S4 and S9 were isolated from the 30S ribosomal subunit of Escherichia coli to greater than 95% purity and characterized in the reconstitution buffer . Neither of the proteins indicated any tendency to self associate at 3 degrees C in the concentration range studied . At higher temperatures (greater than 20 degrees C), protein S9 forms a significant amount of a soluble aggregate as seen from the sedimentation velocity and sedimentation equilibrium experiments . From an analysis of the solution mixture of S4 and S9 at 1:1.08 molar concentration ratio by sedimentation velocity experiment, an s20,w value of 1.77 +/- 0.02S was obtained . A fast moving component which accounts for approximately 20% of the mass was also observed . Increasing the concentration of S9 does not alter the observed s20w value significantly for that component which could be followed . A detailed analysis of the data obtained at 3 degrees C from sedimentation equilibrium experiments on mixtures of the proteins indicated that a species of molecular weight greater than either of the two proteins was present . The proteins were found to interact with a mean equilibrium constant of association of 3.66 +/- 2.39 x 10(4) M-1 and a Gibbs free energy of interaction, delta Go = -5.8 kcal/mole at 3 degrees C in TMKD buffer . This information helps in understanding the energetics of the 30S ribosomal subunits of E . coli. Indian J Biochem Biophys, 1991 Oct-Dec, 28(5-6), 369 - 73 Polyclonal antibodies as probes to distinguish between tight and loose couple 50S ribosomes of Escherichia coli; Dey DN et al.; Antibody has been raised in rabbit against L7/L12 protein of E . coli 50S ribosomes and purified, finally through affinity column . A sensitive assay method using ELISA technique has also been standardised . LC 50S ribosomes react more with the antibody than TC 50S ribosomes . This supports the earlier physical data {Burma D P, Srivastava A K, Srivastava S, Tewari D S, Dash D & Sengupta S K, (1984), Biochem Biophys Res Commun, 124, 970} indicating that L7/L12 stalk region is protruded in medium in LC ribosomes and folded towards the body in TC ribosomes. Indian J Ophthalmol, 1991 Oct-Dec, 39(4), 148 - 50 Clinico-biochemical study of experimental complicated cataracts; Sihota R et al.; Clinically observed complicated cataracts, generally do not have a definite causal factor . We studied the effects of E . coli toxin injected suprachoroidally, to simulate the effect of toxins released by extraocular organisms on the lens . 79.2% of eyes had a definable cataract at the end of the 6th week of observation . The biochemical changes portrayed an increased oxidative activity in the lens, evidenced by a fall in glutathione concentration, and the consequent tertiary reorientation of proteins to increase insoluble proteins, forming a cataract. J Struct Biol, 1991 Oct, 107(2), 189 - 95 The structure of murine interleukin-1 beta at 2.8 A resolution; van Oostrum J et al.; The three-dimensional structure of recombinant murine interleukin-1 beta has been solved by X-ray crystallographic techniques to 2.8 A resolution and refined to a crystallographic R factor of 0.192 . Although murine interleukin-1 beta crystallizes in the same space group as human interleukin-1 beta with almost identical unit cell dimensions, the packing of the molecules is quite different . The murine interleukin-1 beta structure was solved by molecular replacement using the refined structure of human interleukin-1 beta as trial structure, and found to be related to the human structure by a nearly perfect twofold rotation about the crystallographic y-axis and a 14 degrees rotation about the z-axis, with no translation . The folding of murine interleukin-1 beta is similar to that found for the human variant, consisting of 12 beta strands wrapped around a core of hydrophobic side chains in a tetrahedron-like fashion . Significant differences with respect to the human structure are seen at the N terminus and in 4 of the 11 loops connecting the 12 beta strands. Chem Pharm Bull (Tokyo), 1991 Oct, 39(10), 2590 - 6 Synthesis of optically active lipopeptide analogs from the outer membrane of Escherichia coli; Kurimura M et al.; The synthesis of optically active lipopeptide derivatives has been accomplished by the use of chiral glycerol derivatives . Lipopeptide derivatives with (R)-glycerol moieties showed higher mitogenic activities than those with the (S)-configuration . N-2,2,2-Trichloroethoxycarbonyl lipopeptide derivatives increased mitogenic activity. Biull Eksp Biol Med, 1991 Oct, 112(10), 411 - 3 {Genetic region of incompatibility of the F-like plasmid pAP-18-1}; Buianova NI et al.; With help of molecular cloning the genetic region controlling incompatibility of plasmid pAP18-1 (Inc FXI) was localized in EcoR1-fragment f5 (3.6 MD) . The genetic region of incompatibility of its derepressed mutant pAP18-1drd (Inc FVII) is situated in EcoR1-fragment f2 (7,2 MD). J Protein Chem, 1991 Oct, 10(5), 495 - 501 Recombinant human hemoglobin: expression and refolding of beta-globin from Escherichia coli; Fronticelli C et al.; A plasmid analogous to the one described by Nagai and Thogersen (Nature, 309, 810-812, 1984) has been constructed for the expression of globins in E . coli . Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX-beta-globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg . This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when the pH is adjusted to 8.6 . Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic beta-globin which can be folded in the presence of cyanohemin and native alpha-chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin. Arzneimittelforschung, 1991 Oct, 41(10), 1108 - 12 {Immunomodulating effect of killed, apathogenic Escherichia coli, strain Nissle 1917, on the macrophage system}; Hockertz S; The influence of formaldehyde-killed Escherichia coli strain Nissle 1917 (SK 22) on macrophages of C57BL/6 mice was investigated in vitro . It has been shown that SK 22 activated macrophages derived from bone marrow produced Interleukin-6 with high efficiency . In addition, SK 22 stimulated macrophages to secrete tumor necrosis factor, as measured by a bioassay . Furthermore, macrophages were activated by SK 22 to produce a 3 fold amount of oxygen radicals compared to the spontaneous oxygen radical production . In contrast to this finding, the phagocytic capacity of these macrophages was only slightly increased . The specific lysis of P 815 tumor cells by peritoneal macrophages after coincubation with SK 22 was measured using tumor cells prelabelled with radioactive 51Cr . The results of the in vitro experiments presented clearly show that the E . coli preparation SK 22 is an efficient immunomodulator of the unspecific immune system. Arzneimittelforschung, 1991 Oct, 41(10), 1065 - 8 Inhibition of Escherichia coli DNA polymerase I catalysed DNA polymerization by trans-imidazolium-bisimidazoletetrachlororuthenate(III); Holler E et al.; The tumor-inhibiting metal complex trans-imidazolium-bisimidazoletetrachlororuthenate(III) (ICR) reacts with DNA and inhibits template-primer properties for DNA synthesis catalysed by Escherichia coli DNA polymerase I . The reaction with DNA depends on the aging (half-life 6.8 h) of the aqueous solution containing ICR . The kinetics of the reaction with DNA are reminiscent of those for cisplatin. Protein Eng, 1991 Oct, 4(7), 843 - 7 A new family of sugar-inducible expression vectors for Escherichia coli; Cagnon C et al.; A set of 11 expression vectors was constructed, each of them harbouring a cloning cassette under the control of the araB promoter . Some of these vectors enable expression of foreign proteins in the cytoplasm, while others include a synthetic sequence coding for a very efficient secretion signal sequence . Other features are an f1 origin of replication (in plus or minus orientation) and a promoter(up) mutation that enhances the already very high level of expression from these vectors . With such a versatile vector family, cloning, sequencing and site-directed mutagenesis can be performed on the same vector, and the level of expression can be defined according to the specific constraints of a given protein. Protein Eng, 1991 Oct, 4(7), 837 - 41 An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli; Takkinen K et al.; Single-chain antibodies consist of the variable, antigen-binding domains of antibodies joined to a continuous polypeptide by genetically engineered peptide linkers . We have used the flexible interdomain linker region of a fungal cellulase to link together the variable domains of an anti-2-phenyloxazolone IgG1 and show here that the resulting single-chain antibody is efficiently secreted and released to the culture medium of Escherichia coli . The yield of affinity-purified single-chain antibody is 1-2 mg/l of culture medium and its affinity and stability are comparable to those of the corresponding native IgG. Protein Eng, 1991 Oct, 4(7), 801 - 4 Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat; Mandecki W et al.; The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis . Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102 . When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat . Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity . In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0 . Up to 6-fold increases of the kcat/Km ratio were observed. Appl Biochem Biotechnol, 1991 Oct, 31(1), 37 - 41 Immunological and molecular genetic analysis of the cellulase component from Penicillium funiculosum; Sahasrabudhe NA; Following immunization of rabbits, the antiserum was initially analyzed for antiendoglucanase activity using dot-blot ELISA methods . When compared with the preimmune serum, the antiserum showed strong response even at 25 ng concentration . The specificity of the polyclonal antibodies, raised against the partially purified endoglucanase component of Penicillium funiculosum, was determined by Ouchterlony double diffusion . Rocket electrophoresis confirmed the presence of only two types of antigens in the serum . The two rockets obtained were attributable to endo I and the merging of immunologically identical (but mobility wise different) endo II and endo III . This antibody preparation was used as a probe . The deduced M1 of the cloned E . coli endo I was found to be 58 Kd by Western blotting. Virus Genes, 1991 Oct, 5(4), 377 - 80 Nucleotide sequence of pea seed-borne mosaic potyvirus coat protein gene; Johansen E et al.; cDNA of pea seed-borne mosaic potyvirus (PSbMV) RNA was synthesized and cloned in E . coli . Four overlapping clones that cover the complete PSbMV genome, except the extreme 5' terminus, were identified by restriction enzyme mapping, hybridization analysis, and partial sequencing . Overlapping cDNA clones covering 1386 nucleotides of the 3' terminus were sequenced . The nucleotide sequence contains one open reading frame (ORF), followed by an untranslated region of 163 nucleotides and a poly(A)-tract . The deduced amino acid sequence was found to include the C-terminus of the predicted RNA-dependent RNA polymerase and the coat protein . A glutamine-alanine dipeptide was identified as a putative 49-kD proteinase cleavage site at the N-terminus of the coat protein. Mol Microbiol, 1991 Oct, 5(10), 2557 - 68 Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or beta-galactosidase fused to the Hly C-terminal signal domain; Kenny B et al.; Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant . A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export . Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium . This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids . In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides . A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD . This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane . The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E . coli cytoplasmic proteins) . In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased . This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences . Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion . This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain. Mol Microbiol, 1991 Oct, 5(10), 2541 - 5 Role of plasmid multimers in mutation to tetracycline resistance; Boe L et al.; As an additional system for analysing mutations that appear to be specifically induced or directed, we have used a plasmid that contains the mnt repressor gene inserted as an operon fusion with the tet gene of the plasmid pBR322 . Thus, the mnt gene product acts as a negative transcriptional regulator of tet gene expression . Mutations inactivating the Mnt repressor are recessive while those destroying operator recognition (Oc) are dominant in conferring tetracycline resistance on the host . When resistance mutations were isolated on plates with high levels of tetracycline they were preferentially mnt- and the plasmids were monomers . Pre-exposure to low concentrations increased the frequency of resistant mutants by 100- to 1000-fold, and the mutations were now mostly Oc, located on one unit of a plasmid multimer . Recessive repressor mutations on one unit would not have been selected . We suggest that the high frequency of mutation in tandem multimeric plasmids may be caused by the formation of single-stranded and hence highly mutable regions by homologous pairing out of register . The role of tetracycline in promoting mutations is discussed. Mol Microbiol, 1991 Oct, 5(10), 2519 - 27 The hydrogenase structural operon in Rhodobacter capsulatus contains a third gene, hupM, necessary for the formation of a physiologically competent hydrogenase; Cauvin B et al.; The hupM gene, previously called ORFX, found downstream from and contiguous with the structural hydrogenase genes hupS and hupL in Rhodobacter capsulatus, is shown here to form a single hupSLM transcription unit with the two other genes . The hupM gene was inactivated by interposon mutagenesis . The two selected mutants, BCX1 and BCX2, which contained the kanamycin-resistance gene in opposite orientation, still exhibited hydrogenase activity when assayed with the artificial electron acceptors benzylviologen and methylene blue . However, the hydrogenase was not physiologically active in these mutants, which could not grow autotrophically and were unable to recycle electrons to nitrogenase or to respire on H2 . The hupM gene starts nine base pairs downstream from the TGA stop codon of hupL gene, which encodes the large subunit of the {NiFe}hydrogenase of Rhodobacter capsulatus . The three contiguous genes hupS, hupL and hupM were subcloned downstream from the promoter of hupSL, either with the promoter in the correct orientation (plasmid pBC8) or with the promoter in the opposite orientation (plasmid pBC9), then the constructs were introduced into the mutant strains . Only plasmid pBC8 could restore the formation of a competent hydrogenase in mutants BCX1 and BCX2, indicating that the hupM gene is expressed only from the hupSL promoter. Mol Microbiol, 1991 Oct, 5(10), 2481 - 91 Structural and genetic analysis of the bvg locus in Bordetella species; Arico B et al.; The bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence-associated genes in Bordetella species by a system similar to the two-component systems used by a variety of bacterial species to respond to environmental stimuli . We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis . The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA-binding activities . In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability . In total, we find 198 base-pair changes in the bvg loci of B . parapertussis and B . bronchiseptica relative to the bvg locus of B . pertussis . One hundred and seventy-three of these base-pair changes are identical in B . parapertussis and B . bronchiseptica . This confirms our previous observation that B . parapertussis and B . bronchiseptica are more related to each other than to B . pertussis . We have mapped the mutations that cause phase changes in B . bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene . The wild-type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B . bronchiseptica to wild-type levels, but it can do so when the bvgA gene on the plasmid is inactivated . This suggests that hyperexpression of bvgA down-regulates the bvg system. Mol Microbiol, 1991 Oct, 5(10), 2405 - 15 Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector; Blanco DR et al.; Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange . In order to provide a system for the identification of T . pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition . The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region . Library construction with Sau3A-digested T . pallidum genomic DNA resulted in the creation of functional T . pallidum-AP fusion proteins . Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences . Triton X-114 detergent phase partitioning of individual T . pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase . Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following {3H}-palmitate labelling, indicating their lipoproteinaceous nature . DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site . The DNA sequence of Tp75 also indicates that this is a previously unreported T . pallidum lipoprotein . T . pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified . DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain . Immunoblot analysis of T . pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa . {35S}-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein . DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites. Mol Microbiol, 1991 Oct, 5(10), 2391 - 403 Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli haemolysin; Stanley P et al.; We have carried out an extensive mutational analysis of the C-terminal signal which targets the export of the 1024-residue haemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium . Over 60 variants of the HlyA C-terminal 53-amino-acid sequence were created by oligonucleotide-directed mutagenesis and fused to the HlyA N-terminal 830 residues . Transport of the HlyA derivatives by the HlyB/HlyD system was compared with the wild-type level and the data indicate that the HlyA C-terminal export signal lies within the last 48 amino acids and comprises three functional domains: an amphipathic, charged helix between residues 1,977 and R,996; a 13-amino-acid uncharged region from residue T,997 to S,1009; and an 8-amino-acid hydroxylated tail at the extreme C-terminus . Analogous features were found in the C-terminal sequences of an extended family of haemolysins, leukotoxins and proteases which are secreted by HlyB/HlyD-type translocators . In particular, all nine proteins which are secreted into the extracellular medium possess potential extended amphipathic helices . These results suggest a possible role for multiple regions of the HlyA C-terminal export signal in which the first two domains span the membranes and the third domain remains in the cytoplasm. Mol Microbiol, 1991 Oct, 5(10), 2371 - 6 deoP1 promoter and operator mutants in Escherichia coli: isolation and characterization; Dandanell G et al.; Plasmid DNA containing deoP1, one of the two major promoters of the deo operon, has been mutagenized using hydroxylamine, and promoter down-mutations and operator mutations were selected . The isolated mutants are all located within a 16 bp palindromic sequence containing the -10 region of deoP1 . The results show that RNA polymerase and DeoR repressor compete for the same DNA target . The deoP1 promotor activity is dependent on a TG motif one base pair upstream of the -10 consensus sequence . The sequence of the deo operator site was further verified by use of a synthetic linker. Avian Dis, 1991 Oct-Dec, 35(4), 937 - 40 Escherichia coli challenge in chickens selected for high or low antibody response and differing in haplotypes at the major histocompatibility complex; Dunnington EA et al.; Relative sensitivity to Escherichia coli challenge was evaluated in white leg-horn chickens that had been selected for high antibody (HA) or low antibody (LA) response and that differed in haplotypes at the major histocompatibility complex (MHC) . Assessments were made of relative body-weight change and of heart and air-sac lesions after inoculation of 10(6), 10(5), or 10(4) E . coli via the posterior thoracic air sac . As has previously been reported, chicks from line HA were more sensitive to E . coli than those from line LA . Lesion scores were 1.58 +/- 0.12 in line HA and 1.02 +/- 0.12 in line LA (mean +/- S.E.), and ranged from 0.99 +/- 0.14 with the lowest dose of E . coli to 1.79 +/- 0.15 for the highest dose . Relative body-weight change to 72 hours after inoculation was greater in line LA (7.5 +/- 0.5) than in line HA (4.4 +/- 0.8) . There was no apparent resistance or susceptibility conferred to chickens in either the HA or LA genetic background as a result of haplotypes present at the MHC. Trends Biochem Sci, 1991 Oct, 16(10), 382 - 7 The structure of Ras protein: a model for a universal molecular switch; Wittinghofer A et al.; X-ray crystallography has revealed the molecular architecture of the cellular and oncogenic forms of p21Ha-ras, the protein encoded by the human Ha-ras gene, in both its active (GTP-bound) and in its inactive (GDP-bound) forms . From comparison of these two structures, a mechanism is suggested for the GTPase hydrolysis reaction that triggers the conformational change necessary for signal transduction . The structures have also allowed identification of the structural consequences of point mutations and the way in which they interfere with the intrinsic GTPase activity of p21ras . The p21ras structure is similar to that of the G-domain of elongation factor Tu (EF-Tu) from Escherichia coli, suggesting that p21ras can serve as a good model for other guanine nucleotide binding proteins. Trends Biochem Sci, 1991 Oct, 16(10), 358 - 62 Investigating protein conformation, dynamics and folding with monoclonal antibodies; Goldberg ME; Monoclonal antibodies with thoroughly characterized target specificities can be used as powerful probes of protein conformation . In addition to providing information on the relative arrangement of the domains in the native molecule, they can also be used to monitor both early and late stages of protein folding and conformational changes related to enzyme action. J Biochem (Tokyo), 1991 Oct, 110(4), 628 - 34 Synthesis of human big endothelin-1 by sequence-specific proteolysis of a fusion protein in Escherichia coli; Ohashi H et al.; Three DNA constructs, pETB-40, 41, and 42, encoding human big endothelin-1 (ET-1) preceded