Escherichia coli

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 155 - 9
Serogroups of Escherichia coli strains producing cytotoxic necrotizing factors CNF1 and CNF2; Blanco J et al.; The serogroups of 396 necrotizing Escherichia coli of human and bovine origin isolated in Spain between 1979 and 1991 have been determined . The 270 cytotoxic necrotizing factor strains belonged to 22 different O serogroups; however, 84% (226 of 270) were of one of seven serogroups (O2, O4, O6, O14, O22, O75 and O83) . Although necrotizing E . coli producing cytotoxic necrotizing factor 2 belonged to 28 different serogroups, only six of them (O1, O3, O15, O55, O88 and O123) accounted for 60% (76 of 126) of cytotoxic necrotizing factor 2 strains . Furthermore, only 3% (4 of 126) of cytotoxic necrotizing factor 2 strains belonged to serogroups most common among strains producing cytotoxic necrotizing factor 1 . The majority of necrotizing E . coli producing cytotoxic necrotizing factor 1 were obtained from human extraintestinal infections, whereas cytotoxic necrotizing factor 2 strains were isolated from stools of healthy and diarrhoeic calves.

Eur J Biochem, 1992 Sep 15, 208(3), 699 - 704
Precursor of mitochondrial aspartate aminotransferase synthesized in Escherichia coli is complexed with heat-shock protein DnaK; Schmid D et al.; On expression of the cDNA encoding the precursor of chicken mitochondrial aspartate aminotransferase (pmAspAT) in Escherichia coli, the bulk of pmAspAT was found to be associated with the 70-kDa heat-shock protein DnaK which is closely related to mitochondrial 70-kDa heat-shock protein (HSP70) . Purification protocols for the DnaK/pmAspAT complex and its individual components were elaborated . The complex dissociated on treatment with MgATP or at pH 5.5 . Like the mature enzyme, pmAspAT is a dimer (2 x 47 kDa) and exhibits about a third of its enzyme activity . In the DnaK/pmAspAT complex, one DnaK molecule is bound to each subunit of pmAspAT; this tetramer may further aggregate to an octamer . The complex is catalytically almost as active as free pmAspAT . It could be reconstituted from isolated DnaK and pmAspAT . No complex was formed with mAspAT . Apparently, DnaK binds to the solvent-exposed presequence of folded pmAspAT without significantly changing the structure and functional properties of its mature moiety.

Eur J Biochem, 1992 Sep 15, 208(3), 635 - 42
Binding of the competitive inhibitor dCDP to ribonucleoside-diphosphate reductase from Escherichia coli studied by 1H NMR . Different properties of the large protein subunit and the holoenzyme; Allard P et al.; Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two nonidentical subunits, proteins R1 and R2 . The binding of the product dCDP to protein R1 and to the holoenzyme R1R2 has been studied by means of 1H-NMR spectroscopy . In presence of the effector dTTP at 25 degrees C, dCDP was found to be in rapid exchange between the binding sites and the solvent which results in a broadening of the dCDP resonances . When both proteins R1 and R2 are present, so that the complex R1R2 is formed, a smaller broadening is observed than with protein R1 alone . No further linewidth decrease was observed when the {R2}/{R1} ratio exceeded 1 . The binding constant of dCDP to R1 or R1R2 is the same, Kd = 0.9 mM . The smaller broadening of the dCDP resonances observed with the complex R1R2 as compared with R1 may be explained by the combination of two effects: (a) the overall tumbling time of the protein will increase when going from R1 to R1R2, which will cause the broadening to increase correspondingly, and (b) a twofold decrease of the number of binding sites in rapid exchange, which will decrease the broadening by a factor of 0.5 . The effect of R2 without iron (apoR2) is reduced compared with native R2, probably because of some denatured proteins, while a C-terminal peptide from R2 did not cause any narrowing at all.

Eur J Biochem, 1992 Sep 15, 208(3), 631 - 4
Studies on ribonucleoside-diphosphate reductase from Escherichia coli . The product dCDP is a competitive inhibitor and functions as a spectroscopic probe for the substrate binding site; demonstration by enzyme kinetics and 1H NMR; Shen B et al.; Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two protein subunits, R1 of 171.5 kDa and R2 of 86.8 kDa, and catalyzes the reduction of all four common ribonucleoside diphosphates . In a search for ligands that bind weakly to the enzyme active site and may be in fast exchange suitable for NMR studies, we have found that the product dCDP is a competitive inhibitor . Kinetics with CDP as substrate shows Km = 4.8 x 10(-5) M and dCDP inhibits with Ki = 1.6 x 10(-4) M . With an assumed diffusion limited binding rate approximately less than 10(9) M-1s-1, the dissociation rate of dCDP would be approximately less than 10(5) s-1 . In 1H-NMR experiments studying linewidths, i.e . spin-spin relaxation, dCDP is indeed demonstrated to be in fast exchange . Enzyme subunit R1 causes a line broadening of dCDP resonances . Unexpectedly less broadening was observed when subunit R2 combined with R1 . No paramagnetic interaction from the tyrosyl radical of R2 could be detected . It is concluded that dCDP is a promising NMR probe for studies of active-site properties of the enzyme.

Biochemistry, 1992 Sep 15, 31(36), 8648 - 53
Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex; Kumar S et al.; The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity . The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography . The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste . M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor . The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet . From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined . The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique . Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units . The crystals diffract beyond 2.5 A and are suitable for structure determination.

Biochemistry, 1992 Sep 15, 31(36), 8516 - 22
Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments; Adams JA et al.; Viscosogenic agents were used to test the diffusion limits of the reaction catalyzed by the catalytic subunit of the cAMP-dependent protein kinase . The effects of glycerol and sucrose on the maximum rate (kcat) and the apparent second-order rate constants (kcat/Kpeptide) for the phosphorylation of four peptidic substrates were measured at their pH optima . The agents were found to have moderate to no effect on kcat/Kpeptide for good and poor substrates, respectively . Conversely, kcat was highly sensitive to solvent viscosity for three of the four peptides at high concentrations of ATP . Taken together, these data indicate that enzymatic phosphorylation by the catalytic subunit proceeds with rapid or near rapid equilibrium binding of substrates and that all steps following the central substrate complex (i.e., chemical and conformational events) are fast relative to the rate-determining dissociation of product, ADP, when ATP levels are high . Under saturating concentrations of peptide I, LRRASLG, an unproductive form of the enzyme is populated . The observed phosphorylation rate from this complex is involved in rate limitation owing to a slow step separating unproductive and productive enzyme forms . The data are used to establish a kinetic mechanism for the catalytic subunit that predicts initial reaction velocities under varying concentrations of ATP and substrate.

Biochemistry, 1992 Sep 15, 31(36), 8508 - 15
Effects of replacement of active site residue glutamine 231 on activity and allosteric properties of aspartate transcarbamoylase; Peterson CB et al.; Since crystallographic studies on Escherichia coli aspartate transcarbamoylase (ATCase) indicate that Gln 231 is in the active site of the enzyme and participates in the binding of the substrate, aspartate, it seemed of interest to examine mutant enzymes in which Gln 231 was replaced by Asn or Ile . The two mutant forms containing amino acid substitutions were characterized by a combination of steady-state kinetics, hydrodynamic measurements, and equilibrium ligand binding techniques . Both mutant forms exhibited a dramatic reduction in the affinity of the protein for substrates and substrate analogues as well as a very large decrease in catalytic activity . Moreover, the amino acid substitutions introduced within the active site of the enzyme led to unusual allosteric properties in the mutant enzymes . Although the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate promotes the characteristic global conformational change in the mutant forms that is observed with the wild-type enzyme, the combination of substrate and substrate analogue does not . Cooperativity with respect to substrate binding is largely reduced compared to wild-type ATCase . Also, the effector molecules ATP and CTP which bind to the regulatory chains have dramatic effects on the activity of the mutant enzymes containing replacements for Gln 231 in the catalytic chains . In stark contrast to the wild-type enzyme, in which effects of nucleotides are manifested primarily by changes in the K0.5 of the enzyme, ATP and CTP have large effects on the Vmax of the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Sep 15, 31(36), 8482 - 7
Segmental movement: definition of the structural requirements for loop closure in catalysis by triosephosphate isomerase; Sampson NS et al.; To determine what drives the closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase, several residues involved in hydrogen bonding between the loop and the bulk of the protein have been altered . It was known from earlier work that the loop serves two functions: to stabilize the reaction intermediate (and the two transition states that flank it) and to prevent the loss of this unstable species into free solution . To discover what elements of the protein are necessary for proper closure of the loop, selective destabilization of the "open" and the "closed" forms of the enzyme with respect to one another has been attempted . The mutant Y164F isomerase has been prepared to evaluate the importance of the structure of the "open" form, and the mutant E129Q, Y208F, and S211A enzymes have allowed investigation of the "closed" form . The integrity of the loop itself has been destabilized by making the T172A isomerase . We have found that only those mutations that destabilize the "closed" form of the enzyme significantly perturb the catalytic properties of the isomerase . The second-order rate constants (kcat/Km) of the S211A and E129Q enzymes are reduced 30-fold, and that of the mutant Y208F enzyme is reduced 2000-fold, from the level of the wild-type enzyme . The dramatic drop in activity of the Y208F enzyme is accompanied by a 200-fold increase in the dissociation constant of the intermediate analogue phosphoglycolohydroxamate . The most important property of the mobile loop of triosephosphate isomerase lies, therefore, in the stability of the system when the active site contains ligand and the loop is closed.

Biochemistry, 1992 Sep 15, 31(36), 8437 - 41
Effect of 5-deazaflavin on energy transduction during catalysis by Escherichia coli DNA photolyase; Ramsey AJ et al.; DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate . The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 (EdFADH2) matched its absorption spectrum after correction for the presence of a small amount of inactive 5-deazaFADox . The quantum yield for dimer repair with EdFADH2 (phi EdFADH2 = 0.110) was 6-fold lower than that observed with apoenzyme reconstituted with FADH2 . Excited-state redox potential calculations indicate that 5-deazaFADH2 singlet is a better one-electron donor (E = -3.5 V) than FADH2 singlet (E = -2.7 V) . Other studies indicate that the quantum yield for electron transfer from reduced flavin singlet to pyrimidine dimer (0.88) is unaffected when FADH2 is replaced by 5-deazaFADH2 . Enhanced back electron transfer from pyrimidine dimer radical to flavin radical may account for the decreased quantum yield observed with EdFADH2 since, in the ground state, 5-deazaFADH . is a better oxidant than FADH. . The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 plus 5,10-CH(+)-H4folate (EPtedFADH2) matched the absorption spectrum determined for enzyme-bound 5-deazaFADH2, indicating that the pterin chromophore was inactive as a sensitizer . This differs from results obtained with native enzyme, where pterin acts as a sensitizer via efficient singlet-singlet energy transfer to FADH2 . The quantum yield for dimer repair by 5-deazaFADH2 bound to EPtedFADH2 (phi EPtedFADH2 = 0.0318) was 28.9% of that observed for EdFADH2 . Spectroscopic studies indicate that singlet-singlet energy transfer in EPtedFADH2 is very efficient but only occurs in the "wrong" direction, i.e., from excited 5-deazaFADH2 to pterin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Sep 15, 31(36), 8429 - 36
Differences and similarities in the repair of two benzo{a}pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products; Tang MS et al.; We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo{a}pyrene diol epoxide isomers . Using the phi X174 RF DNA-E . coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells . In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells . Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts . Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate . In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions . Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding . These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts . It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1992 Sep 15, 267(26), 18953 - 60
Determination of the 1-ethyl-3-{(3-dimethylamino)propyl}-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase; Dallmann HG et al.; The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-{(3-dimethylamino) propyl}-carbodiimide (EDC), has been determined at the amino acid level . Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide . A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels . Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon . Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis . A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis . epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC . An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431 . Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108 . Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC . Both mutant forms of epsilon supported growth of an uncC-deficient E . coli strain and inhibited F1-ATPase . These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108 . As these residues must be located immediately adjacent to one another in F1-ATPase, our results define a site of subunit-subunit contact between beta and epsilon.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8779 - 83
Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on A.C and A.G mispairs; Tsai-Wu JJ et al.; In Escherichia coli the mutY (or micA)-dependent DNA mismatch repair pathway can convert A degrees G and A degrees C mismatches to C.G and G.C base pairs, respectively, through a short repair-tract mechanism . The MutY protein has been purified to near homogeneity from an E . coli overproducer strain . Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities . The N-glycosylase removes the mispaired adenines of A degrees G and A degrees C mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites . The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurified through multiple chromatographic steps without a change in relative specific activities . Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel . Renaturation required the presence of iron and sulfide . These findings suggest that the MutY protein, like endonuclease III, is an iron-sulfur protein . DNA fragments with A degrees C mismatches were 20-fold less active than DNA with A degrees G mispairs as a substrate for purified MutY.

J Biol Chem, 1992 Sep 15, 267(26), 18255 - 8
Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1; Hizi A et al.; We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) . These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities . Three groups of mutants were studied . 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT . The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme . The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained) . 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT . This mutant of HIV-1 RT lost practically all catalytic activities . 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8696 - 700
Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants; Bertsch U et al.; Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES) . In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP . The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10 . A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro . The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide . Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem . Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined . Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa . N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8803 - 7
Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA; Kuspa A et al.; Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants . We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced . Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations . About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development . The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells . Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes . This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes.

J Biol Chem, 1992 Sep 15, 267(26), 18683 - 8
A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs . Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase; Repaske DR et al.; Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues . To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains . Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes . One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J . K., Kadlecek, A., Sherbert, C . H., Seger, D., Sonnenburg, W . K., Charbonneau, H., Novack, J . P., and Beavo, J . A . (1992) J . Biol . Chem . 267, 18676-18682) . This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone . The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE . Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin . Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line . These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes . Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.

FEBS Lett, 1992 Sep 14, 309(3), 363 - 70
Studies on antisense inhibition of translation in vitro . Anomalies and re-evaluation; Ricker RD et al.; Experiments were carried out to better characterize antisense control of translation . Results in an E . coli system confirmed specific inhibition of poly(U) translation . At low concentrations, certain homopolymers (including poly(rA)) stimulated translation . Oligo(dA(n)) was inhibitory at n less than or equal to 8 . Translation of globin mRNA in reticulocyte lysates indicated that ssDNA 15-mers targeted at beta-globin mRNA inhibited both alpha- and beta-globin production . Sequences targeted immediately downstream of the AUG were the least effective in inhibition . These and other anomalies are discussed here in relation to those of others, emphasizing caution in performing antisense experiments.

Biochemistry, 1992 Sep 1, 31(34), 7815 - 25
Roles of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: kinetic evidence for a second open complex requiring Mg2+; Suh WC et al.; Comparative studies of the effects of Mg2+ vs Na+ and of acetate (OAc-) vs Cl- on the kinetics of formation and dissociation of E . coli RNA polymerase (E sigma 70)-lambda PR promoter open complexes have been used to probe the mechanism of this interaction . Composite second-order association rate constants ka and first-order dissociation rate constants kd, and their power dependences on salt concentration SKa (SKa identical to d log ka/d log {salt}) and Skd (Skd identical to d log kd/d log {salt}), were determined in MgCl2 and NaOAc to compare with the results of Roe and Record (1985) in NaCl . Replacement of NaCl by MgCl2 reduces the magnitude of Ska 2-fold (Ska = -11.9 +/- 1.1 in NaCl; Ska = -5.2 +/- 0.3 in MgCl2) and (by extrapolation) drastically reduces the magnitude of ka at any specified salt concentration (e.g., approximately 10(6)-fold at 0.2 M) . Replacement of NaCl by NaOAc does not significantly affect Ska (Ska = -12.0 +/- 0.7 in NaOAc) and (by extrapolation) increased ka by approximately 80-fold at any fixed {Na+} . In the absence of Mg2+, replacement of NaCl by NaOAc is found to increase the half-life of the open complex by approximately 560-fold at fixed {Na+} without affecting Skd {Skd = 7.6 +/- 0.1 in NaOAc; in NaCl, Skd = 7.7 +/- 0.2 (Roe & Record, 1985)} . Replacement of NaCl by MgCl2 drastically reduces both Skd and the half-life of the open complex at any salt concentration below approximately 0.2 M . Strikingly, Skd = 0.4 +/- 0.1 in MgCl2, indicating that the net uptake of Mg2+ ions in the kinetically significant steps in dissociation of the open complex is much smaller than that expected by analogy with the uptake of approximately 8 Na+ ions in the corresponding steps in NaCl . In NaCl/MgCl2 mixtures, at a constant {NaCl} in the range 0.1-0.2 M, initial addition of MgCl2 (0.5 mM less than or equal to {MgCl2} less than or equal to 1 mM) increases the half-life of the open complex; further addition of MgCl2 causes the half-life to decrease, though the effect of {MgCl2} on kd is always less than that predicted by a simple competitive model . The observed effects of MgCl2 on Skd and kd differ profoundly from those expected from the behavior of kd and Skd in NaCl and NaOAc and indicate that the role of Mg2+ in dissociation is not merely that of a nonspecific divalent competitor with RNAP for interactions with DNA phosphates and of a DNA helix-stabilizer, both of which should cause kd to increase monotonically with increasing {Mg2+}.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Biol Evol, 1992 Sep, 9(5), 787 - 805
Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase; Campbell JW et al.; The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs . Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins . To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA . Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals . A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal . Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity . Both could have resulted from third-base-codon substitutions . A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme . The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme . The complete sequence of chicken retinal GS is also identical to that of the liver enzyme . GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).

Nucleic Acids Res, 1992 Sep 11, 20(17), 4599 - 606
Integration of image analysis and robotics into a fully automated colony picking and plate handling system; Jones P et al.; We describe here the integration of image analysis and robotics to produce a fully automated colony picking/plate handling system . Biological tests were performed to verify its performance in terms of sterilisation and accuracy of picking . The machine was then used by a single operative to pick a 36,000 clone cDNA library in approximately 42 hrs over 5 days.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4591 - 8
Construction and use of lambda PL promoter vectors for direct cloning and high level expression of PCR amplified DNA coding sequences; Cheng X et al.; A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed . The vectors contain the phage lambda PL promoter, a synthetic translation initiation region (TIR), and convenient cloning sites . The cloning sites provide all or part of an AUG translation initiation codon and facilitate the precise fusion of target DNA sequences to vector transcriptional and translational signals . The vectors were constructed with synthetic TIRs because there is evidence which suggests that the efficiency of the phage lambda cII gene TIR present in the parental vector depends strongly on information contained within the cII N-terminal coding sequence . Bovine brain 14-3-3 eta chain cDNA was PCR-amplified and used to demonstrate the expression capacity of the newly constructed vectors . A significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR . Expression levels vary from 15% to 48% of total cell protein . The effects of a reported translational enhancer from phage T7 on expression of the 14-3-3 protein are also discussed . The vectors should be generally useful for high level heterologous protein expression in Escherichia coli.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4547 - 51
Chemical and enzymatic incorporation of N2-(p-n-butylphenyl)-2'-deoxyguanosine into an oligodeoxyribonucleotide; Misra HK et al.; An 18mer oligodeoxyribonucleotide containing a N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG) residue at the 3' end has been synthesized by both chemical and enzymatic methods . Chemical synthesis involved attachment of 5'-DMT-BuPdG as the 3'-H-phosphonate to uridine-controlled pore glass (CPG), followed by extension via H-phosphonate chemistry . After oxidation of the backbone, deprotection of bases, and removal from CPG, the uridine residue was removed by periodate cleavage and beta-elimination . The resulting oligomer 3'-phosphate was digested with alkaline phosphatase to give the free BuPdG-18mer . E.coli DNA polymerase I (Klenow) incorporated BuPdGTP at the 3' end of the corresponding 17mer primer annealed to a complementary 29mer template, and the properties of this product were identical to those of chemically synthesized BuPdG-18mer . E.coli DNA polymerase I (Klenow) was unable to extend the BuPdG-18mer, and the 3' to 5' exonuclease activity of the enzyme was unable to remove the modified nucleotide.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4507 - 13
Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies; Nikonowicz EP et al.; A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence . Uniformly labelled nucleotide 5'triphosphates (NTPs) were prepared from E.coli grown on 13C and/or 15N isotopically enriched media . These procedures routinely yield 180 mumoles of labelled NTPs per gram of 13C enriched glucose . The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription . Several 13C and/or 15N labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC) . Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively . These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues . The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs . Examples of several such heteronuclear NMR experiments are shown.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4417 - 21
Structure of the human DNA repair gene HAP1 and its localisation to chromosome 14q 11.2-12; Robson CN et al.; Apurinic/apyrimidinic (AP) sites are pre-mutagenic DNA lesions which occur spontaneously and following exposure of cells to ionising radiation or chemical mutagens . HAP1 (Human AP endonuclease 1), the major enzyme in human cells initiating repair of AP sites, shows strong sequence homology to DNA repair enzymes from bacteria, Drosophila and other mammalian species . We have cloned the HAP1 gene and determined its complete nucleotide sequence . The site of transcription initiation has been mapped to 452 bp upstream of the ATG initiation codon in the genomic DNA . The HAP1 gene consists of five exons and is unusually small (less than 2.6 kb from transcription initiation site to polyadenylation sequence) with 54% of the protein coding region and the entire 3' untranslated region contained within a single exon . The first exon is non-coding . Regions of three exons show sequence homology to the E.coli xth (exonuclease III) gene . Using in situ hybridisation, the HAP1 gene has been localised to human chromosome 14q 11.2-12.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4397 - 400
Conformity of RNAs that interact with tetranucleotide loop binding proteins; Zwieb C; A group of RNA binding proteins, termed tetraloop binding proteins, includes ribosomal protein S15 and protein SRP19 of signal recognition particle . They are primary RNA binding proteins, recognize RNA tetranucleotide loops with a GNAR consensus motif, and require a helical region located adjacent to the tetraloop . Closely related RNA structures that fit these criteria appear in helix 6 of SRP RNA, in helices 22 and 23A of 16 S ribosomal RNA, and, as a pseudoknot, in the regulatory region of the rpsO gene.