by the specific recognition sequence (Ile-Glu-Gly-Arg) for the activated blood coagulation factor Xa (FXa), fused in frame to the N-terminal portion of beta Gal, were expressed in Escherichia coli . The fusion proteins, pETB-40P, 41P, or 42P, consisted of the 55-, 51-, or 42-aa N-terminal peptide of beta Gal and the 38-aa of big ET-1, and had 1, 0, or 0 Cys residues and 5, 5, or 1 Arg residues in the N-terminal peptide of beta Gal, respectively . Enzymatic cleavage of the purified fusion proteins by FXa or trypsin allowed the recovery of authentic human big ET-1 . The rates of conversion of pETB-40P, 41P, and 42P to big ET-1 by FXa digestion were 5.6, 11.2, and 30.0%, respectively . pETB-40P with a deletion of one Cys residue and four Arg residues in the N-terminal part was a better substrate than the other two for FXa or trypsin in the production of big ET-1. Biokhimiia, 1991 Oct, 56(10), 1832 - 9 {Oligomeric forms of recombinant interleukin-2}; Meriin AB et al.; Human recombinant interleukin 2 produced by Escherichia coli gives rise to oligomeric forms that are stable to complete denaturation . The appearance of these forms is preceded by the formation of oligomers sensitive to reduction . These processes depend on the cell status and seem to be associated with aggregation of the reaction product. Biokhimiia, 1991 Oct, 56(10), 1731 - 47 {Biochemical kinetics of plasmid replication processes}; Varfolomeev SD et al.; Two groups of plasmid replication reactions occurring during the growth of cell populations were kinetically characterized: 1) short-term intermediate processes coupled with changes in plasmid concentrations in host cells and occurring in the first few generations; 2) long-term processes resulting in the loss of plasmids and occurring during prolonged cultivation of cells . It was shown that first group processes are determined by the ratios of plasmid replication rates relative to cell growth . Using growing populations of E . coli as examples, it was shown that the shape of kinetic curves reflecting the plasmid copy number depends on the nature of their replicon . In this case plasmids with ColE1 replicon exhibit kinetic behaviour of the first type (temporal loss of plasmid copy number in the cells), whereas plasmids with p15 replicon display the second type behaviour consisting in the decrease of the plasmid copy number followed by its short-term drastic increase . For both types of kinetic behaviour a kinetic description adequately reflecting these processes is given . It is supposed that the loss of plasmids during long-term cultivation of cell populations may be associated with both segregation (lower growth rates of plasmid-containing cells in comparison with cells containing no plasmids) and mutation mechanisms . Kinetic descriptions of both mechanisms are proposed and principles of their discrimination are formulated . Practical recommendations for the prevention of the plasmid loss by the cells that are based on theoretical data are given. Nutr Clin Pract, 1991 Oct, 6(5), 193 - 6 Total parenteral nutrition in a premature rhinoceros calf; Herrmann VM et al.; A female black rhinoceros calf developed significant hypoglycemia (blood glucose, 30 mg/dL) and hypothermia (97 degrees F) within 48 hours of birth and refused to nurse . Normal gestation of the black rhinoceros is 15 months, but elongated hoof slippers and low birth weight (30 kg) suggested prematurity in this calf . Clinical symptoms of neonatal sepsis including lassitude and poor sucking continued in spite of the aggressive use of antibiotics, and the calf required mechanical ventilatory support on day 7 . Nutritional support including enteral gavage feedings (Pedialyte/4 ounces of SMA {Wyeth Ayerst} with sucraflox) had been instituted and was supplemented with total parenteral nutrition on day 5 . Central venous access was obtained via a jugular cutdown . The total parenteral nutrition included appropriate electrolytes and vitamins for the neonatal calf but did not include trace elements . The use of total parenteral nutrition by our zoos for therapeutic purposes is increasing . Experience with total parenteral nutrition in exotic animals such as the black rhinoceros is limited, yet this may be an important therapeutic modality in these animals, particularly those in danger of extinction. J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2409 - 14 Cloning and expression in Escherichia coli of a recA homologue from Mycobacterium tuberculosis; Nair S et al.; A 3.8 kb PstI fragment of Mycobacterium tuberculosis was cloned in a recA-deleted Escherichia coli by selecting transformants with increased EMS resistance . The cloned fragment restored homologous recombination in Hfr crosses and conferred resistance to long wave (302 nm) but not short wave (254 nm) UV light . E . coli containing the 3.8 kb PstI fragment produced a 38-40 kDa protein which cross-reacted with antibodies raised against the E . coli RecA protein . The cloned DNA thus probably encodes a RecA homologue. Lymphokine Cytokine Res, 1991 Oct, 10(5), 343 - 6 Enhanced release of TNF-alpha, but not IL-1 beta, from uremic blood after endotoxin stimulation; Powell AC et al.; Aberrant immunologic host defenses associated with uremia may be a cause of the high incidence of sepsis in chronic hemodialysis (CHD) patients . This investigation determined the cytokine response of blood from five nondialyzed chronic renal failure (CRF) patients, five CHD patients, and five healthy controls (HC) after in vitro stimulation with 1 ng/ml Escherichia coli 0113 endotoxin . Concentrations of the cytokines TNF-alpha and IL-1 beta were determined by ELISA and were similar in all baseline and unspiked samples . TNF-alpha concentrations in CRF and CHD spiked samples were similar to each other but significantly greater (p less than 0.01) than in HC spiked samples . IL-1 beta concentrations in CRF, CHD, and HC-spiked samples were not significantly different . We conclude that CRF and CHD patients have enhanced TNF-alpha response, which may be related to uremia and not dialysis-related factors . Uremia does not potentiate IL-1 beta release. Mol Biochem Parasitol, 1991 Oct, 48(2), 121 - 30 Cloning and characterisation of an immunodominant major surface antigen of Echinococcus multilocularis; Frosch PM et al.; A lambda gt11 cDNA expression library from mRNA of Echinococcus multilocularis protoscolices has been constructed in Escherichia coli Y1090 . Immunoscreening with pooled sera obtained from patients suffering from E . multilocularis disease revealed 5 reactive clones . By partial DNA sequence comparison all clones proved to encode the same gene . The complete cDNA sequence of the clone pEM10 with the largest insert of 2.2 kb was determined and an open reading frame of 1.7 kb could be described . The derived amino acid sequence shares 42.6% identity with human microvillar cytovillin found in the membranes of placenta and carcinoma tissues . The coding region of the cDNA of pEM10 was amplified by polymerase chain reaction (PCR) and cloned in frame into expression vector pGEX-3X . Immunoblot analysis revealed the expression of a recombinant antigen of 65 kDa and a protein with the same molecular weight was also found in the lysate of E . multilocularis protoscolices . In contrast, the protein was absent from hydatid fluid or larvae of Echinococcus granulosus . By means of immunofluorescence studies this immunodominant antigen could be located in the germinal layer of brood capsules and in the tegument of E . multilocularis protoscolices . The fusion protein was purified and used for diagnostic purposes in immunoblot . The diagnostic value of this antigen is discussed. J Comp Pathol, 1991 Oct, 105(3), 323 - 30 Increased susceptibility of aged rats to haemorrhage and intravascular hypercoagulation following endotoxin administered in a generalized Shwartzman regime; Carthew P et al.; Ageing rats are known to have an increased incidence of myocardial fibrosis and dyspnoea caused by pulmonary intravascular coagulation . In order to determine whether endotoxin can be responsible for such responses in ageing rats we have exposed rats of differing ages (2 months, 16 months and 24 months) to single or repeated (two doses 24 h apart; generalized Shwartzman regime) intravenous doses of endotoxin (E . coli 0111 B4) . Only the 2-year-old rats reacted adversely . Two doses of endotoxin produced death, with focal myocardial necrosis, haemorrhage and pulmonary and hepatic intravascular coagulation . The increased susceptibility of aged rats to the toxic effects of endotoxin explains some of the changes found in the tissues of old rats . The sporadic nature of both cardiac failure and dyspnoea as a cause of morbidity and mortality in ageing rats may be related to the need for two endotoxin episodes in a period of 24 h to provoke a generalized Shwartzman reaction, an occurrence likely to be relatively uncommon under natural conditions. Vaccine, 1991 Oct, 9(10), 715 - 22 Physicochemical and immunological characterization of recombinant host-protective antigen (VP2) of infectious bursal disease virus; Azad AA et al.; Small fusions to the N-terminal end of the host-protective antigen (VP2) of infectious bursal disease virus lead to stable expression of VP2 in Escherichia coli and yeast, and reduce the levels of inclusion body formation in E . coli in comparison to VP2 constructs with larger N-terminal fusions . VP2 produced with small N-terminal fusions, like native viral VP2, can be fractionated into a high molecular weight 'multimeric' form and a monomeric form . A virus-neutralizing monoclonal antibody that only recognizes undenatured VP2 preferentially reacts with multimeric forms of recombinant VP2 . Both native and recombinant monomeric forms of VP2 are non-immunogenic . The multimeric forms of viral and yeast-derived VP2 are highly immunogenic, while those produced in E . coli are not. Mol Gen Mikrobiol Virusol, 1991 Oct, (10), 19 - 22 {Mutagenesis, induced by phosphotriester analogs of oligonucleotides and directed to the cleavage site of double-spiral DNA}; Petrenko VA et al.; The mutagenic properties of phosphotriester analogues revealed in course of interaction with linearized plasmid DNA were studied . The plasmid-based model system permitting one to test reliably the induced mutations is proposed . The efficiency of mutagenesis was shown to depend on the length of the oligonucleotide-mutagen and the genotype of the transformed Escherichia coli strain . The possible mechanisms involved in mutagenesis are discussed. J Appl Physiol, 1991 Oct, 71(4), 1376 - 81 Thromboxane synthase inhibition and cardiopulmonary function during endotoxemia in sheep; Fujioka K et al.; We studied the cardiopulmonary response to endotoxin (lipopolysaccharide, LPS) in sheep with and without the administration of a thromboxane synthase inhibitor, OKY-046 . The animals were instrumented for crystalographic dimension analysis of the left ventricle (LV) and for measurement of LV, aortic, left atrial, and pulmonary arterial pressures and cardiac index, as well as lung lymph flow . They received 1.0 micrograms/kg of Escherichia coli LPS with (n = 8) and without (n = 8) OKY-046 (10 mg/kg bolus, then 10 micrograms.kg-1.min-1) . OKY-046 prevented the increase of pulmonary arterial pressure and the decrease of cardiac index that occurred during the early phase of endotoxemia . Between 8 and 12 h after LPS, cardiac index increased from 6.8 +/- 0.7 to 8.9 +/- 0.51.min-1.m-2 . Concomitantly, the end-systolic pressure-diameter relationship (ESPDR, sensitive myocardial contractility index) significantly decreased from 14.7 +/- 0.6 to 7.7 +/- 0.7 . Other indexes of the LV contractility (+dP/dtmax) were also reduced . OKY-046 prevented the decreases of ESPDR and +dP/dtmax . OKY-046 also attenuated the increased lung lymph flow changes seen with LPS. Bioessays, 1991 Oct, 13(10), 515 - 25 New enzymes for old: redesigning the coenzyme and substrate specificities of glutathione reductase; Perham RN et al.; A set of amino acid side chains that confer specificity for the coenzyme NADPH and the substrate glutathione in the flavoprotein disulphide oxidoreductase, glutathione reductase, has been identified . Systematic replacement of these amino acid residues in the coenzyme-binding site switches the specificity of the enzyme from its natural strong preference for NADPH to a marked preference for NADH . The amino acids replaced all lie in a structural motif within the dinucleotide-binding domain of the protein . Since this domain is a feature common to most dehydrogenases (reductases) that use nicotinamide coenzymes, it may be that the coenzyme specificities of all such enzymes can be manipulated in this way . Similarly, amino acid residues involved in the selective recognition of trypanothione by trypanothione reductase, an enzyme related to glutathione reductase and exclusive to trypanosomatids, were identified . Suitable mutation of the corresponding residues in E . coli glutathione reductase switched its substrate specificity towards trypanothione . A better understanding of the substrate specificity of these enzymes could open up a route to the chemotherapy of trypanosomal infections. Dtsch Tierarztl Wochenschr, 1991 Oct, 98(10), 395 - 8 The influence of colostral leukocytes on the immune system of the neonatal calf . IV . Effects on bactericidity, complement and interferon; synopsis; Riedel-Caspari G et al.; The influence of colostral leukocytes on the bactericidity of whole blood of calves against a strain of E . coli and on the activities of haemolytic complement and interferon-alpha (the antiviral activity of sera resisting an acidic treatment at pH 2 for 6 h) in the serum was investigated during a period of 4 weeks using 4 experimental groups . The calves received either complete colostrum (COL+, n = 16), cell-depleted colostrum (COL-, n = 16), cell-supplemented milk substitute (MS+, n = 7) or pure milk substitute (MS-, n = 6) during their first three days of life . The bactericidity of whole blood of the COL+ group was significantly higher on the second and third days of life while the activity of haemolytic complement was lower after the first week as compared to the COL- group . No interferon-alpha was detectable in the sera of both COL groups . The bactericidity of the MS groups was significantly lower than that of the COL groups after the first day of life . It was significantly lower in the MS+ group after one week of life while the activity of haemolytic complement was higher than that of the MS- group . Three out of 5 MS- and only one out of 7 MS+ calves had low titres of interferon-alpha in their sera on the third day . Three out of 6 MS- calves died and 5 out of 7 MS+ animals . The mean day of death was 4.0 in the MS- and 8.4 in the MS+ group . Based on the in vitro results of this and the previous three communications it can be concluded that leukocytes which are an integral part of normal bovine colostrum, influence immunological reactions of the calf and that they may enhance its defence against infection . Colostral leukocytes in the absence of humoral components of the colostrum are not able to prevent fatal losses in the calves due to natural infection, although their influence on immune responses of the calves was detectable in vitro. J Am Soc Nephrol, 1991 Oct, 2(4), 885 - 93 Chloride and membrane potential dependence of sodium ion-proline symport; Chesney RW et al.; Proline accumulation by renal proximal tubule brush border membrane vesicles is Na+ dependent, but little is known about the role of anions or membrane potential on proline uptake . Recent studies in a variety of transport systems, including rat renal brush border membrane vesicles, indicate that halide anions chloride (Cl-) and bromide (Br-) are essential for glycine, beta-alanine, gamma-aminobutyric acid, and taurine uptake, so the possibility that Na(+)-proline symport is Cl- dependent was explored . Also, the role of membrane potential on transport was assessed by determining the effect of external anions with different membrane permeabilities . The ratio of initial rate Cl- stimulated to thiocyanate (SCN)(-)-stimulated uptake values serves to measure Cl- dependence . The initial rate of proline uptake to equilibrium value was 3.11 +/- 0.5 (SE) in the presence of Cl- versus SCN- . The ratio for D-glucose, whose uptake is governed only by electrogenic status of the membrane, was 0.61 +/- 0.47 (P less than 0.001 versus proline) . In another series of experiments, uptake values for various anions as a percent of equilibrium (I/E x 100) were: SCN-, 84.9 +/- 10.9; NO3, 49.9 +/- 11.0; SO4(2-), 27.3 +/- 4.4; F-, 68.5 +/- 18.3; Cl-, 164.1 +/- 44.6; Br-, 150.6 +/- 30.2; I-, 56.7 +/- 13.5 . The stoichiometry of uptake by Hill plot analysis of proline uptake in the presence of varying concentrations of Na+ (0 to 100 mM) and Cl- (0 to 100 mM) was 2Na+:1Cl-:1 proline.(ABSTRACT TRUNCATED AT 250 WORDS) Comput Appl Biosci, 1991 Oct, 7(4), 447 - 56 Improved algorithms for searching restriction maps; Miller W et al.; We present algorithms for searching a DNA restriction enzyme map for a region that best matches a shorter 'probe' map . Our algorithms utilize a new model of map alignments, and extensive experiments prove our model superior to earlier approaches for certain applications . Let M be the number of map sites and P be the number of probe sites . Our first algorithm, which optimizes only over a restricted class of alignments, requires O(MP log P) worst-case time and O(M + P) space . Our second algorithm, which optimizes over all alignments, runs in O(MP3) time and O(M + P2) space, under reasonable assumptions about the distribution of restriction enzyme cleavage sites . Combining the algorithms gives a map-searching method that optimizes over all alignments in O(MP log P) time in practice . The algorithms' effectiveness is illustrated by searches involving a genomic restriction map of Escherichia coli. Appl Environ Microbiol, 1991 Oct, 57(10), 2995 - 9 Deletion of pgi alters tryptophan biosynthesis in a genetically engineered strain of Escherichia coli; Mascarenhas D et al.; Deletion of the structural gene for phosphoglucose isomerase (pgi) of Escherichia coli dramatically alters the path of glucose catabolism by diverting carbon into the hexose monophosphate shunt . The effect of this genetic alteration on the conversion of glucose to tryptophan by strains optimized for the biosynthesis of this amino acid was determined by using 13C-nuclear magnetic resonance spectroscopy in vivo . Pgi- strains converted glucose to tryptophan almost twice as efficiently as did their Pgi+ counterparts. Appl Environ Microbiol, 1991 Oct, 57(10), 2896 - 900 Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides; Bunkers GJ; The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene . The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E . coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa . A lower transformation rate was obtained with the bml system than with the hph system . Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker . The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation . The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants . Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number . Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity . These results form the basis for construction of a versatile and sensitive reporter gene system for P . herpotrichoides. Appl Environ Microbiol, 1991 Oct, 57(10), 2888 - 90 Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants; Lee N et al.; A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells . Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized . By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated . The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites . Escherichia coli cells transformed by these plasmids were subject to large-scale analysis . One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence. Vet Microbiol, 1991 Oct, 29(2), 195 - 7 Isolation of enterotoxigenic Escherichia coli from camels with diarrhoea; Chauhan RS et al.; E . coli serogroups 02, 08, 083, 0103 and 0120 were isolated from seven camels with diarrhoea of which 02, 08, and 083 were found to be enterotoxigenic on rabbit ligated ileal loop test . Out of 125 apparently healthy camels, 75 strains of E . coli were isolated . The majority of isolates were susceptible to gentamycin, nitrofurantoin, trimethoprim plus sulphonamide, neomycin, kanamycin and chloramphenicol. J Dairy Sci, 1991 Oct, 74(10), 3407 - 11 Reduced lactational performance following intravenous endotoxin administration to dairy cows; Shuster DE et al.; Nonpregnant lactating cows were given 100 micrograms of endotoxin via the jugular vein to determine effects of intravenous endotoxin administration on mammary inflammation and lactational performance . At the first milking (11 h) posttreatment, milk yield was reduced 33% . Milk fat percentage was elevated at this time, but lactose concentration was decreased . Milk yield and composition returned to pretreatment levels within 2 d . Clinical mastitis was not induced by endotoxin treatment, but milk SCC, NAGase, serum albumin, and lactoferrin were increased by 50% . This increase was small compared with increases during mastitis and may have resulted from lower milk volume . These results support the hypothesis that part of the reduced lactational performance during endotoxin mastitis is mediated by systemic pathophysiological responses and indicate that intravenous endotoxin administration may be a useful model to study adverse effects of infectious disease on lactational performance. Int J Food Microbiol, 1991 Oct, 14(1), 27 - 41 Assessment of the hygienic adequacy of a commercial hot boning process for beef by a temperature function integration technique; Reichel MP et al.; The hygienic performance of a commercial hot boning process for beef carcasses was assessed by a temperature function integration technique . The potential proliferation of Escherichia coli was calculated from 50 temperature histories for the persistently warmest, microbially contaminated regions of product passing through both the carcass cutting and carton cooling phases of the process . The maximum calculated proliferation was similar to, but the average proliferation was more than, the respective values previously obtained for a beef side cooling process that complied with Good Manufacturing Practice . After upgrading of the carton cooling facility the process was re-assessed . Then, for a sample of 50 temperature histories, the maximum proliferation was less than, and the average proliferation was similar to, the