Gene, 1992 Sep 10, 118(2), 239 - 45
Production of mature bovine pancreatic ribonuclease in Escherichia coli; Tarragona-Fiol A et al.; The coding sequence for the bovine pancreatic ribonuclease (RNase) precursor has been cloned and produced in Escherichia coli using the polymerase chain reaction (PCR) technique . A PCR amplification has been carried out utilizing as template the recombinant plasmid, pQR138, which contains the coding sequence for the RNase precursor, and primers that allow for the addition of new sequences at the 5' and 3' ends of the coding sequence . The resultant fragment contains two coding sequences, one for a hexapeptide and the other for pre-RNase . This fragment has been cloned into the expression vector, pKK223.3, under the control of the tac promoter, to form a two-cistron vector . Upon induction with IPTG, E . coli cells harboring this construct generate a bicistronic mRNA which upon translation produces a hexapeptide and pre-RNase . The RNase precursor is efficiently translocated into the periplasmic space of E . coli . Upon translocation, the signal sequence is removed generating mature RNase . Formation of the disulfide bridges in RNase is facilitated by the oxidative environment of the periplasm and a fully active protein is obtained . RNase produced in E . coli has been purified to homogeneity by cation-exchange chromatography, and the removal of the signal sequence has been verified by N-terminal sequencing . The total process from inoculation of media to obtaining pure and fully active recombinant RNase is achieved in 48 h.

Gene, 1992 Sep 10, 118(2), 171 - 9
Identification of a genomic DNA fragment containing the Drosophila melanogaster ovarian tumor gene (otu) and localization of regions governing its expression; Comer AR et al.; We have identified a genomic DNA fragment which restores fertility to mutants of the ovarian tumor locus (otu) of Drosophila melanogaster . Germ-line transformants bearing this fragment express otu mRNA with the same tissue specificity as, and at levels comparable to, the wild-type otu gene . Transcription from the otu promoter, P(otu), which lacks a TATA element, appears to be initiated at multiple transcription start points (tsp) within an 80-bp region . Deletion of sequences upstream of the tsp indicates that a region between nucleotides -190 and -310 is required for proper expression from the otu gene . A DNA fragment containing 452 bp upstream and 126 bp downstream from the tsp is able to direct expression of the Escherichia coli lacZ gene in the germ cells of the ovary and testis, indicating that cis-acting regulatory elements governing these expression patterns are located in a 578-bp region surrounding the multiple tsp.

Gene, 1992 Sep 10, 118(2), 153 - 62
The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product; Fabry S et al.; Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells . These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport . By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f . nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga . The 1538-bp yptV1 gene of V . carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon . The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions . Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose . Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V . carteri extract and is expressed throughout the whole life-cycle of the alga . Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring . The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed.

Biochemistry, 1992 Sep 8, 31(35), 8196 - 200
Enhanced catalysis by active-site mutagenesis at aspartic acid 153 in Escherichia coli alkaline phosphatase; Matlin AR et al.; Bacterial alkaline phosphatase catalyzes the hydrolysis and transphosphorylation of phosphate monoesters . Site-directed mutagenesis was used to change the active-site residue Asp-153 to Ala and Asn . In the wild-type enzyme Asp-153 forms a second-sphere complex with Mg2+ . The activity of mutant enzymes D153N and D153A is dependent on the inclusion of Mg2+ in the assay buffer . The steady-state kinetic parameters of the D153N mutant display small enhancements, relative to wild type, in buffers containing 10 mM Mg2+ . In contrast, the D153A mutation gives rise to a 6.3-fold increase in kcat, a 13.7-fold increase in kcat/Km (50 mM Tris, pH 8), and a 159-fold increase in Ki for Pi (1 M Tris, pH 8) . In addition, the activity of D153A increases 25-fold as the pH is increased from 7 to 9 . D153A hydrolyzes substrates with widely differing pKa's of their phenolic leaving groups (PNPP and DNPP), at similar rates . As with wild type, the rate-determining step takes place after the initial nucleophilic displacement (k2) . The increase in kcat for the D153A mutant indicates that the rate of release of phosphate from the enzyme product complex (k4) has been enhanced.

Biochemistry, 1992 Sep 8, 31(35), 8221 - 8
Conformational changes of HIV reverse transcriptase subunits on formation of the heterodimer: correlation with kcat and Km; Anderson SF et al.; The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) is initially expressed as a 66-kDa protein and is subsequently proteolytically processed in vivo to form a 66-kDa/51-kDa heterodimer . Comparison of circular dichroism spectra of the 66-kDa, 51-kDa, and heterodimeric forms of RT indicates that the conversion is accompanied by dramatic changes in subunit conformation . The mean residue ellipticity per subunit at 220 nm decreases from -10.7 x 10(3) deg cm2 dmol-1 for the 66-kDa protein to -6 x 10(3) deg cm2 dmol-1 for the heterodimer . The same loss of ellipticity is observed whether the heterodimer is produced by proteolysis or by mixing a separately-expressed cloned 51-kDa subunit with the 66-kDa protein . Comparison with the spectrum of the cloned 51-kDa protein suggests that much of the conformational change arises from formation of the 51-kDa subunit but substantial changes occur in the remaining 66-kDa subunit as well . A kinetic analysis was performed to correlate these conformational changes with changes in enzyme function . Application of an integrated Michaelis-Menten equation to the catalysis of poly(dT) formation using a d(pT)20-poly(rA) primer-template shows that the kcat for the heterodimer is approximately half that of the 66 kDa enzyme, decreasing from 2.9 to 1.2 nucleotides/s upon formation of the heterodimer . However, km values for the primer-template decrease from 0.54 to 0.12 microM upon heterodimer formation . Thus, kcat/Km is 2-fold larger for the heterodimer, giving it a distinct catalytic advantage at undersaturating concentrations of enzyme and primer-template.(ABSTRACT TRUNCATED AT 250 WORDS)

Thromb Haemost, 1992 Sep 7, 68(3), 306 - 9
Effect of DDAVP on endotoxin-induced intravascular coagulation in rabbits; Paloma MJ et al.; We have evaluated the effect of 1-Deamino-8D-arginine vasopressin (DDAVP) on an experimental model of intravascular coagulation (DIC) induced in rabbits by injection of 20 micrograms kg-1 h-1 during 6 h of E . coli lipopolysaccharide . DDAVP significantly ameliorated the platelet drop and fibrinogen decrease (p less than 0.01) induced by endotoxin in control animals . A significant reduction in factor XII consumption (p less than 0.01) and a decrease in the generation of endotoxin induced PAI-1 activity in rabbits circulation was also observed (p less than 0.005) . Moreover, fibrin deposition in kidneys of rabbits receiving DDAVP was significantly reduced as compared to control animals . Finally, the mortality rate in the control group was significantly higher than in DDAVP-treated rabbits (p less than 0.01) . The hemostatic changes induced by DDAVP correlated with lower fibrin deposition and reduction in mortality rates.

FEBS Lett, 1992 Sep 7, 309(2), 157 - 60
Identification of heme macrocycle type by near-infrared magnetic circular dichroism spectroscopy at cryogenic temperatures; Peng Q et al.; The electron paramagnetic resonance (EPR) and near-infrared magnetic circular dichroism (MCD) spectra of the azide and cyanide adducts of nitrimyoglobin and hydroperoxidase II from Escherichia coli have been measured at cryogenic temperatures . For the first time, ligand-to-metal charge-transfer transitions in the near-infrared have been observed for an Fe(III)-chlorine system . It is shown that near-ultraviolet-to-visible region electronic spectra of 'green' hemes such as these are an unreliable indicator of macrocycle type . However, the combined application of EPR and near-infrared MCD spectroscopies clearly distinguishes between the porphyrin-containing nitrimyoglobin and the chlorine-containing hydroperoxidase II.

FEBS Lett, 1992 Sep 7, 309(2), 127 - 9
The formate complex of the cytochrome bo quinol oxidase of Escherichia coli exhibits a 'g = 12' EPR feature analogous to that of 'slow' cytochrome oxidase; Calhoun MW et al.; The cytochrome bo quinol oxidase of Escherichia coli is homologous in sequence and in structure to cytochrome aa3 type cytochrome oxidase in subunit I, which contains the catalytic core . The cytochrome bo enzyme forms a formate complex which exhibits 'g = 12' and 'g = 2.9' EPR signals at X band; similar signals have previously been observed only in association with the 'slow' and formate-ligand states of cytochrome oxidase . These signals arise from transitions within integral spin multiples identified with the homologous heme-copper binuclear catalytic centers in both enzymes.

J Mol Biol, 1992 Sep 5, 227(1), 72 - 80
Effect of terminal non-homology on intramolecular recombination of linear plasmid substrates in Escherichia coli; Luisi-DeLuca C et al.; Circular dimer plasmids linearized with a restriction endonuclease undergo intramolecular recombination to yield recombinant circular monomers at high efficiency by a recA-independent mechanism in Escherichia coli recB recC sbcA mutants . The rate of this reaction is at least 1000-fold higher than the recombination rate observed for circular plasmid recombination substrates in the same mutants . Three potential models have been previously proposed to explain the recombination events observed . The validity of these models was tested in recA recB recC sbcA mutants using additional recombination substrates . These substrates, when linearized by incubation with an appropriate restriction enzyme, contain non-homologous adenovirus 2 DNA on one or both ends . The data indicate that terminal non-homology does not significantly affect the efficiency of recovering recombinants . In contrast to many recombination models proposed that involve the invasion of homologous duplex DNA by single-stranded DNA ends, the intramolecular recombination reaction studied here does not appear to involve direct pairing from the end(s) of the substrate DNA . Furthermore, the results are consistent with a model proposing that pairing and strand exchange occur between two homologous duplex regions within the linear dimer molecule.

J Mol Biol, 1992 Sep 5, 227(1), 347 - 51
Crystallization and crystallographic characterization of the iron-sulfur-containing DNA-repair enzyme endonuclease III from Escherichia coli; Kuo CF et al.; Endonuclease III from Escherichia coli is an iron-sulfur enzyme possessing both DNA N-glycosylase and apurinic/apyrimidinic lyase activities . It could serve to repair damaged thymine residues in DNA via base excision-repair . We have crystallized endonuclease III by a combination of dialysis and seeding techniques after exploration of a wide variety of precipitants which failed to yield macroscopic crystals . Important features of the optimized crystallization include: the use of 5 to 10% glycerol, a temperature of 15 degrees C, controlled dialysis to decrease ionic strength and macroseeding using a 200 mM-NaCl transfer buffer to dissolve microcrystalline contamination . The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 48.5 A, b = 65.8 A, c = 86.8 A, alpha = beta = gamma = 90 degrees, have one 23 kDa monomer per asymmetric unit, and diffract to 1.84 A . A native anomalous Patterson map located the iron-sulfur cluster and reaffirmed its existence . The reported crystallization procedures ensure an ample supply of crystals for the extensive heavy-atom derivative search necessary for this labile iron-sulfur enzyme . The elucidation of endonuclease III structure will facilitate not only the understanding of glycosylase and lyase mechanisms but also the structure and function of this new class of iron-sulfur proteins.

J Mol Biol, 1992 Sep 5, 227(1), 334 - 46
Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible; Yu X et al.; We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli . The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity . The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix . Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state . The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds . A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal . This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.

J Mol Biol, 1992 Sep 5, 227(1), 283 - 92
Crystal structure of glycinamide ribonucleotide transformylase from Escherichia coli at 3.0 A resolution . A target enzyme for chemotherapy; Chen P et al.; The atomic structure of glycinamide ribonucleotide transformylase, an essential enzyme in purine biosynthesis, has been determined at 3.0 A resolution . The last three C-terminal residues and a sequence stretch of 18 residues (residues 113 to 130) are not visible in the electron density map . The enzyme forms a dimer in the crystal structure . Each monomer is divided into two domains, which are connected by a central mainly parallel seven-stranded beta-sheet . The N-terminal domain contains a Rossmann type mononucleotide fold with a phosphate ion bound to the C-terminal end of the first beta-strand . A long narrow cleft stretches from the phosphate to a conserved aspartic acid, Asp144, which has been suggested as an active-site residue . The cleft is lined by a cluster of residues, which are conserved between bacterial, yeast, avian and human enzymes, and likely represents the binding pocket and active site of the enzyme . GAR Tfase binds a reduced folate cofactor and glycinamide ribonucleotide for the catalysis of one of the initial steps in purine biosynthesis . Folate analogs and multi-substrate inhibitors of the enzyme have antineoplastic effects and the structure determination of the unliganded enzyme and enzyme-inhibitor complexes will aid the development of anti-cancer drugs.

J Mol Biol, 1992 Sep 5, 227(1), 197 - 213
Domain closure in mitochondrial aspartate aminotransferase; McPhalen CA et al.; The subunits of the dimeric enzyme aspartate aminotransferase have two domains: one large and one small . The active site lies in a cavity that is close to both the subunit interface and the interface between the two domains . On binding the substrate the domains close together . This closure completely buries the substrate in the active site and moves two arginine side-chains so they form salt bridges with carboxylate groups of the substrate . The salt bridges hold the substrate close to the pyridoxal 5'-phosphate cofactor and in the right position and orientation for the catalysis of the transamination reaction . We describe here the structural changes that produce the domain movements and the closure of the active site . Structural changes occur at the interface between the domains and within the small domain itself . On closure, the core of the small domain rotates by 13 degrees relative to the large domain . Two other regions of the small domain, which form part of the active site, move somewhat differently . A loop, residues 39 to 49, above the active site moves about 1 A less than the core of the small domain . A helix within the small domain forms the "door" of the active site . It moves with the core of the small domain and, in addition, shifts by 1.2 A, rotates by 10 degrees, and switches its first turn from the alpha to the 3(10) conformation . This results in the helix closing the active site . The domain movements are produced by a co-ordinated series of small changes . Within one subunit the polypeptide chain passes twice between the large and small domains . One link involves a peptide in an extended conformation . The second link is in the middle of a long helix that spans both domains . At the interface this helix is kinked and, on closure, the angle of the kink changes to accommodate the movement of the small domain . The interface between the domains is formed by 15 residues in the large domain packing against 12 residues in the small domain and the manner in which these residues pack is essentially the same in the open and closed structures . Domain movements involve changes in the main-chain and side-chain torsion angles in the residues on both sides of the interface . Most of these changes are small; only a few side-chains switch to new conformations.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1992 Sep 5, 267(25), 17809 - 19
Membrane topology of the pBR322 tetracycline resistance protein . TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion; Allard JD et al.; The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter . Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA . We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA . In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm . However, several TetA-PhoA fusions have unexpected properties . One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity . However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C . F . (1989) J . Biol . Chem . 264, 11663-11670) . PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity . In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287) . We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5 . (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions . We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance . The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e . long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.

J Biol Chem, 1992 Sep 5, 267(25), 17766 - 72
Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs) . Molecular basis for differences in requirements for activity of mammalian ARFs; Price SR et al.; ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase . Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid . Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified . To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized . Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity . Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate . In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP . rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding . These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.

J Biol Chem, 1992 Sep 5, 267(25), 17679 - 87
Escherichia coli serine hydroxymethyltransferase . The role of histidine 228 in determining reaction specificity; Stover P et al.; Serine hydroxymethyltransferase has a conserved histidine residue (His-228) next to the lysine residue (Lys-229) which forms the internal aldimine with pyridoxal 5'-phosphate . This histidine residue is also conserved at the equivalent position in all amino acid decarboxylases and tryptophan synthase . Two mutant forms of Escherichia coli serine hydroxymethyltransferase, H228N and H228D, were constructed, expressed, and purified . The properties of the wild type and mutant enzymes were studied with substrates and substrate analogs by differential scanning calorimetry, circular dichroism, steady state kinetics, and rapid reaction kinetics . The conclusions of these studies were that His-228 plays an important role in the binding and reactivity of the hydroxymethyl group of serine in the one-carbon-binding site . The mutant enzymes utilize substrates and substrate analogs more effectively for a variety of alternate non-physiological reactions compared to the wild type enzyme . As one example, the mutant enzymes cleave L-serine to glycine and formaldehyde when tetrahydropyteroylglutamate is replaced by 5-formyltetrahydropteroylglutamate . The released formaldehyde inactivates these mutant enzymes . The loss of integrity of the one-carbon-binding site with L-serine in the two mutant forms of the enzyme may be the result of these enzymes not undergoing a conformational change to a closed form of the active site when serine forms the external aldimine complex.

J Biol Chem, 1992 Sep 5, 267(25), 17920 - 4
An active covalently linked dimer of human interferon-gamma . Subunit orientation in the native protein; Lunn CA et al.; We have constructed and expressed a covalently linked head to tail dimer of human interferon-gamma (IFN-gamma) in which two monomers are joined head to tail via a rigid peptide hinge using genetic engineering techniques . The hinge was derived from the human immunoglobin IgA1 sequence (Hallewell, R.A., Laria, I., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., Cousens, L.S., and Mullenbach, G.T . (1989) J . Biol . Chem . 264, 5260-5268) . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the polypeptide produced by this construction migrates as a 30,000 polypeptide species . The protein elutes as a single species by molecular sieve chromatography under native conditions . The covalently linked dimer exhibits one-half the antiviral activity of native dimeric IFN-gamma; receptor binding assays show the covalently linked dimer binds to the IFN-gamma receptor with one-half the avidity of native IFN-gamma . This difference is not due to conformational differences between the two molecules, as the aromatic region of the NMR spectrum of the purified covalently linked dimer is identical with that of the wild type protein . From these data, we suggest that human IFN-gamma associates in a head to tail dimer in its active configuration . Regions of IFN-gamma are contiguous with the amino and carboxyl termini and are obscured by the hinge peptide in the covalently linked dimer . Our studies demonstrate that these regions may be important for receptor-ligand interaction.

J Biol Chem, 1992 Sep 5, 267(25), 17693 - 700
Active site of (A)BC excinuclease . II . Binding, bending, and catalysis mutants of UvrB reveal a direct role in 3' and an indirect role in 5' incision; Lin JJ et al.; UvrB plays a central role in (A)BC excinuclease . To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized . All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro . However, three mutants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity . All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically . The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA . However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3' site and promoted near-normal incision by UvrC at the 5' site . We propose that D478 is involved in bending DNA and catalysis of the 3' incision and that the 3' incision precedes the 5' incision . UvrB which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3' incision suggesting that it is missing a residue involved in catalysis . This residue was identified to be E639 by site-specific mutagenesis.

J Biol Chem, 1992 Sep 5, 267(25), 17688 - 92
Active site of (A)BC excinuclease . I . Evidence for 5' incision by UvrC through a catalytic site involving Asp399, Asp438, Asp466, and His538 residues; Lin JJ et al.; (A)BC excinuclease of Escherichia coli removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 15th phosphodiester bond 3' to the modified base . The activity results from the ordered action of UvrA, UvrB, and UvrC proteins . The role of UvrA is to help assemble the UvrB.DNA complex, and it is not involved in the actual incision reactions which are carried out by UvrB and UvrC . To investigate the role of UvrC in the nuclease activity a subset of His, Asp, and Glu residues in the C-terminal half of the protein were mutagenized in vitro . The effect of these mutations on UV resistance in vivo and incision activity in vitro were investigated . Mutations, H538F, D399A, D438A, and D466A conferred extreme UV sensitivity . Enzyme reconstituted with these mutant proteins carried out normal 3' incision but was completely defective in 5' incision activity . Our data suggest that UvrC makes the 5' incision by employing a mechanism whereby the three carboxylates acting in concert with H538 and a Mg2+ ion facilitate nucleophilic attack by an active site water molecule.

J Biol Chem, 1992 Sep 5, 267(25), 18175 - 81
Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase; Luo J et al.; Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit . Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity . This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit . In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase . It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase . In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C . N., Shin, Y . K., Slutsky, M., nd Goldfarb, A . (1990) Gene 89, 7-12) . The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting . The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876 . This region may be important in enzyme assembly or subunit-subunit interaction . The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093 . This region may be involved in the catalytic function of RNA polymerase.

J Biol Chem, 1992 Sep 5, 267(25), 17773 - 9
Effects of the chaperonin GroE on the refolding of tryptophanase from Escherichia coli . Refolding is enhanced in the presence of ADP; Mizobata T et al.; The refolding of the tetrameric enzyme tryptophanase was facilitated by the chaperonin GroE . Maximum refolding yield of tryptophanase molecules (about 80%) was attained in the presence of a 15-fold excess of GroE 21-mer over tryptophanase monomer . The GroEL subunit was required for this improvement in refolding yield, whereas the GroES subunit was not . Light scattering experiments of the refolding reaction revealed that GroE bound to tryptophanase folding intermediates and suppressed their aggregation . The presence of ATP was required for the efficient dissociation of tryptophanase from GroEL . However, our experiments indicated that tryptophanase dissociated readily from GroEL in the presence of not only ATP, but also in the presence of non-hydrolyzable ATP analogues such as ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) and AMP-PNP (adenyl-5'-yl imidodiphosphate) as well . Surprisingly, the release of tryptophanase from GroEL was facilitated in the presence of ADP as well . We concluded that the binding of nucleotides such as ATP and ADP changed the conformation of GroEL and facilitated the dissociation of tryptophanase molecules . The conformation formed in the presence of ADP was distinct from the conformation formed in the presence of ATP, as shown by the selective dissociation of various folding proteins from the two conformations.

J Biol Chem, 1992 Sep 5, 267(25), 17631 - 4
Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures; Mendoza JA et al.; The chaperonin protein cpn60 from Escherichia coli protects the monomeric, mitochondrial enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) against heat inactivation . The thermal inactivation of rhodanese was studied for four different states of the enzyme: native, refolded, bound to cpn60 in the form of a binary complex formed from unfolded rhodanese, and a thermally perturbed state . Thermal stabilization is observed in a range of temperatures from 25 to 48 degrees C . Rhodanese that had been inactivated by incubation at 48 degrees C, in the presence of cpn60 can be reactivated at 25 degrees C, upon addition of cpn10, K+, and MgATP . A recovery of about 80% was achieved after 1 h of the addition of those components . Thus, the enzyme is protected against heat inactivation and kept in a reactivable form if inactivation is attempted using the binary complex formed between rhodanese folding intermediate(s) and cpn60 . The chaperonin-assisted refolding of urea-denatured rhodanese is dependent on the temperature of the refolding reaction . However, optimal chaperonin assisted refolding of rhodanese observed at 25 degrees C, which is achieved upon addition of cpn10 and ATP to the cpn60-rhodanese complex, is independent of the temperature of preincubation of the complex, that was formed previously at low temperature . The results are in agreement with a model in which the chaperonin cpn60 interacts with partly folded intermediates by forming a binary complex which is stable to elevated temperatures . In addition, it appears that native rhodanese can be thermally perturbed to produce a state different from that achieved by denaturation that can interact with cpn60.

J Mol Biol, 1992 Sep 5, 227(1), 38 - 53
ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange; Konforti BB et al.; When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed . This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases . Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired . According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA . Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease . The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis . The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis.

Biochemistry, 1992 Aug 25, 31(33), 7736 - 40
Pertussis toxin-catalyzed ADP-ribosylation of G(o) alpha with mutations at the carboxyl terminus; Avigan J et al.; The guanine nucleotide-binding protein G(o alpha) has been implicated in the regulation of Ca2+ channels in neural tissues . Covalent modification of G(o alpha) by pertussis toxin-catalyzed ADP-ribosylation of a cysteine (position 351) four amino acids from the carboxyl terminus decouples G(o alpha) from receptor . To define the structural requirements for ADP-ribosylation, preparations of recombinant G(o alpha) with mutations within the five amino acids at the carboxyl terminus were evaluated for their ability to serve as pertussis toxin substrates . As expected, the mutant in which cysteine 351 was replaced by glycine (C351G) was not a toxin substrate . Other inactive mutants were G352D and L353 delta/Y354 delta . Mutations that had no significant effect on toxin-catalyzed ADP-ribosylation included G350D, G350R, Y354 delta, and L353V/Y354 delta . Less active mutants were L353G/Y354 delta, L353A/Y354 delta, and L353G . ADP-ribosylation of the active mutants, like that of wild-type G(o alpha), was enhanced by the beta gamma subunits of bovine transducin . It appears that three of the four terminal amino acids critically influence pertussis toxin-catalyzed ADP-ribosylation of G(o alpha).

J Biol Chem, 1992 Aug 25, 267(24), 16841 - 7
The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase; Li SJ et al.; We report characterization of the component proteins and molecular cloning of the genes encoding the two subunits of the carboxyltransferase component of the Escherichia coli acetyl-CoA carboxylase . Peptide mapping of the purified enzyme component indicates that the carboxyltransferase component is a complex of two nonidentical subunits, a 35-kDa alpha subunit and a 33-kDa beta subunit . The alpha subunit gene encodes a protein of 319 residues and is located immediately downstream of the polC gene (min 4.3 of the E . coli genetic map) . The deduced amino acid composition, molecular mass, and amino acid sequence match those determined for the purified alpha subunit . Six sequenced internal peptides also match the deduced sequence . The amino-terminal sequence of the beta subunit was found within a previously identified open reading frame of unknown function called dedB and usg (min 50 of the E . coli genetic map) which encodes a protein of 304 residues . Comparative peptide mapping also indicates that the dedB/usg gene encodes the beta subunit . Moreover, the deduced molecular mass and amino acid composition of the dedB/usg-encoded protein closely match those determined for the beta subunit . The deduced amino acid sequences of alpha and beta subunits show marked sequence similarities to the COOH-terminal half and the NH2-terminal halves, respectively, of the rat propionyl-CoA carboxylase, a biotin-dependent carboxylase that catalyzes a similar carboxyltransferase reaction reaction . Several conserved regions which may function as CoA-binding sites are noted.

Science, 1992 Sep 4, 257(5075), 1392 - 5
Cloning of the 62-kilodalton component of basic transcription factor BTF2; Fischer L et al.; Cloning of the mammalian basic transcription factors serves as a major step in understanding the mechanism of transcription initiation . The 62-kilodalton component (p62) of one of these transcription factors, BTF2 was cloned and overexpressed . A monoclonal antibody to this polypeptide inhibited transcription in vitro . Immunoaffinity experiments demonstrated that the 62-kilodalton component is closely associated with the other polypeptides present in the BTF2 factor . Sequence similarity suggests that BTF2 may be the human counterpart of RNA polymerase II initiation factor b from yeast.

Science, 1992 Sep 4, 257(5075), 1395 - 8
Modulation of the dimerization of a transcriptional antiterminator protein by phosphorylation; Amster-Choder O et al.; The transcriptional antiterminator protein BglG inhibits transcription termination of the bgl operon in Escherichia coli when it is in the nonphosphorylated state . The BglG protein is now shown to exist in two configurations, an active, dimeric nonphosphorylated form and an inactive, monomeric phosphorylated form . The migration of BglG on native polyacrylamide gels was consistent with it existing as a dimer when nonphosphorylated and as a monomer when phosphorylated . Only the nonphosphorylated dimer was found to bind to the target RNA . When the dimerization domain of the lambda repressor was replaced with BglG, the resulting chimera behaved like an intact lambda repressor in its ability to repress lambda gene expression, which suggests that BglG dimerizes in vivo . Repression by the lambda-BglG hybrid was significantly reduced by BglF, the BglG kinase, an effect that was relieved by conditions that stimulate dephosphorylation of BglG by BglF . These results suggest that the phosphorylation and the dephosphorylation of BglG regulate its activity by controlling its dimeric state.

Eur J Pharmacol, 1992 Sep 4, 219(3), 445 - 50
5-HT receptor antagonists and heat-stable Escherichia coli enterotoxin-induced effects in the rat; Beubler E et al.; The effect of heat-stable E . coli enterotoxin on intestinal fluid secretion is commonly considered to be mediated by stimulation of mucosal cyclic guanosine monophosphate (cGMP) . It was demonstrated recently that 5-hydroxytryptamine (5-HT) acts as an important mediator in cholera toxin-induced fluid secretion . To elucidate the possible involvement of 5-HT in the secretory response to heat-stable E . coli enterotoxin, in vivo experiments were performed in the rat jejunum . The inhibitory effects of the 5-HT2 receptor antagonist ketanserin, the 5-HT3 receptor antagonist tropisetron and indomethacin were studied in heat-stable E . coli enterotoxin-induced fluid secretion . Tropisetron and ketanserin (100 micrograms/kg each) alone only partially reduced the secretory effect of the toxin . However, in combination, the two blockers (100 plus 100 micrograms/kg) significantly reduced and at 200 plus 200 micrograms/kg totally abolished heat-stable E . coli enterotoxin-induced secretion without influencing the enterotoxin-induced increase in cGMP . Pretreatment with indomethacin (10 mg/kg) reduced the secretory response to the enterotoxin by about 50% . These results support the concept that 5-HT is an important mediator in intestinal fluid secretion induced by heat-stable E . coli enterotoxin . The enterotoxin may use 5-HT to stimulate prostaglandin formation via 5-HT2 receptors and to activate neuronal structures via 5-HT3 receptors.

Bull Math Biol, 1992 Sep, 54(5), 785 - 812
Poisson, compound Poisson and process approximations for testing statistical significance in sequence comparisons; Goldstein L et al.; DNA and protein sequence comparisons are performed by a number of computational algorithms . Most of these algorithms search for the alignment of two sequences that optimizes some alignment score . It is an important problem to assess the statistical significance of a given score . In this paper we use newly developed methods for Poisson approximation to derive estimates of the statistical significance of k-word matches on a diagonal of a sequence comparison . We require at least q of the k letters of the words to match where 0 less than q less than or equal to k . The distribution of the number of matches on a diagonal is approximated as well as the distribution of the order statistics of the sizes of clumps of matches on the diagonal . These methods provide an easily computed approximation of the distribution of the longest exact matching word between sequences . The methods are validated using comparisons of vertebrate and E . coli protein sequences . In addition, we compare two HLA class II transplantation antigens by this method and contrast the results with a dynamic programming approach . Several open problems are outlined in the last section.

Virology, 1992 Sep, 190(1), 522 - 6
Introduction of foreign DNA into the vaccinia virus genome by in vitro ligation: recombination-independent selectable cloning vectors; Merchlinsky M et al.; Homologous recombination has been the exclusive means of introducing foreign DNA into the genomes of large DNA viruses . Here we demonstrate that direct in vitro ligation can be used to efficiently insert DNA fragments of up to 26,000 bp into the genome of vaccinia virus modified to contain a single NotI site either in the Escherichia coli lacZ gene or in the vaccinia virus thymidine kinase gene . Viruses containing chimeric genomes can be identified by chromogenic screening or thymidine-kinase-negative selection.

Virology, 1992 Sep, 190(1), 510 - 4
Release of a 22-kDa protein derived from the amino-terminal domain of the 49-kDa NIa of turnip mosaic potyvirus in Escherichia coli; Laliberte JF et al.; The coding region for the precursor 6K-small nuclear inclusion a (NIa) protein and for the NIa protein of turnip mosaic potyvirus (TuMV) were introduced into the plasmid pET-11d for high-level expression in Escherichia coli . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses of E . coli proteins showed that the NIa protein underwent endoproteolysis and released a 22-kDa polypeptide . NH2-terminal amino acid sequencing of the recombinant 22-kDa protein was performed and was identical to the predicted amino end of the NIa protein . Site-directed mutagenesis confirmed that the hydrolysis was associated with the NIa proteolytic activity and that the proteinase recognized a Glu residue within an amino acid sequence found in the NIa protein which fitted the TuMV consensus cleavage site sequence . Fusion of the 6K protein with the NIa protein partially inhibited the hydrolytic reaction . The recombinant 22-kDa protein is likely the VPg of TuMV.

FEMS Microbiol Lett, 1992 Sep 1, 75(1), 37 - 42
Chemotaxis of Leptospirillum ferrooxidans and other acidophilic chemolithotrophs: comparison with the Escherichia coli chemosensory system; Acuna J et al.; Ni2+, Fe2+ and Cu2+ were attractants and aspartate was an apparent repellent for Leptospirillum ferrooxidans, a behaviour opposite to that for Escherichia coli . Membranes from L . ferrooxidans contained proteins with a molecular mass in the range of 80 kDa which were methylated in vitro . Methylation was stimulated in the presence of a membrane-free extract from E . coli, showing the response pattern expected for L . ferrooxidans, increased methylation by Ni2+, and demethylation by aspartate . This suggests the existence of sensory transducers having a common methylation domain with the E . coli methyl-accepting chemotaxis proteins . Total chromosomal DNA digests from L . ferrooxidans, Thiobacillus ferrooxidans and T . thiooxidans hybridized with probes containing different domains of the tar gene from E . coli, implying the presence of tar type genes in the acidophilic bacteria studied.

Clin Chem, 1992 Sep, 38(9), 1824 - 9
Immunochemical detection of group I and group II phospholipases A2 in human serum; Nevalainen TJ et al.; Time-resolved fluoroimmunoassay was developed for the detection of synovial-type phospholipase A2 (s-PLA2) in human serum . This solid-phase, sandwich assay uses a polyclonal rabbit antibody raised against synovial-type group II PLA2 produced in Escherichia coli . No cross-reactions were detected between s-PLA2 and PLA2 from human or porcine pancreas, human ascitic fluid, or bee or cobra venom . In healthy individuals, the average concentration of s-PLA2 is 3.7 micrograms/L, with a 95% reference interval from 1.3 to 10.8 micrograms/L . We investigated pancreatic PLA2, which is a group I PLA2, and synovial-type group II PLA2 in sera of patients with hematological malignancies and septic fever . The concentration of s-PLA2 was increased in patient sera and correlated significantly with the catalytic activity of PLA2 and the concentration of C-reactive protein . No correlation with the concentration of pancreatic PLA2 was found . The results suggest that the increased catalytic activity of PLA2 in sera of patients with septic fever results from synovial-type group II PLA2.

J Steroid Biochem Mol Biol, 1992 Sep, 42(8), 803 - 12
Association of the 90-kDa heat shock protein does not affect the ligand-binding ability of androgen receptor; Nemoto T et al.; An N-terminal truncated androgen receptor with putative DNA- and ligand-binding domains (AR438) and that with a ligand-binding domain (AR612) were expressed under control of the T7 promoter in E . coli or translated in vitro with rabbit reticulocyte lysate, and their ligand-binding properties and the interaction with HSP90 were investigated . Bacterially expressed AR438 and AR612 bound a synthetic androgen, {3H}R1881, with apparent dissociation constants of 2.6 +/- 0.2 and 3.1 +/- 0.7 nM, respectively, values which are comparable to those of androgen receptor in target tissues . The recombinant androgen receptors sedimented at the 4-5 S region irrespective of the presence of 10 mM tungstate, indicating that the receptor exists free from HtpG, which is the bacterial homolog of eukaryotic HSP90 . The apparent dissociation constant of truncated androgen receptors translated in vitro was 0.1 nM for AR438 and 0.2 nM for AR612 . Sedimentation coefficients of in vitro translated molecules were converted from 7-8 S in the presence of tungstate to 3 S in the absence of tungstate . Both AR438 and AR612 translated in vitro were retained by anti-rat HSP90 antibody-protein A Sepharose . Exposure to 0.3 M NaCl in the presence of ligand caused dissociation of AR438 and AR612 from HSP90, and concomitantly, the DNA-cellulose binding ability of AR438 was enhanced . Thus, we conclude that the androgen receptor associates with HSP90 through the ligand-binding domain and that this association prevents the interaction of the androgen receptor with DNA . However, HSP90 seems to have little effect on the ligand-binding characteristics of the androgen receptor.

J Protozool, 1992 Sep-Oct, 39(5), 609 - 12
Amphotericin B-induced carbohydrate changes on the Trypanosoma cruzi surface membrane; Haido RM et al.; Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities . Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe . Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated . Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCl3/methanol/H2O soluble fraction . These sugars in 1.4:1 ratio were the major hexose components of control cells . The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T . cruzi cell surface receptors for fimbriated E . coli K12 . Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results . Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.

J Bacteriol, 1992 Sep, 174(18), 5961 - 70
Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100; Dallas WS et al.; The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced . The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons . Increased expression of the enzyme was realized by using a T7 expression system . The enzyme was purified and crystallized . A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole . The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences.

J Bacteriol, 1992 Sep, 174(18), 5953 - 60
Determination of the mechanism of retrotransfer by mechanistic mathematical modeling; Top E et al.; Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that is, the ability of conjugative plasmids to mobilize genes into the cell containing the conjugative plasmid, were developed . This study deals with retromobilization of nonconjugative plasmids (Tra-Mob+) . Plasmid transfer was modeled by two mass action models . The first is based on the hypothesis that retromobilization of the Tra-Mob+ vector occurs in one step, by means of the pilus formed by the Tra+ plasmid in the original host . In the second model, retromobilization is considered to be a two-step process involving two transfer events . The first step involves the transfer of the Tra+ plasmid from the recipient cell to the donor of the nonconjugative vector, and during the second encounter the nonconjugative vector is mobilized toward the recipient . Since the relationships between the number of transconjugants and the number of recipients for the two models are different, filter matings were performed for short time periods with different initial densities of the recipient population . Comparison of the numbers of transconjugants with the results of the mathematical equations confirmed the hypothesis that retromobilization is a one-step conjugation process.

Eur J Biochem, 1992 Sep 1, 208(2), 475 - 80
Site-specific mutagenesis of Escherichia coli asparaginase II . None of the three histidine residues is required for catalysis; Wehner A et al.; Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids . The following enzyme variants were studied: {H87A}EcA2, {H87L}EcA2, {H87K}EcA2, {H183L}EcA2 and {H197L}EcA2 . None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding . The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values . These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis . Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II . A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87 . The fact that {H183L}EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer . 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.

Eur J Biochem, 1992 Sep 1, 208(2), 443 - 9
Mechanistic studies of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase from Escherichia coli; Kohen A et al.; The anomeric specificity and the steady-state kinetic mechanism of homogeneous 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase were investigated . The open-chain 4-deoxy analogue of arabinose-5-phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase . It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate . The kcat/Km value for the natural substrate is seven-times greater than that of the 4-deoxy analogue . However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4-deoxy analogue and Ara5P in solution, then the 'true' Km values must be in the range 31.5 microM and 0.26 microM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4-hydroxyl group of Ara5P . The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate . The steady-state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and D-ribose-5-phosphate as a dead-end inhibitor . First, intersecting lines in double-reciprocal plots of initial-velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme-catalyzed reaction . The inhibition by D-ribose-5-phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P-pyruvate) . These inhibition patterns are consistent with the model wherein P-pyruvate binding precedes that of Ara5P binding . Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P-pyruvate binding, supporting the concept that KDO8P and P-pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P . In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P-pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P . In conclusion, these data suggest a steady-state kinetic mechanism for KDO8P synthase where P-pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P.

Eur J Biochem, 1992 Sep 1, 208(2), 351 - 7
Protein structure of pig liver 4-aminobutyrate aminotransferase and comparison with a cDNA-deduced sequence; De Biase D et al.; The amino acid sequence of pig liver 4-aminobutyrate aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments . The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work {Kwon, O., Park, J . & Churchich, J . E . (1992) J . Biol . Chem . 267, 7215-7216} . Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107-142 . Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work . The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli.

Eur J Biochem, 1992 Sep 1, 208(2), 251 - 7
Site-directed mutagenesis of elongation factor Tu . The functional and structural role of residue Cys81; Anborgh PH et al.; A Cys residue located in the second consensus sequence element (DCPG) of the GTP-binding region is highly conserved in bacterial elongation factors (EF) Tu . Chemical modification of this Cys81 in EF-Tu from Escherichia coli by N-tosyl-L-phenylalanine chloromethane {Jonak, J., Petersen, T . E., Clark, B . F . C . & Rychlik, I . (1982) FEBS Lett . 150, 485-488}, and of homologous Cys residues in other bacterial EF-Tu, selectively blocks the binding of Xaa-tRNA . We have substituted Cys81 with Gly using site-directed mutagenesis of the EF-Tu-encoding tuf A gene . This substitution induces a partial inhibition (20-70%) of: (a) poly(U)-directed poly(Phe) synthesis; (b) EF-Tu/Xaa-tRNA interaction, determined as protection by EF-Tu of the non-enzymic deacylation of Xaa-tRNA; (c) EF-Tu-dependent binding of Xaa-tRNA to the mRNA/ribosome complex and (d) the intrinsic GTPase reaction, that is also less sensitive to stimulation by Xaa-tRNA . Our results thus provide evidence that Cys81, though important, is not essential for the binding of Xaa-tRNA to EF-Tu . The accuracy in poly(Phe) synthesis, measured as misincorporation of Leu, was increased . Both the binding affinity of {C81G}EF-Tu for the nucleotide and the resistance against thermal denaturation are more strongly decreased in the case of the GDP-bound state than in the case of the GTP-bound state, suggesting that Cys81 plays a more specific role in the former conformation . The sensitivity to N-tosyl-L-phenylalanine chloromethane is decreased by 80% but not totally lost . The inhibition by N-tosyl-L-phenylalanine chloromethane treatment of the function of EF-Tu appears to be a consequence of steric hindrance and/or of an altered conformation of EF-Tu.GTP . The lower activities of {C81G}EF-Tu are probably due to long-range effects, mediated by an overall destabilization of the molecule that is particularly pronounced for the GDP-bound state.