respective values for the side cooling process . Observed proliferations of E . coli inocula in cooling cartons of product were compared with the proliferations calculated from temperature histories obtained from sites close to inocula . The pairs of calculated and observed values mostly agree within +/- 1 generation. J Biomol Struct Dyn, 1991 Oct, 9(2), 233 - 8 Conformation of DNA-DNA polymerase I complex observed by scanning tunneling microscopy; Lu CD et al.; The conformation of a complex of a 41 mer/31 mer DNA fragment and the Klenow fragment of DNA polymerase I of Escherichia coli was studied by scanning tunnelling microscopy (STM) . The results shows that near two turns of double helix of this DNA fragment was outside of enzyme while another part containing more than one turn of helix and 10 nucleotides single strand was combined with enzyme . The dimension and shape of DNA polymerase I (KF) in complex were different from that of free enzyme . The conformation of DNA-DNA polymerase I (KF) complex and the application of STM in studying structure of complex of DNA polymerase with DNA were discussed. J Struct Biol, 1991 Oct, 107(2), 136 - 45 A common channel-forming motif in evolutionarily distant porins; Pauptit RA et al.; Four new crystal packings of Escherichia coli porins are presented (phosphoporin, maltoporin, and two crystal forms of matrix porin) . These were determined by molecular replacement methods using a polyalanine trial model acquired from the refined coordinates of porin from Rhodobacter capsulatus . The successful molecular replacement shows that the dominant motif found in R . capsulatus porin (a 16-stranded antiparallel beta-barrel) also applies to the E . coli porins, despite the lack of significant amino acid sequence homology . A 30 degrees-40 degrees tilt of the beta-strands with respect to the membrane normal was derived from the intensity distributions in the X-ray diffraction patterns for each porin studied, stressing their similarity . In view of the evolutionary distance between enteric and photosynthetic bacteria, the antiparallel beta-barrel may have significance as a basic structural motif for the formation of bacterial membrane channel structures. Mol Microbiol, 1991 Oct, 5(10), 2511 - 8 Analysis of the topology of the cytochrome d terminal oxidase complex of Escherichia coli by alkaline phosphatase fusions; Newton G et al.; The cytochrome d complex of Escherichia coli is a heterodimer located in the bacterial cytoplasmic membrane, where it functions as a terminal oxidase of the aerobic respiratory chain . The topology of each of the two subunits of the cytochrome d complex was analysed by the genetic method involving alkaline phosphatase gene fusions . These fusions were generated by both an in vivo method using the transposon TnphoA and an in vitro method of construction . A total of 48 unique fusions were isolated and the whole-cell alkaline phosphatase-specific activities were determined . Data from these fusions, in combination with information from other studies, provide the basis for two-dimensional models for each of the two subunits, defining the way in which the subunits fold in the inner membrane of E . coli. J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2361 - 74 Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry; Mackey BM et al.; Thermograms of whole cells of Escherichia coli obtained by differential scanning calorimetry contained ten main peaks (denoted f, l, m1, m2, m3, n, p, q, r and s) occurring at temperatures of approximately 25, 54, 61, 71, 76, 81, 95, 105, 118 and 124 degrees C, respectively . After cooling to 5 degrees C and reheating, peaks denoted fr, mr and pr were observed at 23, 73 and 94 degrees C, respectively . By examining thermograms of different cell fractions we have identified the following thermal denaturation events . During primary heating there is a broad endotherm (f) beginning below 20 degrees C and extending to just above 40 degrees C that is caused by melting of membrane lipids . Superimposed on this is an exothermic process associated with a change of state of the peptidoglycan . The first irreversible denaturation event occurs just above 47 degrees C, associated with the onset of denaturation of the 30S ribosomal subunit and soluble cytoplasmic proteins . Ribosome melting is a complex process occurring between 47 and 85 degrees C and is characterized by peaks m1, m2 and n . Peak m3 at 75-76 degrees C is of unknown identity but may possibly represent melting of tRNA . Peak p at 95 degrees C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component . Peak q at 105 degrees C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component . The complex events denoted r and s at 118 and 125 degrees C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA . Following cooling and reheating there is a broad endotherm with a maximum at 23 degrees C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and 100 degrees C caused by the remelting of ribosomal RNA . Peak pr at 94 degrees C is caused by the melting of reannealed DNA . Additional features not appearing in whole cells were evident in some cell fractions . These observations should allow us to distinguish events that may lead to loss of viability from those that do not. Mol Reprod Dev, 1991 Oct, 30(2), 90 - 4 Expression of SV40-lacZ gene in mouse preimplantation embryos after pronuclear microinjection; Takeda S et al.; In order to study the expression of an exogenous gene in developing mouse embryos during the preimplantation period, DNA carrying the SV40 early promoter fused with the Escherichia coli beta-galactosidase gene (lacZ) was microinjected into the pronucleus of fertilized mouse eggs . Expression of lacZ gene was detected by staining embryos with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a substrate at pH 7.2 . The embryos expressing the lacZ gene showed various intensities of blue staining, all showing a mosaic pattern . The exogenous gene was expressed from the 4-cell stage until the blastocyst stage . The proportion of embryos expressing the lacZ gene was maximal (38%) at the morula stage, and the expression was dependent on the presence of the SV40 promoter. Mol Gen Genet, 1991 Oct, 229(3), 453 - 9 Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12; Hogg RW et al.; The nucleotide sequence of the Escherichia coli K12 beta-methylgalactoside transport operon, mgl, was determined . Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence . The operon contains three open reading frames (ORF) . The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide . Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids . The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site . The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore . The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E . coli K12 chromosome. J Clin Invest, 1991 Oct, 88(4), 1370 - 8 LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase; Manns MP et al.; LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase . The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli . 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD . PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1) . Antibodies for HSV-1 proteins were detected by ELISA in 17 of 20 LKM-1 sera tested . An immobilized, synthetic peptide, DPAQPPRDC, was used to purify LKM-1 antibodies . Affinity purified LKM-1 autoantibodies react on immunoblots with a protein in BHK cells after infection with HSV-1 . 11 of 24 LKM-1 sera, including 3 that recognize DPAQPPRD, also exhibit antibodies to the hepatitis C virus (HCV) protein, C100-3 . Affinity purified LKM-1 antibodies did not recognize C100-3 . However, partial sequence identity was evident between portions of the immunopositive 33-amino acid segment of P450IID6 and other portions of the putative HCV polyprotein . Immune cross-recognition of P450IID6 and HCV or HSV-1 proteins may contribute to the occurrence of LKM-1 autoantibodies. J Bacteriol, 1991 Oct, 173(19), 6249 - 57 Physiological effects of the fructose-1,6-diphosphate aldolase ts8 mutation on stable RNA synthesis in Escherichia coli; Singer M et al.; The conditional lethal mutations ts8 and h8 are located in fda, the gene encoding aldolase, and they inhibit RNA synthesis upon shift to the nonpermissive temperature . We demonstrate that both mutations preferentially inhibit stable RNA synthesis and that this inhibition occurs at the level of transcription initiation . The susceptibility of a promoter to the inhibitory effects of ts8 is correlated with the ability of the promoter to be growth rate regulated . This effect is independent of relA and spoT function . Inhibition is dependent upon glucose metabolism past the generation of glucose-6-phosphate; however, the mechanism of this effect is unknown. J Clin Endocrinol Metab, 1991 Oct, 73(4), 857 - 60 Thyroperoxidase, but not the thyrotropin receptor, contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector; Libert F et al.; The sequential epitopes on the human thyroperoxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease were investigated using a recombinant DNA technique . Previous studies led to the isolation of two overlapping cDNA clones that encode polypeptides of TPO (85 residues, C2; 100 residues, C21) recognized by sera from several patients with autoimmune disease that contained antimicrosomal autoantibodies . In this report the vector pUEX1 was used to clone and express small random fragments of TPO cDNA in Escherichia coli as a beta-galactosidase fusion protein . Colonies were screened with a serum from a patient with Hashimoto's thyroiditis, and immunoreactive peptides were identified by sequencing the corresponding DNA inserts . Two linear epitopes of human TPO (amino acids 590-622 and 710-722) were recognized by the autoantibodies . This confirmed our previous results and provide a more precise localization of the antigenic determinants involved . The same approach has been applied in an attempt to identify the binding site(s) for autoantibodies on the human TSH receptor . In contrast to the data obtained with TPO, sera from patients with blocking (from idiopathic myxoedema) or stimulating (from Graves' disease) activity did not recognize the linear TSH receptor peptide fragments generated in our libraries. Virology, 1991 Oct, 184(2), 805 - 7 Localization of a VP3 epitope of infectious bursal disease virus; Jagadish MN et al.; An immunodominant region of VP3, one of the two structural proteins of infectious bursal disease virus (IBDV strain 002-73), has been mapped by restriction site-specific deletion analysis and subcloning in Escherichia coli, followed by immunoblot analysis of the synthesized products . The epitope located within 58 amino acids reacted very strongly with a mouse monoclonal antibody (MAb 17/80) raised against IBDV 002-73 . This immunodominant region may be useful in serodiagnosis of IBDV infection in poultry. J Biochem (Tokyo), 1991 Oct, 110(4), 583 - 7 Molecular chaperon produced by an intracellular symbiont; Kakeda K et al.; Symbionin, that is selectively produced by an intracellular symbiont harbored by the aphid bacteriocyte, is structurally homologous to the Escherichia coli groEL protein, a heat shock protein functioning as a molecular chaperon . It was shown that symbionin has ATPase activity and, in the presence of Mg-ATP, is converted into lower molecular mass species . Like the groEL protein, symbionin was able to reconstitute dimeric ribulose 1,5-bisphosphate carboxylase/oxygenase holoenzyme from its unfolded subunits in vitro, suggesting that this protein functions as a molecular chaperon in the endosymbiont . The groES-homologous protein did exist in the endosymbiont, but its amount was small relative to that of symbionin. Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8779 - 83 High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme; Wang YH et al.; A rat cDNA containing the complete coding sequence for rat tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) was isolated from a rat PC12 cDNA library and subcloned in a bacterial expression plasmid, and large amounts of functional enzyme were produced in Escherichia coli . The recombinant enzyme was purified approximately 20-fold to a final specific activity of 1.8 mumol/min per mg of protein, with a yield of 30% . As much as 1 mg of pure protein could be obtained from 1 g of wet bacterial cells . The purified hydroxylase was shown to be homogeneous by denaturing polyacrylamide electrophoresis and isoelectric focusing . Amino acid analysis of the N terminus (25 residues) revealed 100% identity with rat PC12 tyrosine hydroxylase, as deduced from its cDNA sequence . Several of the kinetic properties of the recombinant enzyme resembled those of the native PC12 hydroxylase . However, in contrast to the native enzyme, the purified recombinant hydroxylase was shown to be in an activated form . Phosphorylation with cAMP-dependent protein kinase resulted in stoichiometric incorporation of phosphate, but the kinetic profile of the recombinant enzyme was unaffected . Several clues to these differences are considered that may provide insight into the structural features important to the regulation of tyrosine hydroxylase. Mol Gen Genet, 1991 Oct, 229(2), 285 - 91 Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli; Pedersen PA et al.; Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli . The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein . Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon . The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae . One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein . Such fusion proteins might be useful in the design of recombinant vaccines. J Bacteriol, 1991 Oct, 173(20), 6383 - 9 Cloning, sequence analysis, and functional expression of the acetyl coenzyme A synthetase gene from Methanothrix soehngenii in Escherichia coli; Eggen RI et al.; In the acetoclastic methanogen Methanothrix soehngenii, acetate is activated to acetyl coenzyme A by acetyl coenzyme A synthetase (Acs) . The acs gene, coding for the single Acs subunit, was isolated from a genomic library of M . soehngenii DNA in Escherichia coli by using antiserum raised against the purified Acs . After introduction in E . coli, the acs gene was expressed, resulting in the production of an immunoreactive protein of 68 kDa, which is approximately 5 kDa smaller than the known size of purified Acs . In spite of this difference in size, the Acs enzymes are produced in similar quantities in E . coli and M . soehngenii and show comparable specific activities . Upstream from the acs gene, consensus archaeal expression signals were identified . Immediately downstream from the acs gene there was a putative transcriptional stop signal . The amino acid sequence deduced from the nucleotide sequence of the acs gene showed homology with those of functionally related proteins, i.e., proteins involved in the binding of coenzyme A, ATP, or both. J Bacteriol, 1991 Oct, 173(20), 6355 - 63 Role of multiple environmental stimuli in control of transcription from a nitrogen-regulated promoter in Escherichia coli with weak or no activator-binding sites; Schneider BL et al.; Nitrogen regulator I (NRI {or NtrC})-phosphate stimulates transcription from the glnAp2 promoter of the glnALG operon in enteric bacteria . Unlike most activators, NRI-phosphate can stimulate transcription without apparent activator binding sites . We observed that when lacZ was controlled by a minimal glnAp2 promoter (without NRI binding sites) in Escherichia coli, lacZ expression was regulated by two different stimuli, the nitrogen status of the medium and the particular amino acid used as a nitrogen source . The latter stimulus did not affect the activity of the wild-type glnAp2 promoter, which has two high-affinity NRI binding sites . We present several lines of evidence that suggest that the concentration of NRI-phosphate limits the activity of the minimal glnAp2 promoter in vivo . Our results also suggest that nitrogen regulator II-dependent phosphorylation of NRI cannot account for the proposed variations in the concentration of NRI-phosphate . Therefore, to account for the regulation of the minimal glnAp2 promoter by two environmental stimuli, we propose that at least two protein kinases phosphorylate NRI during nitrogen-limited growth . We isolated and characterized mutants in which NRI could not stimulate transcription from the minimal glnAp2 promoter but could activate transcription from the wild-type glnAp2 promoter . These mutants could not utilize arginine or proline as a nitrogen source, suggesting that degradation of some nitrogen sources may require transcription from promoters similar to the minimal glnAp2 promoter. FASEB J, 1991 Oct, 5(13), 2785 - 91 Guanylyl cyclases, a growing family of signal-transducing enzymes; Koesling D et al.; Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases . So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified . These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain . In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1) . Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases . Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme . Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase . Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms . The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase. J Infect Dis, 1991 Oct, 164(4), 693 - 703 Characterization of interactions of enteropathogenic Escherichia coli O127:H6 with mammalian cells in vitro; Francis CL et al.; Previous studies have identified two bacterial factors involved in enteropathogenic Escherichia coli (EPEC) infection . A plasmid-mediated EPEC adherence factor (EAF) is responsible for initial and localized adherence . A chromosomally encoded E . coli attachment and effacement factor (eae) is involved in effacement of the eukaryotic cell surface and characteristic "pedestal" formation . By using isogenic strains deficient in either EAF, eae, or both, the process of EPEC adherence and entry in vitro was examined . While EAF proved necessary and sufficient for efficient bacterial association with HEp-2 cells, both EAF and eae were required for efficient effacement of and entry into these cells and other cultured cell lines . Invasion mediated by eae was markedly inhibited by cytochalasin D and colchicine . Afimbrial adhesion or type I pili from uropathogenic strains of E . coli substituted for EAF in EAF-Eae+ strains to provide initial adherence to HEp-2 cells and to facilitate actin condensation. Biochemistry, 1991 Oct 1, 30(39), 9421 - 9 Differential scanning calorimetry study of reversible, partial unfolding transitions in dodecameric glutamine synthetase from Escherichia coli; Ginsburg A et al.; Partial unfolding of dodecameric glutamine synthetase (GS) from Escherichia coli has been studied by differential scanning calorimetry (DSC) . A single endotherm (tm = 51.6 +/- 0.1 degrees C and delta Hcal = 211 +/- 4 kcal/mol of enzyme) was observed in DSC experiments with Mn.GS in the presence of 1.0 mM free Mn2+ and 100 mM KCl at pH 7 . The dodecameric structure of Mn.GS was retained throughout heating cycles, and thermal transitions were reversible as shown by rescans {with 6-18 mg of GS (Mr 622,000) from 15 to 68 degrees C at 20-60 degrees C/h} and by greater than 93% recovery of activity . A cooperative ratio delta Hcal/delta HvH of 1.6 +/- 0.1 and deconvolution analysis show two cooperative units (two-state transitions): t1 = 50.4 and t2 = 51.7 degrees C; the ratio of the relative sizes of thermally labile domains is approximately 1:2 as judged by delta H2/delta H1 approximately equal to 2 . However, the thermally induced overall enthalpy change (0.34 cal/g) for GS dodecamer is only 5-10% of that for thermal unfolding of small globular proteins at 50 degrees C . The t1 and t2 values from deconvolutions of DSC data agree with t0.5 values previously calculated from spectral measurements of temperature-induced exposures of approximately 0.7 of 2 Trp and approximately 2 of 17 Tyr per subunit, respectively {Shrake et al . (1989) Biochemistry 28, 6281-6294}, over a 14 degrees C temperature range using both stabilizing and destabilizing conditions for Mn.GS.(ABSTRACT TRUNCATED AT 250 WORDS) Antonie Van Leeuwenhoek, 1991 Oct-Nov, 60(3-4), 373 - 82 Quantitative aspects of glucose metabolism by Escherichia coli B/r, grown in the presence of pyrroloquinoline quinone; Hommes RW et al.; Escherichia coli B/r was grown in chemostat cultures under various limitations with glucose as carbon source . Since E . coli only synthesized the glucose dehydrogenase (GDH) apo-enzyme and not the appropriate cofactor, pyrroloquinoline quinone (PQQ), no gluconate production could be observed . However, when cell-saturating amounts of PQQ (nmol to mumol range) were pulsed into steady state glucose-excess cultures of E . coli, the organisms responded with an instantaneous formation of gluconate and an increased oxygen consumption rate . This showed that reconstitution of GDH in situ was possible . Hence, in order to examine the influence on glucose metabolism of an active GDH, E . coli was grown aerobically in chemostat cultures under various limitations in the presence of PQQ . It was found that the presence of PQQ indeed had a sizable effect: at pH 5.5 under phosphate- or sulphate-limited conditions more than 60% of the glucose consumed was converted to gluconate, which resulted in steady state gluconate concentrations up to 80 mmol/l . The specific rate of gluconate production (0.3-7.6 mmol.h-1.