Am Rev Respir Dis, 1992 Sep, 146(3), 730 - 4
Effects of N-acetylcysteine on diaphragmatic function and malondialdehyde content in Escherichia coli endotoxemic rats; Van Surell C et al.; We evaluated the effects of sublethal Escherichia coli endotoxemia with or without concomitant administration of N-acetylcysteine, an antioxidant agent, on diaphragmatic strength, endurance, and malondialdehyde (MDA) content in rats . One hundred ninety rats were inoculated subcutaneously on 2 successive days with 0.6 and 1.2 mg/100 g body weight of E . coli lipopolysaccharide respectively (E animals, n = 100) or saline (C group, n = 90) . E and C animals were divided into two groups based on administration of endotoxin or saline alone (E group, n = 55; C group, n = 47, respectively) or endotoxin or saline plus N-acetylcysteine (1 g/kg body weight/day intraperitoneally) (E-NAC group, n = 45; C-NAC group, n = 43, respectively) . Diaphragmatic strength was assessed in vivo 48 h after the first endotoxin or saline administration by measuring the transdiaphragmatic pressure (Pdl) generated during electrical stimulation of the phrenic nerves at 0.5, 10, 20, 30, 50, and 100 Hz . Endurance index was calculated as the percent ratio of Pdl generated after 30 s of phrenic stimulation at 10 Hz divided by the initial force . Diaphragmatic MDA (fluorometric technique) was measured 0, 6, 18, 30, 42, and 48 h after the first dose of endotoxin or saline . Pdl for 50 and 100 Hz was significantly reduced in Group E as compared with group C . This phenomenon was associated with a reduced endurance performance as assessed by a lower diaphragmatic endurance index in E as compared with C animals (90.9 +/- 4.2 versus 114.3 +/- 4.1 respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Scand J Immunol, 1992 Sep, 36(3), 371 - 84
Antibodies against secreted and non-secreted antigens in mice after infection with live Mycobacterium tuberculosis; Verbon A et al.; Mice from four different inbred strains were infected with live Mycobacterium tuberculosis and the immune response to M . tuberculosis was followed for 24 weeks, using Western blotting . Nearly all mice, irrespective of H-2 type, reacted with the 38-kDa protein band . Antibodies against this secreted 38-kDa protein were the first to appear, 4 weeks after infection . Thereafter the secreted 19-kDa protein and non-secreted antigens, such as the 65-kDa and 33-kDa proteins, were recognized . The immune response against the non-secreted antigens was influenced by the mouse strain . However, the 33-kDa protein band was recognized by all mouse strains after a second injection with live M . tuberculosis . The specificity of the antibodies was analysed in Western blot using sonicates of M . tuberculosis, M . kansasii, M . avium, M . terrae, M . gordonae and Escherichia coli . Antibodies against the 38-kDa and 33-kDa protein bands seemed to be specific for M . tuberculosis, while antibodies against the 19-kDa protein band showed limited cross-reactivity . Antibodies against the 65-kDa protein were strongly cross-reactive . These results suggest that the 38-kDa protein is secreted in vivo and, therefore, may be available to the humoral immune system at an early stage of infection . The non-secreted 33-kDa protein is only recognized by all mouse strains after prolonged contact with M . tuberculosis.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8073 - 7
A plant cDNA that partially complements Escherichia coli recA mutations predicts a polypeptide not strongly homologous to RecA proteins; Pang Q et al.; A plant (Arabidopsis thaliana) cDNA previously selected for its ability to partially complement the UV sensitivity of Escherichia coli RecA-UvrC-Phr- mutants and designated DRT100 (DNA-damage repair/toleration) was subcloned into a high-copy-number plasmid and expressed via a bacterial promotor . It increased resistance of RecA-UvrB-Phr- bacteria to mitomycin C and methyl methanesulfonate as well as to UV light . This lack of specificity, and its ability to increase resistance in both UvrB- and UvrC- mutants, suggested that Drt100 activity might be complementing RecA- phenotypes . DRT100 partially complemented three RecA- phenotypes thought to reflect deficiencies in homologous recombination--namely, inability to plate lambda red-gam- phages and P1 phages and to recombinationally integrate donor DNA during conjugal crosses--but did not complement inability to induce E . coli SOS functions . The 395-amino acid DRT100 open reading frame encodes an apparent N-terminal chloroplast transit peptide and a putative 322-residue mature protein with a conserved nucleotide binding motif, but otherwise little global homology with bacterial RecA proteins . There are several tandemly repeated leucine-rich motifs . DNA from two closely related plants, but not from maize, hybridized strongly to a DRT100 cDNA probe.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8068 - 72
A homolog of Escherichia coli RecA protein in plastids of higher plants; Cerutti H et al.; Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids . However, no studies have been done of the biochemical pathways involved . Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sativum and Arabidopsis thaliana and in a cDNA library from Arabidopsis . A cDNA was cloned and sequenced, and its predicted amino acid sequence is 60.7% identical to that of the cyanobacterial RecA protein . This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 7890 - 4
Ligand occupancy mimicked by single residue substitutions in a receptor: transmembrane signaling induced by mutation; Yaghmai R et al.; We used mixed, mutagenic oligonucleotides to create single amino acid substitutions in the bacterial chemoreceptor Trg . Mutagenesis was directed at a 20-residue segment of the periplasmic domain implicated in ligand recognition . Transmembrane signaling by the mutant receptors was assayed in vivo by monitoring adaptational covalent modification . Among 20 functionally altered but stable receptors there were two distinct signaling phenotypes . Insensitive receptors did not signal upon stimulation and thus appeared defective in productive ligand interaction . Mimicked-occupancy receptors exhibited transmembrane signaling without ligand . Many mimicked-occupancy receptors produced additional signaling upon ligand binding and in appropriate conditions mediated effective chemotaxis; most insensitive receptors did not . Like normal receptors with one binding site occupied, mimicked-occupancy proteins adapted to persistent transmembrane signaling by increased methylation and thus could respond to other stimuli . Signaling phenotypes were strikingly segregated by residue position . Substitutions mimicking ligand occupancy occurred in half the segment, and those creating insensitive phenotypes occurred in the other half . These observations could be related to the three-dimensional structure of the periplasmic domain of the Tar(s) chemoreceptor . Insensitive substitutions occurred near the distal end of helix 1, where bulky protein ligands could interact; occupancy-mimicking substitutions were on the same helix at positions buried in the subunit interface between helices 1 and 1' . Thus perturbation of the interface induced transmembrane signaling, implicating changes at that interface in signal transduction, a conclusion consistent with differences in crystal structures of unoccupied and ligand-occupied Tar(s).

Genes Dev, 1992 Sep, 6(9), 1679 - 94
Stable synapsis of homologous DNA molecules mediated by the Escherichia coli RecA protein involves local exchange of DNA strands; Adzuma K; Escherichia coli RecA protein promotes stable synapsis between a single-stranded DNA and a homologous duplex DNA, resulting in the formation of a complex of RecA with three DNA strands . To gain insight into the molecular interactions responsible for DNA synapsis, the base-pairing status within the synaptic complex was analyzed by using dimethylsulfate and potassium permanganate as probes . The results indicate that the original base pairs in the parental duplex are disrupted; one strand is displaced and the other strand appears to be involved in Watson-Crick base-pairing with the incoming single-stranded DNA . The state of base-pairing thus resembles that of the end products of strand exchange and not a canonical DNA triple helix involving non-Watson-Crick base-pairing . The results also indicate that this local strand exchange can occur without homology at the ends of the DNA substrates (i.e., when axial rotation of the product heteroduplex with respect to the axis of the parental duplex is obstructed) . Taken together, these results suggest that exchange of DNA strands mediated by RecA occur at or before the stage of stable DNA synapsis by a process that does not require DNA rotation.

J Cell Biol, 1992 Sep, 118(5), 1085 - 95
Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod; Hodge TP et al.; A short nonhelical sequence at the COOH-terminus of vertebrate nonmuscle myosin has been shown to enhance myosin filament assembly . We have analyzed the role of this sequence in chicken intestinal epithelial brush border myosin, using protein engineering/site-directed mutagenesis . Clones encoding the rod region of this myosin were isolated and sequenced . They were truncated at various restriction sites and expressed in Escherichia coli, yielding a series of mutant myosin rods with or without the COOH-terminal tailpiece and with serial deletions from their NH2-termini . Deletion of the 35 residue COOH-terminal nonhelical tailpiece was sufficient to increase the critical concentration for myosin rod assembly by 50-fold (at 150 mM NaCl, pH 7.5), whereas NH2-terminal deletions had only minor effects . The only exception was the longest NH2-terminal deletion, which reduced the rod to 119 amino acids and rendered it assembly incompetent . The COOH-terminal tailpiece could be reduced by 15 amino acids and it still efficiently promoted assembly . We also found that the tailpiece promoted assembly of both filaments and segments; assemblies which have different molecular overlaps . Rod fragments carrying the COOH-terminal tailpiece did not promote the assembly of COOH-terminally deleted material when the two were mixed together . The tailpiece sequence thus has profound effects on assembly, yet it is apparently unstructured and can be bisected without affecting its function . Taken together these observations suggest that the nonhelical tailpiece may act sterically to block an otherwise dominant but unproductive molecular interaction in the self assembly process and does not, as has been previously thought, bind to a specific target site(s) on a neighboring molecule.

J Bacteriol, 1992 Sep, 174(17), 5661 - 8
Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent; Snyder WB et al.; We have used fusions of the outer membrane protein LamB to beta-galactosidase (encoded by lacZ) to study the protein export process . This LamB-LacZ hybrid protein blocks export when synthesized at high levels, as evidenced by inducer (maltose) sensitivity, a phenomenon termed LacZ hybrid jamming . The prlF1 mutation relieves LacZ hybrid jamming and allows localization of the fusion protein to a noncytoplasmic compartment . prlF1 and similar alleles are gain-of-function mutations . Null mutations in this gene confer no obvious phenotypes . Extragenic suppressors of a gain-of-function prlF allele have been isolated in order to understand how this gene product affects the export process . The suppressors are all lon null mutations, and they are epistatic to all prlF phenotypes tested . Lon protease activity has been measured in prlF1 cells and shown to be increased . However, the synthesis of Lon is not increased in a prlF1 background, suggesting a previously unidentified mechanism of Lon activation . Further analysis reveals that prlF1 activates degradation of cytoplasmically localized precursors in a Lon protease-dependent manner . It is proposed that accumulation of precursors during conditions of hybrid protein jamming titrates an essential export component(s), possibly a chaperone . Increased Lon-dependent precursor degradation would free this component, thus allowing increased protein export under jamming conditions.

J Bacteriol, 1992 Sep, 174(17), 5617 - 23
Purification and phosphorylation of the Arc regulatory components of Escherichia coli; Iuchi S et al.; In Escherichia coli, a two-component signal transduction system, consisting of the transmembrane sensor protein ArcB and its cognate cytoplasmic regulatory protein ArcA, controls the expression of genes encoding enzymes involved in aerobic respiration . ArcB belongs to a subclass of sensors that have not only a conserved histidine-containing transmitter domain but also a conserved aspartate-containing receiver domain of the regulator family . 'ArcB (a genetically truncated ArcB missing the two transmembrane segments on the N-terminal end) and ArcA were purified from overproducing cells . Autophosphorylation of 'ArcB was revealed when the protein was incubated with {gamma-32P}ATP but not with {alpha-32P}ATP or {gamma-32P}GTP . When ArcA was incubated in the presence of 'ArcB and {gamma-32P}ATP, ArcA acquired radioactivity at the expense of the phosphorylated protein 'ArcB-32P . When a limited amount of 'ArcB was incubated with excess ArcA and {gamma-32P}ATP, ArcA-32P increased linearly with time . Under such conditions, for a given time period the amount of ArcA phosphorylated was proportional to the concentration of 'ArcB . Thus, 'ArcB acted as a kinase for ArcA . Chemical stabilities of the phosphorylated proteins suggested that 'ArcB-32P contained both a histidyl phosphate and an aspartyl phosphate(s) and that ArcA-32P contained only an aspartyl phosphate(s).

J Bacteriol, 1992 Sep, 174(17), 5597 - 603
Mini-F plasmid mutants able to replicate in Escherichia coli deficient in the DnaJ heat shock protein; Ishiai M et al.; A subset of Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, is required for mini-F plasmid replication, presumably at the step of functioning of the RepE initiator protein . We have isolated and characterized mini-F plasmid mutants that acquired the ability to replicate in the Escherichia coli dnaJ259 . The mutant plasmids were found to replicate in any of dnaJ, dnaK, and grpE mutant hosts tested . In each case, the majority of the mutant plasmids carried a unique amino acid alteration in a localized region of repE coding sequence and showed an increased copy number, whereas the minority contained a common single base change (C to T) in the promoter/operator region and produced an increased amount of RepE . All RepE proteins with altered residues (between 92 and 134) exhibited increased initiator activities (hyperactive), and many showed reduced repressor activities as well, indicating that this region is important for the both major functions of RepE protein . These results together with evidence reported elsewhere indicate that the subset of heat shock proteins serves to activate RepE protein prior to or during its binding to the replication origin and that the mutant RepE proteins are active even in their absence . We also found that a C-terminal lesion (repE602) reduces the initiator activity particularly of some hyperactive mutant RepE proteins but does not affect the repressor activity . This finding suggests a functional interaction between the central and C-terminal regions of RepE in carrying out the initiator function.

J Bacteriol, 1992 Sep, 174(17), 5533 - 9
Anaerobic fumarate transport in Escherichia coli by an fnr-dependent dicarboxylate uptake system which is different from the aerobic dicarboxylate uptake system; Engel P et al.; Escherichia coli grown anaerobically with fumarate as electron acceptor is able to take up C4-dicarboxylates by a specific transport system . The system differs in all tested parameters from the known aerobic C4-dicarboxylate transporter . The anaerobic transport system shows higher transport rates (95 mumol/g {dry weight} per min versus 30 mumol/g/min) and higher Kms (400 versus 30 microM) for fumarate than for the aerobic system . Mutants lacking the aerobic dicarboxylate uptake system are able to grow anaerobically at the expense of fumarate respiration and transport dicarboxylates with wild-type rates after anaerobic but not after aerobic growth . Transport by the anaerobic system is stimulated by preloading the bacteria with dicarboxylates . The anaerobic transport system catalyzes homologous and heterologous antiport of dicarboxylates, whereas the aerobic system operates only in the unidirectional mode . The anaerobic antiport is measurable only in anaerobically grown bacteria with fnr+ backgrounds . Additionally, the system is inhibited by incubation of resting bacteria with physiological electron acceptors such as O2, nitrate, dimethyl sulfoxide, and fumarate . The inhibition is reversed by the presence of reducing agents . It is suggested that the physiological role of the system is a fumarate/succinate antiport under conditions of fumarate respiration.

J Bacteriol, 1992 Sep, 174(17), 5496 - 507
A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome; Kieser HM et al.; The restriction enzymes AseI (ATTAAT), DraI (TTTAAA), and SspI (AATATT) cut the Streptomyces coelicolor A3(2) chromosome into 17, 8, and 25 fragments separable by pulsed-field gel electrophoresis (PFGE) . The sums of their lengths indicated that the chromosome consists of about 8 Mb of DNA, some 75% more than that of Escherichia coli K-12 . A physical map of the chromosome was constructed for AseI and DraI, using single and double digests, linking clones, cross-hybridization of restriction fragments, and locations of genetically mapped genes, insertion sequences, prophages, and the integrated SCP1 and SLP1 plasmids on the physical map . The physical map was aligned with the previously established genetic map, revealing that the two long opposite quadrants of the genetic map that are almost devoid of markers (the silent regions at 3 o'clock and 9 o'clock) are indeed physically long rather than being hot spots for genetic exchange . They must therefore contain long stretches of DNA different in function from the remainder of the genome . Consistent with this conclusion are the locations of significant deletions in both of the silent regions . Of these, a 40-kb deletion in the 9 o'clock region accompanied or followed integration of the SCP1 linear plasmid to produce the NF fertility state . PFGE analysis of Streptomyces lividans 66, a close relative of S . coelicolor A3(2), was hampered by the previously described susceptibility of its DNA to degradation during electrophoresis . However, ZX7, a mutant derivative of S . lividans lacking the DNA modification responsible for this degradation, yielded good PFGE preparations . Not more than 7 of the 17 S . coelicolor AseI fragments could be shared by the S . lividans strain.

Biochemistry, 1992 Sep 1, 31(34), 7855 - 61
Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease; Grasby JA et al.; The stereochemical course of the reaction catalyzed by the EcoRV restriction endonuclease has been determined . This endonuclease recognizes GATATC sequence and cuts between the central T and dA bases . The Rp isomer of d(GACGATsATCGTC) (this dodecamer contains a phosphorothioate rather than the usual phosphate group between the central T and dA residues, indicated by the s) was a substrate for the endonuclease . Performing this reaction in H2 18O gave {18O}dps(ATCGTC) (a pentamer containing an 18O-labeled 5'-phosphorothioate) which was converted to {18O}dAMPS with nuclease P1 . This deoxynucleoside 5'-{18O}phosphorothioate was stereospecifically converted to {18O}dATP alpha S with adenylate kinase and pyruvate kinase {Brody, R . S., & Frey, P . A . (1981) Biochemistry 20, 1245-1251} . Analysis of the position of the 18O in this product by 31P NMR spectroscopy showed that it was in a bridging position between the alpha- and beta-phosphorus atoms . This indicates that the EcoRV hydrolysis proceeds with inversion of configuration at phosphorus . The simplest interpretation is that the mechanism of this endonuclease involves a direct in-line attack at phosphorus by H2O with a trigonal bipyramidal transition state . A covalent enzyme oligodeoxynucleotide species can be discounted as an intermediate . An identical result has been previously observed with the EcoR1 endonuclease {Connolly, B . A., Eckstein, F., & Pingoud, A . (1984) J . Biol . Chem . 259, 10760-10763} . X-ray crystallography has shown that both of these endonucleases contain a conserved array of amino acids at their active sites . Possible mechanistic roles for these conserved amino acids in the light of the stereochemical findings are discussed.

Biochemistry, 1992 Sep 1, 31(34), 7848 - 54
Enzymatic catalysis of prolyl isomerization in an unfolding protein; Mucke M et al.; Prolyl isomerases are able to accelerate slow steps in protein refolding that are limited in rate by cis/trans isomerizations of Xaa-Pro peptide bonds . We show here that prolyl isomerizations in the course of protein unfolding are also well catalyzed . To demonstrate catalysis we use cytoplasmic prolyl isomerase from Escherichia coli as the enzyme and reduced and carboxymethylated ribonuclease T1 as the substrate . This form of ribonuclease T1 without disulfide bonds is nativelike folded only in the presence of moderate concentrations of NaCl . Unfolding can be induced by reducing the NaCl concentration at ambient temperature and in the absence of denaturants . Under these conditions prolyl isomerase retains its activity and it catalyzes prolyl cis/trans isomerization in the unfolding protein . Under identical conditions within the NaCl-induced transition unfolding and refolding are catalyzed with equal efficiency . The stability of the protein and thus the final distribution of unfolded and folded molecules attained at equilibrium is unchanged in the presence of prolyl isomerase . These results demonstrate that prolyl isomerase functions in protein folding as an enzyme and catalyzes prolyl isomerization in either direction.

Biochemistry, 1992 Sep 1, 31(34), 7834 - 40
Complementary perturbation of the kinetic mechanism and catalytic effectiveness of dihydrofolate reductase by side-chain interchange; Wagner CR et al.; The variable residue Leu-28 of Escherichia coli dihydrofolate reductase (DHFR) and the corresponding residue Phe-31 in murine DHFR were interchanged, and the impact on catalysis was evaluated by steady-state and pre-steady-state analysis . The E . coli L28F mutant increased the pH-independent kcat from 11 to 50 s-1 but had little effect on Km(H2F) . An increase in the rate constant for dissociation of H4F from E.H4F.NH (from 12 to 80 s-1) was found to be largely responsible for the increase in kcat . Unexpectedly, the rate constant for hydride transfer increased from 950 to 4000 s-1 with little perturbation of NADPH and NADP+ binding to E . Consequently, the flux efficiency of the E . coli L28F mutant rose from 15% to 48% and suggests a role in genetic selection for this variable side chain . The murine F31L mutant decreased the pH-independent kcat from 28 to 4.8 s-1 but had little effect on Km(H2F) . A decrease in the rate constant for dissociation of H4F from E.H4F.NH (from 40 to 22 s-1) and E.H4F (from 15 to 0.4 s-1) was found to be mainly responsible for the decrease in kcat . The rate constant for hydride transfer decreased from 9000 to 5000 s-1 with minor perturbation of NADPH binding . Thus, the free energy differences along the kinetic pathway were generally similar in magnitude but opposite in direction to those incurred by the E . coli L28F mutant . This conclusion implies that DHFR hydrophobic active-site side chains impart their characteristics individually and not collectively.

Biochemistry, 1992 Sep 1, 31(34), 7826 - 33
Functional role of a mobile loop of Escherichia coli dihydrofolate reductase in transition-state stabilization; Li L et al.; The function of a highly mobile loop in Escherichia coli dihydrofolate reductase was studied by constructing a mutant (DL1) using cassette mutagenesis that had four residues deleted in the middle section of the loop (Met16-Ala19) and a glycine inserted to seal the gap . This part of the loop involves residues 16-20 and is disordered in the X-ray crystal structures of the apoprotein and the NADP+ binary complex but forms a hairpin turn that folds over the nicotinamide moiety of NADP+ and the pteridine moiety of folate in the ternary complex {Bystroff, C., & Kraut, J . (1991) Biochemistry 30, 2227-2239} . The steady-state and pre-steady-state kinetics and two-dimensional 1H NMR spectra were analyzed and compared to the wild-type protein . The kinetics on the DL1 mutant enzyme show that the KM value for NADPH (5.3 microM), the KM for dihydrofolate (2 microM), the rate constant for the release of the product tetrahydrofolate (10.3 s-1), and the intrinsic pKa value (6.2) are similar to those exhibited by the wild-type enzyme . However, the hydride-transfer rate declines markedly from the wild-type value of 950 s-1 to 1.7 s-1 for the DL1 mutant and when taken with data for substrate binding indicates that the loop contributes to substrate flux by a factor of 3.5 x 10(4) . Thus, the mobility of loop I may provide a mechanism of recruiting hydrophobic residues which can properly align the nicotinamide and pteridine rings for the hydride-transfer process (a form of transition-state stabilization).(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1992 Sep, 12(9), 4067 - 75
Kappa B site-dependent activation of the interleukin-2 receptor alpha-chain gene promoter by human c-Rel; Tan TH et al.; The cis-acting control elements of the interleukin-2 receptor alpha-chain (IL-2R alpha) gene contain a potent kappa B-like enhancer whose activity can be induced by various mitogenic stimuli . Recent cloning of the p50 and p65 subunits of the kappa B-binding protein NF-kappa B complex revealed a striking sequence homology of these proteins with the c-rel proto-oncogene product (c-Rel) . On the basis of this homology, we examined the potential role of c-Rel in controlling IL-2R alpha transcription . We now demonstrate that the recombinant human c-Rel protein binds to the kappa B element in the IL-2R alpha promoter and results in alteration of the DNA structure in the adjacent downstream regulatory elements containing the CArG box and the GC box . We found that human c-Rel can activate transcription from the IL-2R alpha promoter, but not the kappa B-containing human immunodeficiency virus type 1 promoter, upon cotransfection into Jurkat T cells . Furthermore, truncation of the carboxyl terminus of c-Rel results in a c-Rel mutant (RelNA) that (i) localizes exclusively in the nucleus and (ii) acts in synergy with wild-type c-Rel in activating transcription from the kappa B site of the IL-2R alpha promoter . Finally, induction of surface IL-2R alpha expression coincides with the induced levels of endogenous c-Rel and induced c-Rel binding to the IL-2R alpha kappa B site . Our study identified c-Rel as one component of the Rel/NF-kappa B-family proteins involved in the kappa B-dependent activation of IL-2R alpha gene expression . Furthermore, our results suggest that a Re1NA-like cellular factor (e.g., NF-kappa B p50 or p49 subunit) acts in synergy with c-Re1 during T-cell activation.

J Immunol, 1992 Sep 1, 149(5), 1666 - 70
Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha; Doherty GM et al.; Current evidence indicates that endogenously produced peptide cytokines, most notably TNF-alpha and IL-1, mediate the lethality of experimental endotoxemia . Because circulating serum levels of IFN-gamma can be detected soon after TNF-alpha and IL-1 in response to endotoxin, we investigated the role of IFN-gamma in endotoxin and TNF-alpha lethality . Specific neutralizing antibodies to murine TNF-alpha (anti-TNF-alpha Ab) or murine IFN gamma (anti-IFN-gamma Ab) produced in our laboratory protected mice against the lethality of Escherichia coli endotoxin (LPS) administered 6 h later . Serum IFN-gamma levels 2 h after i.v . LPS were lower in mice treated with anti-TNF-alpha Ab compared to mice that received nonimmune IgG (median less than 2.5 vs 3.0 U/ml, P2 less than 0.05) . In contrast, serum TNF-alpha levels 1 h after i.v . LPS peaked more than fourfold higher in mice treated with anti-IFN-gamma Ab compared to controls (median greater than 6400 vs 1405 pg/ml, p2 less than 0.05) . Doses of TNF-alpha (300 micrograms/kg) and IFN-gamma (50,000 U) which were well tolerated when given individually were synergistically lethal in combination (0% lethality vs 100% lethality, P2 less than 0.001), and were associated with higher serum levels of IL-6 than with either cytokine alone . Anti-IFN-gamma Ab provided complete protection against exogenous human rTNF-alpha at the LD100 dose (1400 micrograms/kg, p2 less than 0.001), and in fact prevented lethality at doses four- to fivefold greater than the LD100 human rTNF-alpha (up to 6000 micrograms/kg) . We conclude that IFN-gamma is synergistic with TNF-alpha, is essential for the lethality of LPS and TNF-alpha, and may have modulating effects on the negative control of serum levels of TNF-alpha after LPS in mice.

Oncogene, 1992 Sep, 7(9), 1737 - 42
Transcriptional activator Tax of HTLV-1 binds to the NF-kappa B precursor p105; Hirai H et al.; Human T-cell leukemia virus type 1 (HTLV-1) induces adult T-cell leukemia and also a neurological disease, tropical spastic paraparesis . Tax protein (p40tax) of HTLV-1 activates in trans its own transcriptional enhancer in the long terminal repeat and also those in some cellular genes such as interleukin 2 receptor alpha, granulocyte-macrophage colony-stimulating factor, Fos, Jun and MHC class I . Thus, Tax has been proposed to play a critical role in the pathogenesis induced by HTLV-1 infection . Here, we report formation of a complex of Tax protein with the precursor protein p105 of the NF-kappa B p50 subunit . p105 was co-immunoprecipitated with Tax protein from cells infected with HTLV-1 from cells transfected with the Tax expression plasmid, but not from cells transfected with inactive mutants of Tax . Furthermore, a GST-p105 fusion protein produced in Escherichia coli bound to Tax protein . These results strongly suggest that the trans-activator Tax protein forms a complex with precursor NF-kappa B p105 and plays a role in trans-activation of transcriptional initiation.

J Virol, 1992 Sep, 66(9), 5425 - 31
Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates; Sumiyoshi H et al.; Japanese encephalitis virus (JEV) is a positive-stranded enveloped RNA virus that belongs to the family Flaviviridae . Genomic JEV RNA is approximately 11 kb long and encodes 10 proteins, 3 structural and 7 nonstructural . A full-length cDNA copy of the JEV genome was constructed by in vitro ligation of two cDNA fragments which encode the 5' (nucleotide positions 1 to 5576) and 3' (nucleotide positions 5577 to 10976) halves of the genome . T7 RNA polymerase transcripts of the ligated full-length cDNA template were infectious when transfected into BHK-21 cells . To identify the recombinant virus, a silent mutation was introduced into the clone encoding the 3' half of the genome, which abolished an XbaI site at nucleotide position 9131 . Virus recovered by transfection with the transcripts contained this silent mutation, confirming its identity . Recombinant and parent viruses were identical with respect to growth and plaque production in BHK-21 cells, envelope protein expression in C6/36 cells, and neurovirulence and immunogenicity in mice . Repeated attempts to obtain infectious RNA by transcription from full-length JEV genome cDNA templates cloned into plasmid vectors were unsuccessful . Synthesis of infectious JEV RNA from in vitro-ligated JEV cDNA templates will be useful for molecular and genetic studies of flavivirus replication and virulence.

J Virol, 1992 Sep, 66(9), 5232 - 41
RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication; Hatton T et al.; The hepatitis B virus capsid or core protein (p21.5) binds nucleic acid through a carboxy-terminal protamine region that contains nucleic acid-binding motifs organized into four repeats (I to IV) . Using carboxy-terminally truncated proteins expressed in Escherichia coli, we detected both RNA- and DNA-binding activities within the repeats . RNA-binding and packaging activity, assessed by resolving purified E . coli capsids on agarose gels and disclosing their RNA content with ethidium bromide, required only the proximal repeat I (RRRDRGRS) . Strikingly, a mutant in which four Arg residues replaced repeat I was competent to package RNA, demonstrating that Arg residues drive RNA binding . In contrast, probing immobilized core proteins with 32P-nucleic acid revealed an activity which (i) required more of the protamine region (repeats I and II), (ii) appeared to bind DNA better than RNA, and (iii) was apparently modulated by phosphorylation in p21.5 derived from Xenopus oocytes . Deletion analysis suggested that this activity may depend on an SPXX-type DNA-binding motif in repeat II . Similar motifs found in repeats III and IV may also function to bind DNA . On the basis of these observations, together with a reinterpretation of recent studies showing that capsid protein mutants cause defects in viral genome replication, we propose a model suggesting that hepadnavirus capsid proteins participate directly in the intracapsid reverse transcription of RNA into DNA . In this model, repeat I binds RNA whereas the distal repeats are progressively recruited to bind elongating DNA strands . The latter motifs may be required for replication to be energetically feasible.

Gastroenterology, 1992 Sep, 103(3), 905 - 12
Acute-phase induction of manganese superoxide dismutase in intestinal epithelial cell lines; Valentine JF et al.; Cellular protection from immune-generated oxygen free radicals is initiated by the reduction of oxygen radicals by manganese superoxide dismutase (MnSOD) and copper/zinc superoxide dismutase (Cu/ZnSOD) . Using rat adult (IEC-6) and fetal (IRD-98) intestinal epithelial cell lines, factors involved in the regulation of the SODs at the messenger RNA (mRNA) level were examined . Exposure of IEC-6 and IRD-98 to Escherichia coli lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha) results in a marked increase in MnSOD mRNA as early as at 1 hour . Cotreatment of cells exposed to LPS or TNF-alpha with actinomycin D or cycloheximide showed that de novo transcription but not protein synthesis is required for the LPS- and TNF-alpha-dependent induction in MnSOD mRNA . Treatment with interleukin 1 beta results in a 12-fold increase in MnSOD mRNA, but no change was observed with interleukin 6 or interferon alpha . No change was observed in the level of Cu/ZnSOD mRNA under any condition tested . The results indicate that MnSOD functions as a cytokine-regulated acute phase protein involved in cellular protection from free radical-mediated damage.

Southeast Asian J Trop Med Public Health, 1992 Sep, 23(3), 402 - 5
Molecular cloning of Plasmodium falciparum blood stage antigens and application of the recombinant proteins in serodiagnosis; Liu KY et al.; A Plasmodium falciparum genomic DNA library was established in the expression vector lambda gt11, cloned in Escherichia coli . The library was screened with human hyperimmune sera by in situ hybridization . Twenty clones expressing P . falciparum sequences as polypeptides fused to beta-galactosidase were identified . One, CD3A/9025/60, reacted with all immune sera and expressed polypeptides that were larger than beta-galactosidase as well as reacting with antibodies to beta-galactosidase and to P . falciparum . When the fusion proteins were used as target antigens to diagnose malaria antibodies, a result was obtained which correlated well with indirect fluorescence assay.

Res Microbiol, 1992 Sep, 143(7), 671 - 81
Functional analysis of the pertussis toxin promoter; Gross R et al.; The expression of the pertussis toxin ptx operon is positively regulated in cis by a promoter region of about 170 base pairs and in trans by the bvg locus, which codes for the transcriptional activator protein BvgA . The promoter contains two direct repeats which are essential for its activity . When the position of these direct repeats relative to the transcription start point was changed, the activity of the promoter was strongly impaired . The repeated sequences therefore do not represent enhancer-like elements similar to those which have been identified in other positively regulated promoters; instead, the integrity of the whole promoter region seems to be an important feature of ptx regulation . A transcription interference assay was carried out to analyze in vivo binding of regulatory proteins to the ptx promoter . The results suggest that the direct repeats are the recognition sequence of a protein, which binds to them only under conditions in which the promoter is activated . In vitro DNA binding experiments with BvgA protein purified from an overproducing Escherichia coli strain were performed . However, no binding of BvgA to the ptx promoter was observed under conditions where binding of BvgA to the fha and bvg promoters occurred . This suggests that factors in addition to the bvg system are involved in the regulation of the Bordetella virulence regulon.

Res Microbiol, 1992 Sep, 143(7), 655 - 63
The level of supercoiling affects the regulation of DNA replication in Escherichia coli; von Freiesleben U et al.; The chromosome of Escherichia coli is negatively supercoiled . This favours processes that unwind the two DNA strands, such as DNA replication . In this paper, we have investigated the effect of changed levels of overall chromosomal supercoiling on the initiation of DNA replication . Specifically, we have used flow cytometry to reveal effects on the synchrony of initiations of DNA replication in single cells . An increase in the level of supercoiling moderately reduced initiation synchrony . In contrast, decreased supercoiling led to pronounced asynchrony . We have excluded the possibility that this asynchrony is caused by changes in the level of the Dam methyltransferase or the DnaA protein . We suggest that the global level of supercoiling influences the topology of oriC and thereby the sequence of events leading to initiation of DNA replication in E . coli.

Lik Sprava, 1992 Sep, (9), 86 - 8
{Escherichiosis in adults and the carriage of pathogenic Escherichia in some regions of Ukraine}; Kas'ianenko AM et al.; The role is shown of pathogenic Escherichieae (PE) in the development of escherichiosis in two regions with socioeconomic differences . The epidemiological picture of these infections depending on the age of the examined is analyzed . The seasonal character of circulation of pathogenic Escherichieae serogroups was found to play a major role in the etiology of escherichiosis in adults . The biological properties of the isolated strains were studied.

Biull Eksp Biol Med, 1992 Sep, 114(9), 299 - 302
{Submicroscopic features of cecal cells in experimental Escherichia infection}; Barkhina TG et al.; Ultrastructural changes of the caecum cells were studied for the period from 15 minutes to 2 weeks after inoculation using the model of experimental escherichiosis . Evolution processes in relation to different caecum cell populations were followed up submicroscopically . Ultrastructural changes observed evidence derangement of protein and water-salt cell metabolism, immune trends of experimental escherichiosis.

J Neurosci Methods, 1992 Sep, 44(2-3), 85 - 90
Further refinement of the Escherichia coli brain abscess model in rat; Nazzaro JM et al.; The rat brain abscess model provides a substrate for the modeling of delivery of therapeutic agents to intracerebral mass lesions . We now report refinement of the Escherichia coli brain abscess model in rat . A K1 surface antigen-negative E . coli isolated from human blood culture was stereotaxically inoculated into deep brain sites . Histopathologic analyses and quantitative cultures demonstrated the consistent production of lesions . No animal in this consecutive series developed meningitis, ventriculitis or sepsis . By contrast, prior experience with E . coli abscess production resulted in 25% failure rate of abscess production or death from sepsis . This improvement in the model may be attributable to specific characteristics of the bacteria used, modification of the inoculation method or the intracerebral placement technique . The present work suggests a reliable and consistent brain abscess model, which may be further used to study brain suppuration.

Mol Biol (Mosk), 1992 Sep-Oct, 26(5), 1088 - 98
{Design and expression of a diphtheria toxin hybrid protein and human interleukin-2 gene in Escherichia coli}; Shemiakin IG et al.; Recombinant fusion proteins consist of the N-terminal 488 or 513 amino acids of diphtheria toxin joined to human interleukin 2 . Initially those fusion proteins were expressed in E . coli under the control of the tox promotor . Western blot analyses showed that E . coli strains bearing the hybrid genes produce 68 kDa or 72 kDa fusion proteins that retain the immunological determinants of both the diphtheria toxin component and the interleukin 2 component . The fusion protein with mol . mass 72 kDa was partially purified by affinity chromatography . The expression of the fusion proteins under the control of the strong promotors was increased (100-fold for tac- promotor) compared to that under the control of the tox promotor . DT-IL2 might be a useful cytotoxic agent in the treatment of diseases involving IL2 receptor-positive cells, such as allograft rejection, graft-versus-host disease, multiple sclerosis et al.

Mol Biol (Mosk), 1992 Sep-Oct, 26(5), 1080 - 7
{The effect of intracellular concentrations of tRNA, corresponding to the rare arginine codons AGG and AGA, on the gene expression in Escherichia coli}; Gurskii IaG et al.; Influence of increased arginine concentrations of tRNA's corresponding to rare codons AGG and AGA was studied in the model system constructed earlier . The model system is a chimeric gene consisting of CAT gene fragment, part of the gene encoding for alpha-domain of beta-galactosidase E . coli and a series of synthetic inserts enriched with codons AGG and AGA . In order to increase the intracellular tRNA concentration the natural gene of AGA-specific tRNA and the artificial gene of AGG-specific tRNA were cloned in plasmid under the control of p15A ori compatible with co1EI ori and used for maintaining the model gene . It was shown that the artificial AGG-specific tRNA gene produces a functionally active tRNA . A steep rise in the synthesis of polypeptide encoded by the model template containing rare codons was demonstrated when the genes of tRNAs recognizing these codons were propagated in the multicopy plasmid . It was shown that AGA-specific tRNA efficiently translates both AGA and AGG codons while AGG-specific tRNA - only AGG codons.

Mol Biol (Mosk), 1992 Sep-Oct, 26(5), 1063 - 79
{Rare codons and gene expression in Escherichia coli}; Gurskii IaG et al.; Influence of rare codons upon gene expression in E . coli was investigated . The chimeric gene was created combining CAT gene and a fragment of the gene, encoding for alpha-domain of beta-galactosidase . The synthetic oligonucleotides were inserted in different parts of the chimeric gene . The constructed synthetic oligonucleotides encoded the same amino acid sequences and contained arginine codons AGG, AGA and CGT in various combinations . It was shown that the presence of rare arginine codons AGG and AGA in the template and their mutual arrangement significantly influence the level of gene expression . At the same time the presence of leucine, isoleucine, glycine and proline rare codons does not cause such an effect . Translation of AGGAGG and AGAAGA sequences was found to lead to the formation of a considerable amount of polypeptides of incomplete length . It was shown that the presence of such a cluster of rare codons effects on the length of specific mRNA.

Eur J Clin Invest, 1992 Sep, 22(9), 625 - 9
Effect of lipid A on the deformability, membrane rigidity and geometry of human adult red blood cells; Poschl JM et al.; Lipid A is responsible for the activities of endotoxin and may cause circulatory failure and haemolysis . This study evaluated the effects of different lipid A concentrations on red blood cell (RBC) deformation (rheoscope), the aspiration pressure required to aspirate RBC into 3.3 microns pipettes, the membrane shear elastic modulus (i.e . membrane rigidity) and cellular geometry (micropipette system) after 15 min of incubation . Lipid A concentrations of 10 and 100 micrograms ml-1 of RBCs decreased RBC deformability by 26% and 39%, respectively . The aspiration pressure for RBCs into a 3.3 microns micropipette increased by 235% at a lipid A concentration of 10 micrograms ml-1 and by 586% at a concentration of 100 micrograms ml-1 . The elastic shear modulus almost doubled at a lipid A concentration of 10 micrograms ml-1 and tripled at 100 micrograms ml-1 . At a lipid A concentration of 100 micrograms ml-1, 37% of RBCs showed spicules . These echinocytes were less deformable than discocytes . Mean corpuscular volume, RBC volume and surface area were not affected by lipid A . We conclude that lipid A causes marked reduction of RBC deformability due to increasing membrane rigidity.

Electrophoresis, 1992 Sep-Oct, 13(9-10), 604 - 8
Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media; Wheeler D et al.; Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to "linear" standards . The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards . Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E . coli operon promoter shows in addition that curves of migration distance vs . gel concentration ("Ferguson curves") of such abnormally conformed DNA differ from those of "linear" standards . Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size . In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than "linear" standards from one which shows the same dependence . The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards . Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel . Another authentically "abnormal" DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid . Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biol Rep, 1992 Sep, 16(4), 277 - 84
Effect of deletions 5' to the translation initiation sequence on the expression of an mRNA in animal cells; Ganoza MC et al.; To learn if an mRNA.18S rRNA interaction or a special secondary structure in the mRNA start region is essential for translation in eukaryotic cells, we constructed recombinant plasmids with the SV40 early promoter 5' to part of the Escherichia coli tufB-lacZ gene . Deletion of bases potentially complementary to the 18S rRNA highly increased the transient beta-galactosidase expressed in transfected CHO cells . Deletion of bases that fostered formation of potential hairpins with the mRNA 5'-terminus or altered the structure of the coding region reduced beta-galactosidase activity suggesting that these features of the mRNA secondary structure may be essential for initiation of translation . Computer aided analysis of the potential structure of 290 mRNAs suggests these are conserved features of the initiation region.

Mol Microbiol, 1992 Sep, 6(18), 2733 - 41
gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression; Castano I et al.; We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD . Two open reading frames were identified, the first of which corresponds to gltF . The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT) . gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction . Histidase synthesis was determined as a measure of Ntr function . First, insertions in gltB, gltD or gltF all prevent Ntr induction . Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF . Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene . Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.

Chem Pharm Bull (Tokyo), 1992 Sep, 40(9), 2483 - 6
Effect of metal ions on transcription of the ada gene which encodes O6-methylguanine-DNA methyltransferase of Escherichia coli; Takahashi K et al.; The effect of metal ions on transcription of the ada gene of Escherichia coli which is promoted by Ada protein in the presence of methylated deoxyribonucleic acid (DNA) was examined in a reconstituted system . Their effect on the O6-methylguanine-DNA methyltransferase (MGTase) activity of Ada protein was also examined . Ag+, Cd2+, Cu2+ and Hg2+ severely inhibited transcription of the ada gene at a dose which inhibited neither transcription of the lacUV5 gene nor MGTase activity . Zn2+ inhibited both transcription and MGTase activity in the same dose range . Al3+ and Fe3+ inhibited transcription of both ada and lacUV5 genes without affecting the MGTase activity of Ada protein . Inhibitory mechanisms are discussed.

Biochem Int, 1992 Sep, 27(6), 991 - 1000
Allosteric activation by nucleotides of the inactive by phosphatase ornithine decarboxylase of Escherichia coli; Anagnostopoulos C et al.; ODC was purified to homogeneity from E . coli K12 MG1655 strain transformed with a pBR322 plasmid carrying the ODC gene . This preparation was homogeneous as it was analyzed by SDS-polyacrylamide gel electrophoresis . From this preparation the amino-terminal sequence analysis was obtained . The native ODC of E . coli is activated by ATP, GTP, CTP and UTP at 10(-3) M concentration to around 170-300% . Our results indicate that the recombinant ODC is activated only by GTP and UTP at 10(-3) M 370% and 300%, respectively . When the recombinant ODC was incubated with calf intestine alkaline phosphatase, this inactive ODC can be reversibly activated allosterically only by GTP or UTP at a concentration of 10(-6) or 10(-5) M . That GTP or UTP can allosterically convert the inactive form of ODC to an active form suggests that these analogues may be the in vivo physiological regulators of ODC.