(g dry wt cells)-1) was dependent on the growth rate and the nature of the limitation . The production rate of other overflow metabolites such as acetate, pyruvate, and 2-oxoglutarate, was only slightly altered in the presence of PQQ . The fact that the cells were now able to use an active GDH apparently did not affect apo-enzyme synthesis. Biull Eksp Biol Med, 1991 Oct, 112(10), 409 - 11 {Incompatibility and replication of plasmids}; Grishina EV et al.; The identified basic replicons rep1 and rep2 of plasmid pAP42 belong to different groups of incompatibility (inc FIX and inc FVIII) . The replicons are partly incompatibile with other inc F-groups too . The results indicate connection between plasmid incompatibility and their replication. Ann Ital Med Int, 1991 Oct-Dec, 6(4), 357 - 63 {ACTH of lymphocytic origin under normal and pathological conditions}; Buzzetti R et al.; Evidence has accumulated that human peripheral blood mononuclear cells (PBMC) may release adrenocorticotropic hormone (ACTH) and endorphin-like peptides into the culture medium when stimulated with different substances such as Newcastle disease virus and the lipopolysaccharide of Escherichia coli . However, to our knowledge, no quantitative assessment of ACTH-LIR (like-immunoreactivity) in human PBMC has been reported . We thus utilized a radioimmunoassay for ACTH to find a median of 30 pg of ACTH-LIR in 10(7) PBMC of 11 normal subjects . ACTH-LIR was also detected in 7 different cell lines derived from patients with lymphoid and myeloid malignancies, two of them, JM and U937, showed values of 135 and 108 pg/10(7) cells respectively . Stimulation with IL-1 beta at the concentration of 1000 U/mL induced, after 48 h, a significant increase of intralymphocytic ACTH levels when compared to basal and 24 h values . The chromatographic characterization of this ACTH-LIR showed, at least, three molecular forms of immunoreactive ACTH; molecular weights were 31 kD POMC, 22 kD ACTH and 4.5 kD ACTH . We used northern blotting with human genomic DNA probe for POMC gene to evidence specific mRNA in PBMC; mRNA was also observed in a T lymphocyte cell line derived from a patient with lymphoma . We conclude that PBMC produce ACTH-LIR which may act as a paracrine immunomodulator similar to lymphokine and/or may signal the adrenal gland to secrete glucocorticoids. Protein Eng, 1991 Oct, 4(7), 785 - 91 Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin); Goh CR et al.; Single amino acid substitutions were generated in predicted hydrophilic loop regions of the human tumour necrosis factor beta (TNF-beta) molecule, and the mutant proteins were expressed in Escherichia coli and purified . Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid 50 or tyrosine 108, were found to have greatly reduced receptor binding and cytotoxic activity . These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta . The two distinct loops at positions 31-34 and 84-89 in the known three-dimensional structure of TNF-alpha (equivalent to positions 46-50 and 105-110 respectively in TNF-beta), lie on opposite sides of the TNF-alpha monomer . When the TNF-alpha monomer forms a trimer, the two loops, each from a different subunit of the trimer, come together and lie in a cleft between adjacent subunits . Together, these findings suggest that a TNF receptor binds to a cleft between subunits via surface loops at amino acid residues 31-34 and 84-89 in TNF-alpha, and similarly via surface loops including amino acids aspartic acid 50 and tyrosine 108 in TNF-beta. Mol Cell Endocrinol, 1991 Oct, 81(1-3), 147 - 54 Efficient production of biologically active human prolactin in Escherichia coli; Hiraoka Y et al.; To obtain an adequate amount of human prolactin (hPRL) for elucidation of the structure-function relationship, we have expressed the hPRL cDNA in Escherichia coli (E . coli) by using a high-expression vector . The vector contained a chimeric gene encoding a fusion of protein A, a peptide sensitive to collagenase digestion and hPRL, which was inserted downstream of the right direction promotor of lambda phage . The resulting protein fusion was purified through three column chromatographies of immunoglobulin G-linked Sepharose 4B, DEAE-5PW, and phenyl-5PW . In a typical experiment, a final sample with a purity of more than 80% was obtained with a recovery of more than 40% judged by enzyme-linked immunosorbent assay (ELISA) . The fusion thus obtained was digested with collagenase, and protein reactive to anti-hPRL antibody was purified through phenyl-5PW column chromatography . The hPRL sample was found to be identical to authentic hPRL with respect to the amino acid composition and an N-terminal sequence of 20 residues, except that it contained an additional four amino acids at the N-terminal end . This peptide was presumed to be derived from the collagenase-target sequence . The hPRL thus obtained was found to be as active as the authentic hormone either immunologically judged by ELISA or biologically judged by the growth stimulatory effect on rat Nb2 lymphoma cells. Prostaglandins, 1991 Oct, 42(4), 369 - 78 Dietary selenium effects on milk eicosanoid concentration in dairy cows during coliform mastitis; Maddox JF et al.; The effect of selenium deficiency on the product profile of arachidonic acid oxidation by enzymatic pathways in Holstein cows with experimentally-induced coliform mastitis was investigated . The animals were fed dairy rations containing 0.05 mg Se/kg dry matter, with the supplemented group receiving additional Se to increase the dietary concentration to approximately 0.35 mg Se/kg dry matter . Cows were inoculated intracisternally with 30 colony-forming-units of Escherichia coli at 14-16 weeks of lactation . Eicosanoids and bacteria numbers were recorded at various intervals of time for 60 h postinoculation . Milk from cows fed the Se-depleted diet had significantly higher (p less than 0.05) concentrations of TXB2 between 24 and 48 h and 6-keto-PGF1 alpha between 24 and 60 h postinoculation . Milk PGE2 concentration was significantly higher in the Se-deficient group at 24 h, whereas LTB4 was higher between 36 and 60 h postinoculation in the Se-deficient cows (p less than 0.05) . Milk bacteria numbers were significantly higher between 16 and 24 h postinoculation in the Se-deficient group and three of the four cows in this group required euthanasia, whereas all four cows in the Se-supplemented group recovered without therapeutic intervention . These data indicate marked effects of dietary Se on milk eicosanoid concentrations in response to an E . coli infection . The changes in eicosanoid concentrations may be associated with the altered pathogenesis and outcome of mastitis in a Se-deficient state. Biotechniques, 1991 Oct, 11(4), 432 - 4, 436 A general method to cleave a known DNA sequence at any site; Freije JM et al.; We describe a new method for obtaining DNA fragments starting at a desired point where there is no recognition sequence for any known restriction endonuclease . A single-stranded DNA containing the fragment of interest is annealed to a synthetic oligonucleotide hybridizing at the 5' end of the required fragment . Then, a partially double-stranded DNA is synthesized using the Klenow fragment of DNA polymerase I in the presence of the four deoxynucleoside triphosphates . The remaining single-stranded regions are removed by digestion with a single-strand nuclease, and the resulting 5' blunt-ended fragment is finally released by digestion with a restriction endonuclease at any site downstream its 3' end . The usefulness of the method was exemplified here by insertion of an epidermal growth factor-like African swine fever virus gene immediately downstream of the ribosome binding site of an expression vector. Mol Microbiol, 1991 Oct, 5(10), 2499 - 502 Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis; Martin C et al.; A method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2 . Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M . smegmatis by electroporation . Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome . This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2. FEMS Microbiol Lett, 1991 Oct 1, 67(2), 205 - 11 Molecular cloning and DNA sequence of dniR, a gene affecting anaerobic expression of the Escherichia coli hexaheme nitrite reductase; Kajie S et al.; A gene responsible for increased synthesis of hexaheme nitrite reductase (cytochrome c552) of Escherichia coli K-12 was cloned into pBR322 by the direct immunological screening method using antiserum against the purified enzyme . The cloned gene was mapped at 5 min on the chromosomal linkage map as the dni gene (related to increased synthesis of the dissimilatory nitrite reductase) by conjugation and transduction . The dni gene was subcloned into pUC118 and was shown to be on a 2.6-kilobase-pair PstI-BamHI fragment by immunoblotting analysis of the expressed enzyme . The nucleotide sequence of this fragment was determined . A plausible open-reading frame corresponding to 222 amino acids was detected . Analysis of a dni deletion mutant by immunoblotting demonstrated that this mutant expressed a greatly reduced amount of the nitrite reductase . Thus, the dni gene is suggested to have a positive regulatory action on induced synthesis of the nitrite reductase, and was designated as dniR. Am J Vet Res, 1991 Oct, 52(10), 1692 - 8 Modulation of function of bovine polymorphonuclear leukocytes and lymphocytes by high temperature in vitro and in vivo; Elvinger F et al.; Function of polymorphonuclear leukocytes (PMNL) and proliferation of lymphocytes after stimulation with mitogens were evaluated in vitro at incubation temperatures of 38.5 and 42 C, and after in vivo heat stress of lactating Holstein cows . Cytochrome-c reduction and random migration of PMNL were reduced when cells were preincubated or incubated at 42 C, but high incubation temperature had little or no effect on phagocytosis and killing of Escherichia coli . Proliferation of lymphocytes was reduced when cells were incubated for 60 hours at 42 C after stimulation with phytohemagglutinin, pokeweed mitogen, or concanavalin A . After stimulation with phytohemagglutinin, lymphocytes were most sensitive to high temperature during the first 24 hours of the 60-hour culture period . High incubation temperature had little effect on viability of cells . In vivo heat stress had no significant effect on responses of PMNL in vitro, but the decrease in proliferation of lymphocytes in vitro at high temperature was less when cells were obtained from heat-stressed cows . Total leukocyte counts in blood and somatic cell counts in milk were higher in heat-stressed cows . Results indicate that: exposure to high temperature in vitro can depress responses of PMNL and lymphocytes; apparent adaptive mechanisms induced by in vivo heat stress provide protection from effects of high temperature seen in vitro; and evidence could not be found to support the hypothesis that reduction in immune function is the basis for increases in the incidence of mastitis during the summer. Mol Gen Mikrobiol Virusol, 1991 Oct, (10), 13 - 6 {Detection of herpes simplex virus by DNA-DNA hybridization method}; Diorditsa SV et al.; Eight recombinant clones were obtained by insertion of BamHI fragments of herpes simplex type I viral DNA into a vector plasmid pUC19o . Of the obtained clones 5 were found to hybridize with herpes simplex type I and 2 viral DNA while 3 clones revealed a positive reaction with the Vero cells DNA . A constructed DNA-probe possessing the highest level of activity was selected for further studies . The probe is a BamHI fragment of herpes simplex type I viral DNA labelled with 32P dTTP . Probe sensitivity in blot hybridization is 10 pg for identification of type I viral DNA and 50 pg for type 2 viral DNA . The DNAs of cytomegalovirus and herpes zoster virus do not show positive signals with the probe . The increased sensitivity of the used dot hybridization as compared with biological or IEA antigen identification of the virus was confirmed with the clinical material from 59 patients with the different clinical manifestations of the herpes viral infection. J Bioenerg Biomembr, 1991 Oct, 23(5), 715 - 41 The proton-translocating nicotinamide adenine dinucleotide transhydrogenase; Jackson JB; H(+)-transhydrogenase couples the reversible transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane . There are separate sites