Appl Environ Microbiol, 1992 Sep, 58(9), 3095 - 100
Detection of virulence factors in culturable Escherichia coli isolates from water samples by DNA probes and recovery of toxin-bearing strains in minimal o-nitrophenol-beta-D-galactopyranoside-4-methylumbelliferyl-beta-D-g luc uronide media; Martins MT et al.; A total of 449 Escherichia coli isolates in treated and raw water sources were submitted to DNA-DNA hybridization using seven different DNA probes to detect homology to sequences that code for Shiga-like toxins I and II; heat-stabile and heat-labile toxins, adherence factors EAF and eae, and the fimbrial antigen of entero-hemorrhagic E . coli . Fifty-nine (13%) of the isolates demonstrated homology with one or more specific DNA probes . More than 50% of the isolates in treated water were not recovered in MMO-4-methylumbelliferyl-beta-D-glucuronide media designed for detection of this indicator.

Vet Microbiol, 1992 Sep, 32(2), 149 - 61
Diphenylamine increases cloacin DF13 sensitivity in avian septicemic strains of Escherichia coli; Valvano MA; Thirteen avian septicemic isolates of Escherichia coli were examined for the presence of the aerobactin iron transport system . All of the strains possessed a functional aerobactin system and hybridization experiments showed that the aerobactin genes were located on ColV-type plasmids in all cases . The expression of the aerobactin receptor IutA was also studied by determining the bacterial susceptibility to the bacteriocin cloacin DF13 . Twelve of the 13 isolates were cloacin-resistant but became sensitive to this bacteriocin upon treatment with diphenylamine which caused a reduction in the amount of O-side chain lipopolysaccharide.

Rev Med Panama, 1992 Sep, 17(3), 163 - 72
{Cloning of kDNA minicircles in different species of Leishmania and its use as probes for diagnosis}; Pascale JM et al.; The present study describes the cloning procedure for fragments of kinetoplast DNA minicircles from different Leishmania species and its use for detecting the presence of these parasites . Our methodology was as follow: the DNA of the kinetoplast from Leishmania mexicana amazonensis and Leishmania braziliensis panamensis was extracted, purified and digested with the enzyme Dra I . These fragments were cloned in the site for Hinc II in the plasmid pKS . E . coli was the bacterial strain used for transforming and amplifying the cloned fragments; the selection was carried out in LB medium supplemented with ampicillin . With the clones suspected to be positives we run a Southern blot and total kDNA, from each Leishmania species, was used as hybridization probe . Finally, the cloned purified fragments were tested as diagnostic probes against kDNA from eleven different species of Leishmania and one of Trypanosoma cruzi parasites . After cloning, transforming, amplifying and selecting, we obtained two probes of fragments of kDNA minicircles: one from L . m . amazonensis and the other from L . b . panamensis . Both probes showed high sensitivity for diagnosing cutaneous Leishmania complexes (Mexicana or Braziliensis); however, we observed a low grade crossreaction between some species belonging to the same complex . It is necessary to continue studies in order to obtain subfragments of these probes with a higher grade of specificity at the level of species and subspecies.

Protein Eng, 1992 Sep, 5(6), 583 - 91
Overexpression and structure--function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine; Rock F et al.; A synthetic chimeric IL-2/IL-6 gene was synthesized to engineer a bifunctional lymphokine which was overproduced in Escherichia coli . Following denaturation of the inclusion bodies in 6 M guanidine and refolding and reoxidation in the presence of a redox system, the fusion protein (rIL-2/IL-6) was purified to homogeneity and shown to react with both monospecific anti-IL-2 and anti-IL-6 antisera . A collagen-like spacer was introduced between the two cytokine moieties to generate IL-2 and IL-6 molecules upon collagenase digestion . After cleavage, the two subunits, purified in a single-step procedure, were found to be correctly reoxidized and functionally as active as their native counterparts . Circular dichroism studies of rIL-2/IL-6 revealed that both cytokine subunits refolded independently and exhibited the alpha-helical structures characteristic of the corresponding wild-type lymphokines . The chimera displayed full IL-2 activity in the CTLL-2 cell proliferation assay . It also retained the IL-6 property to enhance IgM synthesis in SKW6.4 cells, induce the proliferation of B-cell hybridomas and stimulate the production of fibrinogen in hepatocytes . Because IL-2 amplifies the cellular immune response and IL-6 up-regulates the humoral response, this bifunctional lymphokine represents a potentially useful therapeutic adduct and may serve as an immunomodulator to enhance the host's response to vaccination.

Protein Eng, 1992 Sep, 5(6), 569 - 75
Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis; Wang J et al.; Site-specific mutagenesis was employed to study structure-function relationships at the substrate binding site of rat tissue kallikrein . Four kallikrein mutants, the Pro219 deletion (P219del), the 34-38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation {YYFG(34-38)IN}, the Trp215----Gly exchange (W215G) and the double mutant with Tyr99----His and Trp215----Gly exchange (Y99H:W215G) were created by site-directed mutagenesis to probe their function in substrate binding . The mutant proteins were expressed in Escherichia coli at high levels and analyzed by Western blot . These mutant enzymes were purified to apparent homogeneity . Each migrated as a single band on SDS-PAGE, with slightly lower molecular mass (36 kDa) than that of the native enzyme, (38 kDa) because of their lack of glycosylation . The recombinant kallikreins are immunologically identical to the native enzyme, displaying parallelism with the native enzyme in a direct radioimmunoassay for rat tissue kallikrein . Kinetic analyses of Km and kcat using fluorogenic peptide substrates support the hypothesis that the Tyr99-Trp215 interaction is a major determinant for hydrophobic P2 specificity . The results suggest an important role for the 34-38 loop in hydrophobic P3 affinity and further show that Pro219 is essential to substrate binding and efficient catalysis of tissue kallikrein.

Protein Eng, 1992 Sep, 5(6), 559 - 67
Analysis of disulphide bridge function in recombinant bovine prolactin using site-specific mutagenesis and renaturation under mild alkaline conditions: a crucial role for the central disulphide bridge in the mitogenic activity of the hormone; Luck DN et al.; We have previously described a method for isolating Escherichia coli-produced methionyl bovine prolactin (Met-bPRL) and its renaturation using thioredoxin . This report describes an alternative renaturation procedure in which extracted Met-bPRL is incubated in air at pH 10 and 20 degrees C . Within 1 h of such treatment essentially all of the reduced Met-bPRL was converted to the oxidized form; this was accompanied by an increase to full mitogenic activity in the Nb2 cell bioassay . It was also found that, to minimize contamination by high mol . wt Met-bPRL derivatives, it is essential to have a reducing agent (dithiothreitol) present during disruption of the bacteria and to extract the protein at neutral pH . The contribution of each of the three disulphide bridges in bPRL to its bioactivity was studied with Met-bPRL variants, prepared via site-specific mutagenesis, in which cysteines were replaced by serines to prevent disulphide bond formation . Variants lacking the C4-C11 bridge, the C191-C199 bridge or both these terminal bridges were as mitogenic as authentic bPRL . (Variants lacking the C191-C199 bridge had markedly increased solubility in the presence of deoxycholate.) In contrast, variants lacking the C58-C174 bridge had greatly reduced bioactivity, indicating that integrity of the large disulphide loop is crucial to the hormone's mitogenic activity.

Protein Eng, 1992 Sep, 5(6), 551 - 7
A structural role of histidine 15 in human glutathione transferase M1-1, an amino acid residue conserved in class Mu enzymes; Widersten M et al.; His15 is a conserved amino acid residue in all known class Mu glutathione transferases . This His residue in human glutathione transferase M1-1 has been mutated into 17 different amino acid residues by means of site-directed random mutagenesis to determine if any substitutions are compatible with catalytic activity . The majority of the mutant proteins appeared unstable and could not be isolated in reasonable quantities by heterologous expression in Escherichia coli . Five mutant enzymes, H15C, H15K, H15N, H15Q and H15S were purified and more extensively characterized . The mutant proteins shared the same size as that of the wild-type enzyme but could be separated from the parental enzyme by reverse phase HPLC . For all the mutant forms except H15N, the sp . act . with 1-chloro-2,4-dinitrobenzene was less than 3% of the wild-type value--the H15N mutant enzyme displayed 29% of the wild-type activity . None of the catalytically active mutant enzymes showed any major alteration of the binding affinity for the substrate analog S-hexylglutathione, suggesting that His15 is not part of the active site of the enzyme . The high activity of the mutant H15N, also reflected in the kcat/Km, V and S0.5 values, rules out the possibility that His15 in the native enzyme contributes to catalysis by serving as a base . The role of His15, largely replaceable by Asn in the same position, appears to be structural, probably involving hydrogen bonds that maintain the protein in a stable and catalytically active conformation . A critical structural role of His15 in a buried position may explain the evolutionary conservation of this residue in the class Mu glutathione transferases.

Mol Endocrinol, 1992 Sep, 6(9), 1502 - 12
Expression and regulation of a proenkephalin beta-galactosidase fusion gene in the reproductive system of transgenic mice; Borsook D et al.; A fusion gene containing 3 kilobases of human proenkephalin 5'-flanking sequences and 1 kilobase of human proenkephalin 3'-flanking sequence and the easily visualized histochemical marker, Escherichia coli beta-galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice . Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice . Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids . In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice . Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct.

J Cell Sci, 1992 Sep, 103 ( Pt 1), 201 - 9
Radial movement of lysosomes along microtubules in permeabilized macrophages; Swanson JA et al.; In murine bone marrow-derived macrophages, lysosomes often form tubulovesicular compartments, whose extended distribution in the cytoplasm depends on the integrity of cytoplasmic microtubules . When macrophages with fluorescently labeled lysosomes were plated onto coverslips opsonized with IgG, they engaged that surface in a phagocytic response (frustrated phagocytosis) . The tubular lysosomal compartment of these cells collected in a central, perinuclear region, despite the continued presence of a radiating array of cytoplasmic microtubules . Using methods developed in the study of melanophores, we permeabilized macrophages engaged in frustrated phagocytosis, then re-activated lysosome extension along microtubules . Permeabilization was selective for plasma membranes, in that high molecular weight probes such as trypan blue or IgG could enter cells, while fluorescent probes previously loaded into lysosomes via endocytosis remained contained therein . Addition of 2 mM ATP, GTP or UTP to these permeabilized cell models produced centrifugal extension of tubular lysosomes . Selective depletion of ATP, using Escherichia coli glycerol kinase, inhibited ATP-dependent extension but not that which occurred with GTP or UTP, indicating that the mechanism of radial movement can use any of these three nucleotide triphosphates . Extension was independent of pH between 6.8 and 7.4, and was inhibited by AMP-PNP and by GMP-PNP . Depolymerization of cytoplasmic microtubules with nocodazole prevented subsequent ATP-inducible lysosome extension, whereas preincubation of cells with cytochalasin D did not inhibit the response . These results are consistent with the in vitro mechanochemical properties of kinesin (Cohn et al., 1989), and support earlier evidence, obtained in living cells, that kinesin is the mechanochemical motor of lysosome extension along microtubules in macrophages.

J Biochem (Tokyo), 1992 Sep, 112(3), 350 - 4
Purification, analysis, and enzymatic activity of recombinant human synovial fluid phospholipase A2 and N-terminal variants; Di Marco S et al.; Recombinant human synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized . The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges . Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded . Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.

J Biochem (Tokyo), 1992 Sep, 112(3), 314 - 20
Biochemical and immunological characterization of the DNA binding protein (RBP-J kappa) to mouse J kappa recombination signal sequence; Hamaguchi Y et al.; We have investigated whether J kappa recombination signal sequence (RS) binding protein (RBP-J kappa) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA . Murine RBP-J kappa protein purified by J kappa-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity . To obtain a large amount of purified RBP-J kappa protein, recombinant RBP-J kappa was synthesized in Escherichia coli as a fusion protein and also in silkworm cells . Although recombinant RBP-J kappa produced in silkworm cells could bind J kappa-RS, it failed to show either ligase or DNA bending activity . Since the DNA affinity-purified RBP-J kappa has the ligase activity, the RBP-J kappa protein may form a complex with a ligase in vivo . We have raised monoclonal antibodies against the RBP-J kappa fusion protein which was synthesized in E . coli and unable to bind J kappa-RS . Using the anti-RBP-J kappa monoclonal antibody we have shown that the RBP-J kappa protein is expressed ubiquitously in mammalian tissues . The ubiquitous expression of the RBP-J kappa protein is consistent with the hypothesis that the RBP-J kappa protein may have dual function {Furukawa et al . (1991) J . Biol . Chem . 266, 23334-23340}.

J Biochem (Tokyo), 1992 Sep, 112(3), 306 - 8
Expression in Escherichia coli and a functional study of a beta-troponin T 25 kDa fragment of rabbit skeletal muscle; Fujita S et al.; A 25 kDa fragment of beta-type troponin T (beta-TnT) was expressed in Escherichia coli, and its function as a component of the regulatory system for actomyosin ATPase was compared with that of the authentic counterpart, the full length alpha-TnT . The expressed species, designated as beta-TnT(N'-208), consists of 208 residues . It lacks the entire variable region at the amino-terminus and, near the carboxyl-terminus, a segment of 14 residues is changed from the alpha-type to the beta-type sequence . Functional tests indicated that the truncated beta-TnT was not distinguishable from the full length alpha-TnT, suggesting that neither deletion of the variable N-terminal region nor alteration of the type has a significant effect on the regulatory action of TnT.

J Antibiot (Tokyo), 1992 Sep, 45(9), 1414 - 9
Reveromycins, new inhibitors of eukaryotic cell growth . II . Biological activities; Takahashi H et al.; Reveromycins A, B, C and D showed inhibitory activity against EGF-stimulated mitogen response in Balb/MK cells . Furthermore reveromycins A, C and D exhibited morphological reversion of srcts-NRK cells, antiproliferative activity against human tumor cell lines and antifungal activity . The effects of reveromycins A, C and D on eukaryotic cells were closely similar to each other, but those of reveromycin B were very weak . In vitro studies revealed that reveromycin A is a selective inhibitor of protein synthesis in eukaryotic cells.

Eur Respir J, 1992 Sep, 5(8), 992 - 6
Lipopolysaccharide (LPS) inhalation in healthy subjects increases neutrophils, lymphocytes and fibronectin levels in bronchoalveolar lavage fluid; Sandstrom T et al.; Bacterial endotoxin has been suggested as responsible for the development of subjective symptoms and transient or chronic lung function impairment seen after exposure to organic dusts in cotton mills, poultry houses, swine confinement buildings and saw mills . Animal experiments have demonstrated bronchoalveolar neutrophilia being the most prominent cell response in animals following bacterial lipopolysaccharide (LPS) inhalation . The present study was conducted to obtain information on some aspects of the early inflammatory response to inhaled LPS in man . Eight healthy nonsmoking subjects, 23-27 yrs old, underwent bronchoalveolar lavage (BAL), 3 h after a provocation test with 100 micrograms LPS from E . coli dissolved in 2 ml isotonic NaCl . The solution was aerosolized with a jet nebulizer and inhaled . The calculated dose delivered to the lung was approximately 25 micrograms, which equals exposure in some occupational settings . The BAL data for each individual subject were compared with data from a control BAL performed at least 6 weeks prior to the LPS challenge . The major cellular response to LPS, reflected in BAL fluid, was an approximately hundredfold increase in neutrophils . The total number of lymphocytes was on average tripled . The alveolar macrophage phagocytosis of opsonized yeast particles in vitro was significantly reduced . A further indicator of an ongoing inflammation was an increase in fibronectin . No changes were seen in the levels of BAL albumin, indicating that the elevated level of fibronectin could not be explained by an increased permeability, but rather by a local production . The results correspond with data from animal studies and further supports the hypothesis that bacterial LPS is important in the pulmonary reaction induced by organic dusts.

Acta Paediatr Suppl, 1992 Sep, 381, 39 - 44
Persistent diarrhea in northeast Brazil: etiologies and interactions with malnutrition; Lima AA et al.; With the improved control of acute diarrheal illness mortality with oral rehydration therapy, persistent diarrhea is now emerging as a major cause of childhood mortality in tropical developing areas like the impoverished populations in Brazil's Northeast . "Graveyard surveillance" in the rural community of Guaiuba in northeastern Brazil revealed fully half of the 70% diarrhea mortality was due to persistent diarrheal illnesses . Furthermore, 11% of 14 or more diarrheal illnesses per child per year in an urban slum in Fortaleza persisted beyond 14 days, a definition that clearly identified the high risk children for heavy diarrhea burdens . Not only did heavy diarrhea burdens ablate the key "catch-up" growth seen in severely malnourished children and in children following previous diarrheal illnesses, but malnutrition significantly predisposed children to a greater incidence and duration of diarrhea as well as a greater incidence of persistent diarrhea . Etiologic studies of 37 children presenting with persistent diarrhea to Hospital das Clinicas in Fortaleza revealed that Cryptosporidium (in 13%) and enteroadherent E . coli (36% with aggregative, 29% with diffuse and 13% with localized adherence to HEp-2 cells) were the predominant potential pathogens found in the stool or upper small bowel . These findings suggest that persistent diarrhea is emerging as an important health problem in Brazil's Northeast, that it identifies a high risk child for heavy diarrhea burdens, that important interactions occur with malnutrition and that Cryptosporidium and enteroadherent E . coli warrant further study as potential etiologies of this major cause of morbidity and mortality.

Acta Paediatr Suppl, 1992 Sep, 381, 124 - 6
Persistent diarrhea in Vietnamese children: a preliminary report; Ngan PK et al.; The clinical and laboratory features of persistent diarrhea were investigated in 83 children under three years of age who were treated in the Gastroenterology Division of the Institute for the Protection of Children's Health, Hanoi from August 1988 to August 1989 . The number of cases of diarrhea was highest in the children aged 4-5 months . The mean age of the children studied was 6.6 +/- 3.4 months . The ratio of males to females was 2.6 and mean age of first episode of diarrhea was 4.3 +/- 3.4 months; persistent diarrhea was more common in children under six months of age than in older children . Persistent diarrhea occurred in the first diarrheal episode in 66.5% of cases . Recent nonenteric infections were found in 30% of the study group . Of the 83 children studied, 36% had stool specimens positive for enteric pathogens; 24% had enterotoxigenic Escherichia coli isolated, 8% had enteropathogenic E . coli, 5% rotavirus, 6% Candida, and 4% Giardia lamblia . The duration of diarrhea was longer in children who received antibiotics than in those who did not (p < 0.01).

Biophys J, 1992 Sep, 63(3), 839 - 53
Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii . A multidimensional time-resolved polarized fluorescence study; Bastiaens PI et al.; Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K . The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme . The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis . The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes . Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme . Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins . The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system . By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated . For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A . vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered . In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved . The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site . The local environment of the prosthetic groups in the deletion mutant of the A . vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range . In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied . Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent . This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.

Plasmid, 1992 Sep, 28(2), 157 - 65
Transfer of mobilizable plasmids to Sorangium cellulosum and evidence for their integration into the chromosome; Jaoua S et al.; Recombinant vectors derived from the broad-host-range mobilizable plasmid pSUP2021 were constructed and transferred by IncP-mediated conjugation from Escherichia coli to Sorangium cellulosum, where they were integrated into the chromosome by homologous recombination and maintained stably . This appears to be the first system of gene transfer to S . cellulosum.

Plasmid, 1992 Sep, 28(2), 123 - 9
Complete nucleotide sequence of a Selenomonas ruminantium plasmid and definition of a region necessary for its replication in Escherichia coli; Attwood GT et al.; A plasmid from Selenomonas ruminantium subspecies lactilytica has been subcloned in Escherichia coli K-12 and completely sequenced . Three open reading frames (ORFs) of 909, 801, and 549 bp were identified and the complete sequence was analyzed by comparison with DNA and protein databases . No significant deoxynucleotide or amino acid sequence homology with other published genes or proteins was detected . The plasmid was shown to replicate independently in E . coli K-12 by a DNA polymerase I-dependent mechanism and deletion analysis defined the DNA sequence responsible for this phenotype.

Mol Gen Genet, 1992 Sep, 234(3), 346 - 52
ATPase activity of SopA, a protein essential for active partitioning of F plasmid; Watanabe E et al.; The sopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli . In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied . {alpha-32P}ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs . In contrast, no ATP binding activity was detected for the SopB protein . The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA . The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein . However, the SopB protein alone failed to stimulate the ATPase activity.

Microbiol Rev, 1992 Sep, 56(3), 412 - 29
Editing of errors in selection of amino acids for protein synthesis; Jakubowski H et al.; All living cells must conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from gene to finished protein product . One crucial "quality control" point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains . During selection of amino acids, synthetases very often have to distinguish the cognate substrate from a homolog having just one fewer methyl group in its structure . The binding energy of a methyl group is estimated to contribute only a factor of 100 to the specificity of binding, yet synthetases distinguish such closely related amino acids with a discrimination factor of 10,000 to 100,000 . Examples of this include methionine versus homocysteine, isoleucine versus valine, alanine versus glycine, and threonine versus serine . Many investigators have demonstrated in vitro the ability of certain aminoacyl-tRNA synthetases to edit, that is, correct or prevent incorrect attachment of amino acids to tRNA molecules . Several major editing pathways are now established from in vitro data . Further, at least some aminoacyl-tRNA synthetases have recently been shown to carry out the editing function in vivo . Editing has been demonstrated to occur in both Escherichia coli and Saccharomyces cerevisiae . Significant energy is expended by the cell for editing of misactivated amino acids, which can be reflected in the growth rate . Because of this, cellular levels of aminoacyl-tRNA synthetases, as well as amino acid biosynthetic pathways which yield competing substrates for protein synthesis, must be carefully regulated to prevent excessive editing . High-level expression of recombinant proteins imposes a strain on the biosynthetic capacity of the cell which frequently results in misincorporation of abnormal or wrong amino acids owing in part to limited editing by synthetases . Unbalanced amino acid pools associated with some genetic disorders in humans may also lead to errors in tRNA aminoacylation . The availability of X-ray crystallographic structures of some synthetases, combined with site-directed mutagenesis, allows insights into molecular details of the extraordinary selectivity of synthetases, including the editing function.

Mol Microbiol, 1992 Sep, 6(17), 2525 - 37
RecA protein of Escherichia coli and chromosome partitioning; Zyskind JW et al.; Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication . This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants . We also find that more than 10% of recA mutant cells contain no DNA . These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell . Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells.

Mol Microbiol, 1992 Sep, 6(17), 2517 - 24
Quantitative determination of FtsA at different growth rates in Escherichia coli using monoclonal antibodies; Wang H et al.; FtsA is an essential cell division protein in Escherichia coli . Its synthesis in low amounts makes the investigation of its functions difficult . Partially purified FtsA protein was obtained by solubilizing cellular inclusion bodies after overexpression of the ftsA gene for the purpose of raising monoclonal antibodies . Mice were immunized with this FtsA protein fraction and their spleen cells were fused to Sp2/0-AG14 mouse myeloma cells . Hybrid cells were screened and two clones were positively identified as FtsA monoclonal antibody producers by enzyme-linked immunosorbent assay and Western blotting . A quantitative assay using these monoclonal antibodies indicated that the average number of FtsA molecules per cell to be between 50 and 200 . However, the concentration of FtsA protein normalized to total cell protein was constant over a wide range of growth rates . This finding is in agreement with the hypothesized role of FtsA protein as a stoichiometric component of the septum.

Mol Microbiol, 1992 Sep, 6(17), 2423 - 8
SecY and integral membrane components of the Escherichia coli protein translocation system; Ito K; Genetic approaches can address the question of how integral membrane Sec factors interact with each other and facilitate protein translocation across the cytoplasmic membrane of E . coli . This review summarizes genetic analyses of SecY, SecE and some other protein translocation factors, utilizing 'prl' mutations, 'sec' mutations, 'suppressor-directed inactivation', 'Sec titration', dominant negative mutations and their suppressors . Evidence suggests that co-ordinate participation of SecY, SecE, SecD, SecF, and probably some other factors, is crucial for the process.

J Med Entomol, 1992 Sep, 29(5), 818 - 26
Modulation of host-immune responses by ticks (Acari: Ixodidae): effect of salivary gland extracts on host macrophages and lymphocyte cytokine production; Ramachandra RN et al.; Ixodid tick infestation induces host acquired resistance, which involves immunoglobulin cell-mediated and complement-dependent effector pathways . Ticks have developed countermeasures to modulate host antiarthropod responses . Ixodid-mediated host immunomodulation results in vitro in reduced responsiveness to T-lymphocyte mitogens for cells obtained from infested hosts and impaired antibody responses to a thymic dependent antigen . Salivary gland extracts from days 0-9 of engorgement from unmated, female Dermacentor andersoni Stiles suppressed lymphocyte proliferative responses (LPS) to the T-cell mitogen Con A up to 68.4%, whereas responsiveness to E . coli LPS was enhanced . Cytokines assessed in this study included interleukin-1, IL-1, and tumor necrosis factor (TNF) alpha produced by macrophages, and interleukin-2, IL-2, and gamma interferon (IFN-G) secreted by T-lymphocytes . Salivary gland extracts prepared from tissues obtained on days 0-5 of engorgement suppressed IL-1 elaboration from 89.8% on day 0 through 37.5% on day 6 . Levels of TNF were reduced from 40.7 to 94.6% throughout the course of the study . Production of IL-2 was suppressed by 14.1-31.9%, and IFN-G was reduced by 8.7-57.0% . Reduced IL-1 levels during the early phases of feeding indicated reduced host ability to activate T-lymphocytes and provide costimulatory, differentiation, and development signals for B-cells . Both IL-1 and TNF are endogenous pyrogens and activate polymorphonuclear leukocytes . Activities of TNF and IFN-G include antiviral properties and induction of expression of class I and II major histocompatibility complex molecules, which are critical components in the recognition of antigen by T-lymphocytes . The autocrine role of IL-2 in proliferation of T-lymphocytes is central to the development of immune reactivity involving T-cell regulation or effector functions or both . Reductions in cytokine levels would suppress immune responses directed toward immunogens introduced into the host during the course of tick feeding . These results indicates that immunomodulation of the host during tick feeding facilitates engorgement and pathogen transmission.

J Gen Virol, 1992 Sep, 73 ( Pt 9), 2429 - 33
The cloning, sequencing and expression of a major antigenic region from the feline calicivirus capsid protein; Guiver M et al.; RNA purified from the feline calicivirus (FCV) F9 vaccine strain was used to prepare a cDNA library in the expression vector lambda gt11 . The library was screened for expression of FCV antigen using a rabbit antiserum prepared against purified FCV . A 330 bp cDNA clone was identified and used as a probe to obtain a larger overlapping clone of 1369 bp . Comparative sequence analysis with the CFI and F4 strains showed that the clones were derived from the 3' open reading frame encoding the capsid protein . The region encoded by the 330 bp clone was shown to be variable in the three strains compared, and therefore the probable location of major antigenic variation . This clone was expressed in a bacterial system and antiserum to the recombinant protein was used in immunoblots to confirm that this clone was derived from the gene encoding the capsid protein . From these immunoblots, several other capsid-related polypeptides were identified . Comparison with immunoblots using post-vaccination cat sera showed the antibody response in the cat was directed mainly against the capsid protein . Antiserum to the recombinant protein was shown to be effective in neutralizing the infectivity of FCV, indicating that at least one major neutralizing epitope had been cloned.

J Gen Virol, 1992 Sep, 73 ( Pt 9), 2245 - 54
Organization of Germiston bunyavirus M open reading frame and physicochemical properties of the envelope glycoproteins; Gerbaud S et al.; We describe the construction of plasmids which express fusion proteins representing various regions of Germiston virus M polyprotein . The fusion proteins were purified and inoculated into rabbits to produce antisera . The N- and C-terminal regions of the polyprotein induced specific antibodies which reacted with glycoproteins G2 and G1, respectively, and the intermediate region induced antibodies against the NSM polypeptide . This enabled us to determine the gene order: G2-NSM-G1 . Glycoproteins G1 and G2 form the spikes on the surface of the virion . We attempted to determine the structural organization of the glycoproteins by using a membrane-permeable cross-linking reagent, dimethyl suberimidate, but were unable to demonstrate that G1 and/or G2 form oligomeric structures . We analysed the glycoproteins further and showed that, like peripheral membrane proteins, the G2 and NSM proteins are almost completely extracted into the aqueous phase of detergent Triton X114-treated cellular extracts, whereas glycoprotein G1 is distributed in almost equal proportions between the aqueous and the detergent fractions . This indicates that G1 is a membrane-associated protein, but its presence in the aqueous phase suggests that it is less hydrophobic than a typical membrane protein . We have also characterized the intracellular transport of the envelope glycoproteins from the endoplasmic reticulum to the Golgi complex . Pulse-chase labelling followed by immunoprecipitation and treatment with endoglycosidase H (endo H) showed that both G1 and G2 are transported from the endoplasmic reticulum to the Golgi complex . Conversion to the endo H-resistant form is a rather slow process which takes more than 2 h . The mature G1 and G2 proteins present in the virion particle contain almost completely endo-H-resistant glycans.

J Appl Physiol, 1992 Sep, 73(3), 1146 - 9
Sensitivity to endotoxin in rabbits is increased after hemorrhagic shock; Mileski WJ et al.; The immunoinflammatory response following trauma and hemorrhage may predispose to the development of sepsis and multiple-organ failure syndrome . Cardiac output (CO), arterial pressure, arterial PO2, and pulmonary permeability index were measured . We examined the sensitivity of rabbits to infusions of lipopolysaccharide (LPS) after hemorrhagic shock . Shock was produced by reducing CO to 40% of baseline for 90 min, followed by resuscitation with shed blood and then with lactated Ringer solution to maintain CO near baseline . Animals were assigned to three groups: 1) hemorrhagic shock only, 2) LPS only, and 3) hemorrhagic shock + LPS . Groups 1 and 3 were subjected to hemorrhagic shock on day 1 . Escherichia coli LPS was infused (1.0 microgram/kg i.v.) into groups 2 and 3 on day 2 . Fluid resuscitation with lactated Ringer solution was continued in an effort to maintain CO at baseline . Five hours after LPS infusion, 125I-albumin was injected intravenously, and rabbits were killed 1 h later for measurement of pulmonary permeability index . LPS infusion after shock (group 3) caused significant decreases in CO, arterial pressure, and PO2 and an increase in pulmonary permeability . These changes were not seen in the groups 1 and 2 . We conclude that hemorrhagic shock and resuscitation result in a proinflammatory state, leading to increased sensitivity to subsequent exposure to LPS.

Invest Radiol, 1992 Sep, 27(9), 698 - 705
Computed tomographic enhancement of the liver, liver abscesses, spleen, and major vessels with perfluorooctylbromide emulsion . Influence of dosage and injection velocity in an animal model; Adam G et al.; RATIONALE AND OBJECTIVES . Computed tomographic (CT) enhancement of the liver, liver abscess, spleen, and major vessels was investigated between 2 and 48 hours after intravenous administration of perfluorooctylbromide (PFOB emulsion) in an animal model of 63 rabbits . METHODS . Twenty-one animals received 3 g/kg PFOB as a fast bolus injection . Using a slow infusion rate, the same number of animals received either the same dose (3 g/kg) or half the dose (1.5 g/kg) . RESULTS . Vascular enhancement was best after bolus injection of 3 g/kg emulsion . The density peak occurred after 2 hours . A continuous enhancement of approximately 100 Hounsfield units (HU) was observed up to 24 hours in the animals receiving 3 g/kg, independent of the injection velocity . A density peak of 70 HU was found 2 hours after the infusion of 1.5 g/kg . The density peak of the liver, the spleen, and the abscess wall was observed 48 hours after emulsion administration in all groups receiving 3 g/kg . The peak was approximately 150 HU for the liver, 400 HU for the spleen, and 150 HU for the abscess wall . In animals receiving only 1.5 g/kg perflubron, the peak density of the abscess wall was 132 HU after 12 hours, approximately 80 HU for the liver between 2 and 48 hours, and approximately 280 HU after 48 hours for the spleen . CONCLUSIONS . PFOB emulsion produces the highest vascular enhancement within the first 2 hours after the bolus injection of 3 g/kg . For spleen and abscess wall imaging, even the relatively low dose of 1.5 g/kg produced a satisfactory enhancement level for a significant length of time, whereas liver enhancement was best after administration of the higher dose.

Circ Shock, 1992 Sep, 38(1), 50 - 4
Effect of endotoxicosis on plasma and tissue levels of calcitonin gene-related peptide; Griffin EC et al.; This study was designed to investigate the changes in tissue content and plasma concentrations of CGRP, a 37 amino acid vasoactive peptide, in male Sprague Dawley rats injected intravenously with a nonlethal dose of 3 mg/kg E . coli endotoxin . Plasma CGRP concentrations in nonendotoxemic animals, measured by a specific RIA, were initially 30.5 +/- 3.3 pg/ml, and were significantly increased to 63.7 +/- 4.6 pg/ml 2 hr after induction of endotoxemia (P less than 0.001; n = 13) . A higher dose of LPS did not further elevate plasma CGRP levels, indicating that the maximal response occurred following a dose of 3 mg/kg LPS . CGRP levels in abdominal aorta, inferior vena cava, stomach, kidney, and left ventricular myocardium (4.11, 8.5, 2.61, 0.69, and 0.25 pmol/g wet weight tissue, respectively) were not changed significantly following the injection of endotoxin . However, in lung and mesenteric artery the levels increased significantly from 1.47 +/- 0.12 and 7.97 +/- 1.32 pmol/g wet weight tissue to 1.96 +/- 0.19 (P less than 0.05, n = 11) and 15.02 +/- 2.3 pmol/g (P less than 0.01; n = 7), respectively . In contrast, CGRP levels in the duodenum were significantly decreased from 11.3 +/- 0.93 pmol/g wet weight tissue to 6.2 +/- 0.68 pmol/g (P less than 0.001; n = 6) . The changes in plasma concentration and tissue content of CGRP suggest that splanchnic organs may be the source of the elevated plasma CGRP levels in endotoxemia and that selective organ CGRP levels reflect a role in the pathogenesis of the response to endotoxemia.

Circ Shock, 1992 Sep, 38(1), 29 - 33
Effect of platelet-activating factor antagonist and leukotriene antagonist on endotoxin shock in the rat: role of the leukocyte; Yoshikawa D et al.; The effects of platelet-activating factor (PAF) antagonist (CV-3988) and leukotrienes (LTs) antagonist (ONO-1078) on endotoxin-induced sequelae in the rat were assessed . Pretreatment with either CV-3988 (6 mg/kg, i.v.) or ONO-1078 (150 mg/kg, p.o.) did not improve survival rate following the administration of Escherichia coli lipopolysaccharide (LPS) compared with that of control rats pretreated with solvents of the drugs . Rats pretreated with both CV-3988 and ONO-1078 exhibited significantly (P less than 0.01) enhanced survival following lipopolysaccharide (LPS) administration . Percentage survivals 48 hr after the administration of LPS were 20%, 32%, 24%, and 68% in the pretreatment with solvents, CV-3988, ONO-1078, and CV-3988 combined with ONO-1078 groups, respectively . Pretreatment with CV-3988 combined with ONO-1078 inhibited the change of plasma transaminase 3 hr after LPS administration . The neutropenia, due to the administration of vinblastine (1 mg/kg, i.v.), increased the survival rate following the administration of LPS without pretreatment with PAF and LT antagonists . Antishock action of CV-3988 and ONO-1078 could not be seen in neutropenic rats . These data suggest that combined pretreatment with PAF antagonist and LT antagonist inhibited leukocyte-mediated tissue injury in LPS-induced endotoxemia.

Carcinogenesis, 1992 Sep, 13(9), 1643 - 50
Inhibition of repairable DNA-damage in Escherichia coli K-12 cells recovered from various organs of nitrosamine-treated mice by vitamin A, phenethylisothiocyanate, oleic acid and triolein; Knasmuller S et al.; The influence of various dietary constituents--phenethylisothiocyanate (PEITC), oleic acid (OA), triolein (TO), and vitamin A (ROL)--on the genotoxic activity of nitrosamines (NDMA, NDELA, NPYR) was investigated . For this purpose differential DNA repair assays with Escherichia coli K-12 strains were performed in vitro and in vivo with mice . Under in vitro conditions (liquid holding), all compounds reduced nitrosamine induced DNA-damage in the indicator bacteria in the dose range 1-10 micrograms/ml, the ranking order of efficiency being PEITC greater than OA greater than ROL greater than or equal to TO . In animal-mediated assays, acute oral treatment with PEITC (17-150 mg/kg), 2 h before nitrosamine administration, resulted in a marked decrease of nitrosamine genotoxicity in liver, kidneys, lungs and in the blood . Also in other organs (spleen, testes) an increase in differential survival (which serves as a measure for repairable DNA damage) occurred . With ROL only a comparatively moderate antigenotoxic effect was obtained at a high dose level (250 mg/kg) under identical experimental conditions . OA (2000 mg/kg) and TO (16,000 mg/kg) were completely inactive . Upon repeated treatment (consecutive oral administration of the putative antigenotoxins over 4 days, a final treatment 24 h before nitrosamine administration) PEITC (150 mg/kg/day), ROL (80 mg/kg/day) and OA (2000 mg/kg/day) had no influence on the genotoxic effects of the nitrosamines . Repeated treatment with TO (4000-16,000 mg/kg/day) resulted in a moderate dose-dependent reduction of NDMA-induced DNA-damage in the indicator bacteria, whereas in combination with NPYR only a marginal effect was observed . Biochemical experiments indicated that the antigenotoxic effects of PEITC seen under in vivo conditions were due to inhibition of alpha-hydroxylation of the nitrosamines, whereas ROL and TO appeared not to interfere strongly with this metabolic activation step . Our results indicate that in vitro assays do only partly reflect the antigenotoxic properties of the different food constituents in vivo and that animal-mediated DNA repair assays with E . coli strains are an appropriate approach to study the effects of modifiers of nitrosamine genotoxicity in the living animal.

Carcinogenesis, 1992 Sep, 13(9), 1615 - 8
Spectrum of mutations in single-stranded DNA phage M13mp2 exposed to sunlight: predominance of G-to-C transversion; Negishi K et al.; Sunlight is regarded to be a cause of skin cancer, though the mechanisms underlying the causation are still unclear . The genotoxic effects of sunlight are believed to be induced by pyrimidine photoproducts produced by the action of the UV portion of sunlight . However, it is not clear whether these pyrimidine modifications are the sole sources for the mutations . In the present study, we have analyzed the mutagenic potential of sunlight on the lacZ alpha region of single-stranded DNA phage M13mp2 using an SOS-deficient recA- strain and an SOS-induced rec+ strain of Escherichia coli as hosts . Exposure to sunlight caused mutations; approximately 10-fold increases in the mutation frequency were observed with the use of both hosts . When SOS functions were induced in the host CSH50, the mutation frequencies increased another 10-fold over those obtained with the host lacking the SOS functions . DNA sequences of the mutants were analyzed by automated DNA sequencers . Sequence changes were identified in 53 mutants from the mutant DNAs obtained using NR9099 as host and in 78 mutant samples obtained using UV-treated CSH50 . Most of the mutations were transversions of guanine, either G to C or G to T . Furthermore, 59% of the identified sequence changes in the SOS- host and 40% of those in the SOS-induced host were G-to-C transversions . These transversions may be caused by unidentified guanine damages or by the effects of damage at pyrimidines distal from guanines to be mutated.

Biochem J, 1992 Sep 1, 286 ( Pt 2), 603 - 6
Changes in the adenine nucleotide and inorganic phosphate content of Escherichia coli F1-ATPase during ATP synthesis in dimethyl sulphoxide; Beharry S et al.; Escherichia coli F1-ATPase contained 2.9 +/- 0.1 mol of adenine nucleotide and 3.1 +/- 0.3 mol of Pi/mol of enzyme . After preincubation with ATP, the nucleotide and phosphate contents were 5.6 and 6.0 +/- 0.5 mol/mol of enzyme respectively . The F1-ATPase was induced to synthesize ATP in the presence of 30% (v/v) dimethyl sulphoxide (Me2SO) . The ATP originated from endogenous bound ADP . The bound adenine nucleotide and Pi contents of the enzyme during the time course of ATP synthesis were investigated by using F1-ATPase which had been preincubated with ATP . We show that the process of ATP synthesis in Me2SO involves (i) an initial rapid loss of nucleotide from the enzyme, the process being facilitated by exogenous Pi, (ii) a rapid loss of Pi from the enzyme, at least in the absence of exogenous Pi, (iii) re-binding of a portion of the lost nucleotide, and (iv) synthesis of ATP from bound ADP and exogenous Pi . It is proposed that transfer of the F1-ATPase to the Me2SO medium induces a change in the conformation of the enzyme to a form favouring ATP synthesis.

J Cell Biol, 1992 Sep, 118(5), 1109 - 20
Cyclin A potentiates maturation-promoting factor activation in the early Xenopus embryo via inhibition of the tyrosine kinase that phosphorylates cdc2; Devault A et al.; We have produced human cyclin A in Escherichia coli and investigated how it generates H1 kistone kinase activity when added to cyclin-free extracts prepared from parthenogenetically activated Xenopus eggs . Cyclin A was found to form a major complex with cdc2, and to bind cdk2/Eg1 only poorly . No lag phase was detected between the time when cyclin A was added and the time when H1 histone kinase activity was produced in frog extracts, even in the presence of 2 mM vanadate, which blocks cdc25 activity . Essentially identical results were obtained using extracts prepared from starfish oocytes . We conclude that formation of an active cyclin A-cdc2 kinase during early development escapes an inhibitory mechanism that delays formation of an active cyclin B-cdc2 kinase . This inhibitory mechanism involves phosphorylation of cdc2 on tyrosine 15 . Okadaic acid (OA) activated cyclin B-cdc2 kinase and strongly reduced tyrosine phosphorylation of cyclin B-associated cdc2, even in the presence of vanadate . 6-dimethylamino-purine, a reported inhibitor of serine-threonine kinases, suppressed OA-dependent activation of cyclin B-cdc2 complexes . This indicates that the kinase(s) which phosphorylate(s) cdc2 on inhibitory sites can be inactivated by a phosphorylation event, itself antagonized by an OA-sensitive, most likely type 2A phosphatase . We also found that cyclin B- or cyclin A-cdc2 kinases can induce or accelerate conversion of the cyclin B-cdc2 complex from an inactive into an active kinase . Cyclin B-associated cdc2 does not undergo detectable phosphorylation on tyrosine in egg extracts containing active cyclin A-cdc2 kinase, even in the presence of vanadate . We propose that the active cyclin A-cdc2 kinase generated without a lag phase from neo-synthesized cyclin A and cdc2 may cause a rapid switch in the equilibrium of cyclin B-cdc2 complexes to the tyrosine-dephosphorylated and active form of cdc2 during early development, owing to strong inhibition of the cdc2-specific tyrosine kinase(s) . This may explain why early cell cycles are so rapid in many species.