on the enzyme for the binding of NAD(H) and of NADP(H) . There are some indications of the position of the binding sites in the primary sequence of the enzymes from mitochondria and Escherichia coli . Transfer of hydride ion equivalents only proceeds when a reduced and an oxidized nucleotide are simultaneously bound to the enzyme . When delta p = 0 the rate of interconversion of the ternary complexes of enzyme and nucleotide substrates is probably limiting . An increase in delta p accelerates the rate of interconversion in the direction of NADH----NADP+ until another kinetic component, possibly product release, becomes limiting . The available data are consistent with either direct or indirect mechanisms of energy coupling. Genetics, 1991 Oct, 129(2), 317 - 26 Spontaneous mutation in the Escherichia coli lacI gene; Schaaper RM et al.; To gain more detailed insight into the nature and mechanisms of spontaneous mutations, we undertook a DNA sequence analysis of a large collection of spontaneous mutations in the N-terminal region of the Escherichia coli lacI gene . This region of circa 210 base pairs is the target for dominant lacI mutations (i-d) and is suitable for studies of mutational specificity since it contains a relatively high density of detectable mutable sites . Among 414 independent i-d mutants, 70.8% were base substitutions, 17.2% deletions, 7.7% additions and 4.3% single-base frameshifts . The base substitutions were both transitions (60%) and transversions (40%), the largest single group being G.C----A.T (47% of base substitutions) . All four transversions were observed . Among the 71 deletions, a hotspot (37 mutants) was present: an 87-bp deletion presumably directed by an 8-bp repeated sequence at its endpoints . The remaining 34 deletions were distributed among 29 different mutations, either flanked (13/34) or not flanked (21/34) by repeated sequences . The 32 additions comprised 29 different events, with only two containing a direct repeat at the endpoints . The single-base frameshifts were the loss of a single base from either repeated (67%) or nonrepeated (33%) bases . A comparison with the spectrum obtained previously in strains defective in DNA mismatch correction (mutH, mutL, mutS strains) yielded information about the apparent efficiency of mismatch repair . The overall effect was 260-fold but varied substantially among different classes of mutations . An interesting asymmetry was uncovered for the two types of transitions, A.T----G.C and G.C----A.T being reduced by mismatch repair 1340- and 190-fold, respectively . Explanations for this asymmetry and its possible implications for the origins of spontaneous mutations are discussed. Pediatr Infect Dis J, 1991 Oct, 10(10), 746 - 51 Potential role of adherence traits of Escherichia coli in persistent diarrhea in an urban Brazilian slum; Wanke CA et al.; We examined stools from 40 children with persistent diarrhea (duration, 14 days or more), from 50 children with acute diarrhea and from 38 control children to determine infectious etiologies for persistent diarrhea in Goncalves Dias, an urban favela (slum) in Fortaleza, Ceara, Brazil . Children with persistent diarrhea and children with acute diarrhea had similar rates of isolation of routine viral, bacterial and parasitic enteric pathogens . Routine pathogens were identified in at least 20% of cultures done more than 14 days into the diarrheal illness . We examined Escherichia coli isolated from these stools for adherence potential . Enteroaggregative E . coli were isolated significantly more often from children with persistent diarrhea than from control children or children with acute diarrhea (P less than 0.05) . E . coli with hemagglutination patterns suggestive of adherence pili were also isolated more often from children with persistent diarrhea than from children with acute diarrhea (38% vs . 18%; P less than 0.05) . Enterotoxigenic E . coli were isolated in combination with rotavirus more often from children with persistent diarrhea than from children with acute diarrhea . E . coli which were hydrophobic or exhibited hemagglutination were also seen more often in association with Giardia in children with persistent diarrhea . These findings suggest that the etiology of persistent diarrhea in children is complex and that the aggregative E . coli are associated with prolonged diarrheal illness . Although routine diarrheal pathogens may be present for more than 14 days, combinations of pathogens, including E . coli with adherence potential, may also contribute to prolonged diarrheal disease. Mol Gen Genet, 1991 Oct, 229(3), 341 - 52 A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA) Dassa J, Fsihi H, Marck C, Dion M, Kieffer-Bontemps M, Boquet PL. The Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new sigma transcription-initiation factor . However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation . The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ . It contains two new genes, appC and appB, which both encode extracytoplasmic proteins . appC and appB are expressed from a promoter (P2) lying just upstream of appC . Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA . Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone . Genes appC and appB encode proteins of Mr 58,133 and 42,377, respectively, which have the characteristics of integral membrane proteins . The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E . coli cytochrome d oxidase (encoded by genes cydA and cydB) . The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants. Mol Gen Genet, 1991 Oct, 229(3), 325 - 33 A new type of insertion mutation in monkey cells: insertion accompanied by long target site duplication; Ohira M et al.; We have developed a system for the detection of a new type of insertion mutation in mammalian cells . We have used a shuttle vector, plasmid pNK1, which contains the SV40 and pBR322 replication origins, and ApR, galK, and neoR genes . This plasmid was introduced into monkey COS1 cells, allowed to replicate, and then recovered plasmids were reintroduced into Escherichia coli HB101 to detect insertion mutations in the galK gene . We selected galK- KmR ApR mutants in order to eliminate galK- KmS deletion mutants . Insertion mutations in the plasmids recovered were then screened by agarose gel electrophoresis . Finally, insertion mutants that had the following characteristics were selected . First, they had the ability to produce gal+ revertants caused by the precise excision of inserted DNA in E . coli, implying that they had a target site duplication on both sides of the insertion . Second, they contained some repetitive sequence(s) as judged by hybridization with a bulk monkey DNA probe . Nucleotide sequence analysis of one of the mutants, 15K-1, showed that it contained alpha-satellite sequences within the coding region of the galK gene . It contained 13 1/2 tandem repeat units of alpha-satellite sequence and was flanked by a 64 bp target site duplication, indicating that the alpha-satellite sequence had been translocated from the monkey genome into the plasmid by illegitimate recombination . Another insertion mutant, N11-1, contained an 11 kb insert which included an unknown repetitive sequence that was also flanked by a target site duplication of 353 bp.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Endocrinol, 1991 Oct, 7(2), 123 - 9 Purification of human c-erb A beta protein; Ichikawa K et al.; Human thyroid hormone receptor (c-erb A protein) produced by Escherichia coli expression vector plasmid was purified sequentially using polyethylenimine precipitation of DNA, hydroxylapatite column chromatography, ammonium sulphate precipitation, Sephacryl S-300 gel filtration and mono Q-Sepharose column chromatography . These column procedures resulted in 41.3-fold purification of 3,5,3'-tri-iodo-L-thyronine (T3) binding activity over the initial E . coli extract . Purified protein as well as crude preparation showed high-affinity binding to T3 . The c-erb A protein enriched by column purification was further purified by electroelution after electrophoresis . Rabbit antibody against the c-erb A protein was prepared and used for the Western blotting analysis . The antibody recognized c-erb A protein but not the bacterial proteins in crude E . coli extract . When partially purified rat hepatic nuclear thyroid hormone receptor was analysed, a 56 kDa receptor was specifically recognized by the antibody. Am J Physiol, 1991 Oct, 261(4 Pt 1), G592 - 601 Characterization of the synergistic interaction of Escherichia coli heat-stable toxin and carbachol; Levine SA et al.; STa, the heat-stable enterotoxin of Escherichia coli, is a specific activator of membrane-bound guanylyl cyclase and stimulates secretion of Cl- in a human colonic carcinoma cell line (T84) . We investigated the effect of the cholinergic agent carbachol on the secretory response to STa . T84 cell monolayers were studied under voltage-clamped conditions in modified Ussing chambers . Simultaneous addition of STa and carbachol resulted in a biphasic synergistic response characterized by a brief peak in short-circuit current (Isc) followed by a prolonged plateau phase lasting up to 90 min . A synergistic response was also seen with sequential addition of the agonists, and was altered by the order and timing of agonist addition . Pretreatment with STa enhanced the synergistic response to carbachol, while the reverse order of additions produced synergy only when STa was added during or immediately after the Isc response to carbachol . Synergy occurred only with a concentration of STa sufficient to produce an Isc response alone . However, a concentration of carbachol that caused neither an increase in Isc nor intracellular Ca2+ mobilization was sufficient to evoke a synergistic response . Addition of 8-bromoguanosine 3',5'-cyclic monophosphate also produced a synergistic Isc response with carbachol, although maximal synergism was seen with simultaneous addition . Augmentation of the intracellular Ca2+ response to carbachol by STa is not the mechanism of synergy . Although the mechanism of synergy is not understood, these studies suggest that STa-induced cGMP interacts with other second messengers to produce the synergistic response, and that multiple intracellular mediators may influence the ability of STa to cause disease. Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8548 - 52 Expression of two human beta-adrenergic receptors in Escherichia coli: functional interaction with two forms of the stimulatory G protein; Freissmuth M et al.; When expressed in Escherichia coli, the human beta 1- and beta 2-adrenergic receptors retain their ligand binding specificity . Their functional integrity was investigated by analyzing receptor-guanine nucleotide-binding regulatory (G) protein coupling by using two splice variants of the alpha subunit of the stimulatory G protein Gs synthesized in E . coli (rGs alpha-S and rGs alpha-L) and the beta gamma subunits of G protein purified from bovine brain . In competition binding experiments with (-)-{125I}iodocyanopindolol and (-)-isoproterenol, rGs alpha-S.beta gamma and rGs alpha-L.beta gamma reconstituted guanine nucleotide-sensitive high-affinity agonist binding with comparable affinities, whereas rGs alpha PT, a mutant of rGs alpha-L with an altered carboxyl terminus, and a r