Gene, 1992 Sep 1, 118(1), 97 - 102
Chimeric HU-IHF proteins that alter DNA-binding ability; Goshima N et al.; Chimeric proteins between Escherichia coli histone-like HU and IHF were constructed by genetic engineering, in which part of the arm region was replaced by the corresponding region of IHF alpha (designated as HupANhimA) or IHF beta (HupANhimD); alternatively, an alpha-helix 2-beta 1 region was replaced by the corresponding region of IHF alpha (HupAXhimA) or IHF beta (HupAXhimD) (symbols N and X indicate NotI and XhoI junctions) . These proteins were synthesized in a hupA-hupB double-deletion mutant . HupANhimA exhibited marked reduction in nonspecific DNA binding in vitro, and a drastic loss of HU activity in replicative transposition of Mu phage in vivo . HupANhimD also showed a significant reduction in the ability for DNA binding, though this protein supported Mu phage development . In contrast, the other two chimeric HU proteins showed only slight changes in nonspecific DNA-binding ability: they retained activities for transposition of Mu phage in vivo . These observations confirm that the flexible arm of HU-2, a domain proposed for DNA binding {Tanaka et al., Nature 310 (1984) 376-381; Goshima et al., Gene 96 (1990) 141-145}, plays an important role in the physiological function of this protein . The results indicate that a unique conformation of the arm structure of HU protein, particularly the N-terminal half of a two-strand antiparallel beta-ribbon of the structure, is important for the DNA-binding ability of this protein.

EMBO J, 1992 Sep, 11(9), 3237 - 44
Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement; Kruse N et al.; Interleukin-4 (IL-4) represents a prototypic lymphokine (for a recent review see Paul, 1991) . It promotes differentiation of B-cells and the proliferation of T- and B-cell, and other cell types of the lymphoid system . An antagonist of human IL-4 was discovered during the studies presented here after Tyr124 of the recombinant protein had been substituted by an aspartic acid residue . This IL-4 variant, Y124D, bound with high affinity to the IL-4 receptor (KD = 310 pM), but retained no detectable proliferative activity for T-cells and inhibited IL-4-dependent T-cell proliferation competitively (K(i) = 620 pM) . The loss of efficacy in variant Y124D was estimated to be greater than 100-fold on the basis of a weak partial agonist activity for the very sensitive induction of CD23 positive B-cells . The substitution of Tyr124 by either phenylalanine, histidine, asparagine, lysine or glycine resulted in partial agonist variants with unaltered receptor binding affinity and relatively small deficiencies in efficacy . These results demonstrate that high affinity binding and signal generation can be uncoupled efficiently in a ligand of a receptor belonging to the recently identified hematopoietin receptor family . In addition we show for the first time, that a powerful antagonist acting on the IL-4 receptor system can be derived from the IL-4 protein.

EMBO J, 1992 Sep, 11(9), 3165 - 74
Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli; Bieker-Brady K et al.; The sec/prl gene products catalyze the translocation of precursor proteins from the cytoplasm of Escherichia coli . Recessive, conditionally lethal mutant alleles of these genes (sec mutations) cause a generalized defect in protein secretion; dominant suppressor mutant alleles (prl mutations) restore export of precursor proteins with altered signal sequences . In prl strains, a precursor protein with a defective signal sequence can be selectively targeted to the suppressor gene product . When a precursor LacZ hybrid protein is used, the targeted prl protein is inactivated by the large, toxic hybrid molecule, a result termed suppressor-directed inactivation (SDI) . Using SDI, two different secretion-related complexes can be generated: a pretranslocation complex that contains a hybrid protein with an unprocessed signal sequence, and a translocation complex in which the hybrid protein is jammed in transmembrane orientation with the signal sequence cleaved . Additional Sec proteins that are contained within, and thus sequestered by, each of these complexes can be identified when their functional levels are lowered using the conditional lethal sec mutations . Results of this genetic analysis suggest a multistep pathway for protein secretion in which the translocation machinery assembles on demand.

Arch Biochem Biophys, 1992 Sep, 297(2), 340 - 4
Catalytic properties of Escherichia coli F1-ATPase depleted of endogenous nucleotides; Senior AE et al.; Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al . (1983, Biochem . J . 215, 343-350) . This enzyme had high rates of steady-state ATPase and GTPase activity . When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites . The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1 . Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1 . Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis . In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E . coli F1 . Such an entity has been reported for chloroplast and mitochondrial F1 . However, our experiments gave no indication for inhibitory MgADP in E . coli F1.

Arch Biochem Biophys, 1992 Sep, 297(2), 334 - 9
F1-ATPase with cysteine instead of serine at residue 373 of the alpha subunit; Lee RS et al.; Escherichia coli strain AN718 contains the alpha S373F mutation in F1F0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F1-ATPase activity . The revertant strain AN718SS2 containing the mutation alpha C373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB . Purified F1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity . Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in alpha S373F mutant F1, were regained in alpha C373 F1 . N-Ethylmaleimide (NEM) inactivated alpha C373 F1 steady-state ATPase potently but had no effect on unisite ATPase . Complete inactivation of alpha C373 F1 steady-state ATPase corresponded to incorporation of one NEM per F1 (mol/mol), in just one of the three alpha subunits . NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence . The data confirm the view that placement at residue alpha 373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F1 . The fact that NEM inhibits steady-state ATPase when only one alpha subunit of three is reacted suggests a cyclical catalytic mechanism.

FEMS Microbiol Immunol, 1992 Sep, 5(1-3), 55 - 62
Haemolysin of Escherichia coli: comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins; Benz R et al.; Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages . One is a prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s . Membranes formed of pure lipids were rather inactive targets for this haemolysin . Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH . The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase . The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm . The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin . Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes . Both types of haemolysin channels have similar properties but different lifetimes.

J Gen Virol, 1992 Sep, 73 ( Pt 9), 2457 - 61
Mapping of sequential epitopes recognized by monoclonal antibodies on the bovine leukaemia virus external glycoproteins expressed in Escherichia coli by means of antipeptide antibodies; Ban J et al.; A lambda gt11 cDNA library prepared from bovine leukaemia virus (BLV)-producing ovine cells was screened with a cocktail of anti-BLV gp51 monoclonal antibodies (MAbs) . Four recombinant phages with inserts of about 2-5 kbp were isolated . One, lambda BLV-gp51-1, was sequenced and shown to encode the C-terminal part of gp51 and all of gp30 . This insert was subcloned into pEV-vrf1 and expressed in Escherichia coli N-4830-1 cells . The BLV product and a series of antipeptide antibodies were used to localize the sequential epitopes defined on BLV envelope glycoprotein gp51 by their reactivity with MAbs . Epitope B was localized to amino acids 180 to 205, B' to residues 195 to 205, D and D' to residues 218 to 237, and A to amino acids 249 to 260 . All the mapped sequential epitopes were localized in the C-terminal half of BLV gp51 . The results of epitope mapping with bacterially produced gp51 confirm the map obtained using native viral glycoprotein.

J Gen Virol, 1992 Sep, 73 ( Pt 9), 2451 - 5
Inhibition of viral replication by monoclonal antibodies directed against human immunodeficiency virus gp120; Niedrig M et al.; Monoclonal antibodies (MAbs) were raised against the glycoprotein gp120 of human immunodeficiency virus type 1 (strain HTLV-IIIB) . The reactivity of five selected MAbs was characterized in several tests: ELISA, immunostaining of Western blots, immunofluorescence, immunoprecipitation, immunoelectron microscopy, alkaline phosphatase-anti-alkaline phosphatase assay and neutralization . The binding region was delimited by sequential overlapping Escherichia coli fusion proteins of the gp120 sequence between amino acids (aa) 49 and 280 . In the ELISA, when using sequential overlapping 15 aa peptides, the binding epitopes were localized between aa 64 and 78 for three MAbs and between aa 114 and 123 for the fourth Mab . The fifth Mab showed multiple reactions with different peptides possibly indicating a reaction with a discontinuous epitope . In virus growth inhibition assays, all five MAbs inhibited the spread of HIV-1 infection in cell cultures after a single or repeated treatment at a concentration of 63 micrograms/ml of the purified MAbs . All MAbs showed low but significant neutralizing activity at concentrations of 100 micrograms/ml.

J Gen Virol, 1992 Sep, 73 ( Pt 9), 2435 - 9
Location of monoclonal antibody binding sites in the capsid protein of feline calicivirus; Milton ID et al.; We report the localization of three monoclonal antibody (MAb) binding sites in the capsid protein of feline calicivirus . Gene fragments were generated by restriction enzyme digestion or the polymerase chain reaction, and expressed as beta-galactosidase fusion proteins in Escherichia coli . These chimeric molecules were screened using three MAbs . A non-neutralizing MAb recognized a region within 36 amino acids of the C terminus . Two neutralizing MAbs bound to a different region of 37 amino acids in the centre of the protein . Comparative sequence analysis shows this area to be the major variable region of the capsid protein.

FEMS Microbiol Lett, 1992 Sep 1, 75(1), 49 - 53
Arg276 of GseA, a Chlamydia trachomatis Kdo transferase, is required for the synthesis of the chlamydial genus-specific epitope in Escherichia coli; Ekpo P et al.; GseA is an enzyme from Chlamydia trachomatis that can catalyse the addition of three 3-deoxy-D-manno-octulosonic acid (Kdo) residues onto lipid A precursors . GseA is similar, and in a few stretches identical, in its amino acid sequence to KdtA, an Escherichia coli Kdo transferase . In this study we altered an amino acid of GseA in a region that is identical between GseA and KdtA to test its importance in the structure or catalytic activity of GseA . We found that when Arg276 was changed to Lys, Ile or Ser, GseA activity was lost, suggesting an enzymatic role for this amino acid residue.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8006 - 10
Targeted cleavage of mRNA by human RNase P; Yuan Y et al.; Ribonuclease P from Escherichia coli can cleave RNAs in simple, hydrogen-bonded complexes of two oligoribonucleotides that resemble the aminoacyl stem and 5' leader sequence of tRNA precursors . RNase P from human (HeLa) cells cannot catalyze the cleavage in vitro of the 5'-proximal oligoribonucleotide that contains the leader sequence in such simple complexes but can do so when the 3'-proximal oligoribonucleotide (external guide sequence) is altered to resemble three-quarters of a tRNA molecule . In such a complex, the efficiency of cleavage of the mRNA for chloramphenicol acetyltransferase, as the 5'-proximal oligoribonucleotide, depends on the structural details of the external guide sequence and on the choice of target site within the mRNA . The presence of the appropriately designed external guide sequence in cells in tissue culture reduces chloramphenicol acetyltransferase activity and the level of the corresponding intact mRNA in the cells . Thus, it appears that the use of such external guide sequences may provide a general technique for gene inactivation.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 7851 - 5
Hydroxyl radical cleavage of tRNA in the ribosomal P site; Huttenhofer A et al.; Hydroxyl radical is a useful probe of the accessibility of the sugar moiety of nucleic acids to solvent . Here we compare the accessibility of free and ribosome-bound yeast tRNA(Phe), Escherichia coli tRNA(Phe), and E . coli tRNA(Leu2) to attack by hydroxyl radicals generated from Fe(2+)-EDTA . When bound to the P site of 30S ribosomal subunits, a discrete region, corresponding almost precisely to the anticodon stem-loop, is strongly protected; weaker protection is observed in the 3' strand of the D stem and in the variable loop . The protected nucleotides constitute a well-defined substructure, corresponding to the lower half of the anticodon-D loop coaxial arm of the tRNA crystal structure . This result suggests that the 30S P site contains a pocket that becomes inaccessible to the Fe(2+)-EDTA complex when tRNA is bound, whose minimum dimensions can be inferred from the boundaries of the protected region of tRNA . When bound to the P site of 70S ribosomes, the entire tRNA backbone becomes inaccessible to hydroxyl radicals . Since previous studies have shown that virtually the entire footprint of a P-site tRNA on 16S and 23S rRNAs is mimicked by the extremities of the tRNA (the anticodon stem-loop plus the 3'-terminal aminoacyl-pentanucleotide), protection of the entire tRNA was unexpected . We conclude that protection of the elbow of tRNA is due either to interactions with ribosomal proteins or to enclosure in an inaccessible site formed by association of the two ribosomal subunits.

Mutat Res, 1992 Sep, 269(1), 27 - 33
Directly acting geno- and cytotoxic agents from a wild mushroom Dermocybe sanguinea; von Wright A et al.; The wild mushroom, Dermocybe sanguinea, contains several anthraquinone pigments, of which emodin (1,3,8-trihydroxy-6-methylanthraquinone) is quantitatively the most important . In our preliminary tests, Dermocybe sanguinea extracts were genotoxic without metabolic activation . The ethanol extract of Dermocybe sanguinea was fractionated by flash chromatography, and the emodin contents of the fractions were determined by HPLC . Their genotoxicities were assayed using a bacterial repair assay and sister-chromatid exchange analysis . The cytotoxicity of the fractions was assayed with mouse hepatoma cells using growth inhibition as the endpoint . The results of the biological tests were compared with those obtained with pure emodin . It was concluded that, in addition to emodin, Dermocybe sanguinea contains several other geno- and cytotoxic compounds.

Biochemistry, 1992 Sep 1, 31(34), 7989 - 97
Structure-function analysis of DNA polymerase-beta using monoclonal antibodies: identification of a putative nucleotide binding domain; Recupero AJ et al.; DNA polymerase-beta was purified from Novikoff hepatoma and used as an antigen in an in vitro immunization system to produce monoclonal antibodies . These reagents surprisingly showed cross-reactivity to a number of proteins, including several DNA polymerases . Nearly all of these proteins possess nucleotide binding sites, which suggested the potential value of using the monoclonals to elucidate structure-function relationships within polymerase-beta . Furthermore, these antibodies were able to partially neutralize (40-50%) polymerase-beta activity, and this effect could be blocked by dNTP1 but not by dNMP or rNTP . The limited neutralization phenomenon is at least partially explained by the weak binding affinity of these antibodies . Scatchard analysis of immunoprecipitation data predicted a Kd of 1.8 x 10(-8) M . Epitope mapping studies showed that the region of polymerase-beta recognized by one of the monoclonal antibodies is within residues 235-335, and sequence homology studies indicated that the epitope is probably located in the region of amino acids 283-320 . At least a portion of this area, namely residues 301-308 and 311-315, appears to be part of a nucleotide binding domain which has sequence homology with a portion of the highly conserved ATP binding site in adenylate kinase.

EMBO J, 1992 Sep, 11(9), 3323 - 35
Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme; Xanthoudakis S et al.; The DNA binding activity of Fos and Jun is regulated in vitro by a post-translational mechanism involving reduction-oxidation . Redox regulation occurs through a conserved cysteine residue located in the DNA binding domain of Fos and Jun . Reduction of this residue by chemical reducing agents or by a ubiquitous nuclear redox factor (Ref-1) recently purified from Hela cells, stimulates AP-1 DNA binding activity in vitro, whereas oxidation or chemical modification of the cysteine has an inhibitory effect on DNA binding activity . Here we demonstrate that the protein product of the ref-1 gene stimulates the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kappa B, Myb and members of the ATF/CREB family . Furthermore, immunodepletion analysis indicates that Ref-1 is the major AP-1 redox activity in Hela nuclear extracts . Interestingly, Ref-1 is a bifunctional protein; it also possesses an apurinic/apyrimidinic (AP) endonuclease DNA repair activity . However, the redox and DNA repair activities of Ref-1 can, in part, be distinguished biochemically . This study suggests a novel link between transcription factor regulation, oxidative signalling and DNA repair processes in higher eukaryotes.

EMBO J, 1992 Sep, 11(9), 3289 - 95
Interaction of the RNA-binding domain of the hnRNP C proteins with RNA; Gorlach M et al.; The hnRNP C proteins are among the most abundant and avid pre-mRNA-binding proteins and they contain a consensus sequence RNA-binding domain (RBD) that is found in a large number of RNA-binding proteins . The interaction of the RBD of the hnRNP C proteins with an RNA oligonucleotide {r(U)8} was monitored by nuclear magnetic resonance (NMR) . 15N and 13C/15N-labelled hnRNP C protein RBD was mixed with r(U)8 and one- and two-dimensional (1D and 2D) NMR spectra were recorded in a titration experiment . NMR studies of the uncomplexed 93 amino acid hnRNP C RBD (Wittekind et al., 1992) have shown that it has a compact folded structure (beta alpha beta beta alpha beta), which is typical for the RBD of this family of proteins and which is comprised of a four-stranded antiparallel beta-sheet, two alpha-helices and relatively unstructured amino- and carboxy-terminal regions . Sequential assignments of the polypeptide main-chain atoms of the hnRNP C RBD-r(U)8 complex revealed that these typical structural features are maintained in the complex, but significant perturbations of the chemical shifts of amide group atoms occur in a large number of residues . Most of these residues are in the beta-sheet region and especially in the terminal regions of the RBD . In contrast; chemical shifts of the residues of the well conserved alpha-helices, with the exception of Lys30, are not significantly perturbed . These observations localize the candidate residues of the RBD that are involved in the interaction with the RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1992 Sep, 11(9), 3255 - 61
Molecular architecture of acetylcholinesterase collagen-tailed forms; construction of a glycolipid-tailed tetramer; Duval N et al.; Asymmetric forms of Torpedo acetylcholinesterase (AChE) are produced in COS cells by the simultaneous expression of collagenic subunits (Q) and catalytic T subunits (AChET) . Truncated AChET delta subunits, from which most of the C-terminal peptide (TC) had been deleted by mutagenesis, did not associate with Q subunits . The TC peptide is therefore necessary for the association of the AChET and Q subunits . In order to determine the orientation of the Q subunit in the collagen-tailed forms, we have developed an antiserum against its non-collagenic C-terminal domain, expressed as a fusion protein in Escherichia coli . This antiserum, which recognized the Q subunit in Western blots, was found to react with intact asymmetric forms, but not with collagenase-treated forms, from which the distal part of the tail had been cleaved, suggesting that the N-terminal non-collogenic domain (QN) is responsible for the interaction with the AChET subunits . This was confirmed by creating a chimeric subunit (QN/HC), in which QN was linked to the C-terminal peptide of the H subunit of Torpedo AChE, which contains the glycophosphatidylinositol (GPI) cleavage/attachment signal: co-expression of AChET and QN/NC produced GPI-anchored tetramers, which were sensitive to PI-PLC and largely exposed to the external surface of the cells . We thus demonstrate that: (i) the HC peptide is sufficient to determine the addition of a glycolipid anchor and (ii) the QN domain is sufficient to bind a catalytic AChET tetramer by interacting with the TC peptide.

Biotechnology (N Y), 1992 Sep, 10(9), 1013 - 9
Polyethylene glycol enhanced protein refolding; Cleland JL et al.; Previous studies on the refolding of recombinant bovine carbonic anhydrase B (CAB) indicated that polyethylene glycol (PEG) significantly enhanced the recovery of active protein by reducing aggregation . To further test the ability of PEG to enhance refolding, three recombinant human proteins, deoxyribonuclease (rhDNAse), tissue plasminogen activator (rhtPA), and interferon-gamma (rhIFN-gamma) were refolded in the presence of PEG (3350 MW) . rhDNAse produced from CHO cells was denatured in 7.2 M urea and refolded by rapid dilution to 4.0 M urea and 0.20 mg/ml protein . When a final PEG to rhDNAse molar ratio of 5 to 1 (0.1 milligram PEG, 3350 MW) was used in the dilution buffer, refolding was improved by 30% to yield complete recovery of active protein . Impure E . coli derived inclusion body preparations of rhDNAse were solubilized in 8 M urea and refolded by dilution to 4 M urea and 0.10 mg/ml protein . Refolding with a dilution buffer which yielded a final PEG to rhDNAse molar ratio of 10 to 1 (0.1 milligram PEG, 3350 MW) resulted in a three-fold increase in the recovery of active protein . When PEG was used in the dilution buffer, aggregation of rhDNAse did not occur during refolding in either case . rhtPA produced from CHO cells was denatured in 5 M guanidine hydrochloride (GuHCl) and refolded by rapid dilution to 0.10 M GuHCl and 0.20 mg/ml protein.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biotechnol, 1992 Sep, 25(3), 307 - 18
Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with Immobiline membranes; Ettori C et al.; Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography . For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers . The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e . equidistant from the pI value of hGH (pI 5.12) . r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field . The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90% . The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40) . The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer . Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82) . This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.

J Biotechnol, 1992 Sep, 25(3), 221 - 30
Production of an enzymatic active protein using a continuous flow cell-free translation system; Endo Y et al.; We have examined the characteristics of protein synthesis in an improved continuous flow cell-free translation system prepared from wheat germ extract with dihydrofolate reductase (dhfr) mRNA as the translated message . Continuous buffer flow and separation of product from the reaction mixture were accomplished by the use of a modified Amicon ultrafiltration chamber as reaction vessel . The system produced protein for more than 20 h, and the product had an activity of dhfr comparable to that of authentic enzyme from E . coli . Analysis of RNA recovered from the filtrate supports the notion that a functionally active protein-synthesizing machinery is superorganized in a dynamic complex.

Hear Res, 1992 Sep, 62(1), 124 - 6
Construction of a cDNA library from microdissected guinea pig organ of Corti; Wilcox ER et al.; Poly (A) RNA was isolated from microdissected guinea pig organ of Corti and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase . After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids were transformed into DH10B E . coli via electroporation . The library was found to have 3.35 x 10(6) independent colonies with ten percent of the colonies lacking an insert . After checking 33 randomly selected colonies for inserts, the average insert size was 1218 base pairs, ranging from 3300 base pairs to 400 base pairs . The library was screened with a beta-actin oligonucleotide probe and 1.4% of the colonies contained an insert hybridizing to the probe.

Infection, 1992 Sep-Oct, 20(5), 273 - 5
S-fimbriae mediated adhesion of Escherichia coli to human buccal epithelial cells is age independent; Schroten H et al.; S-fimbriated Escherichia coli, which cause sepsis and meningitis in the newborn, bind to sialic acid-containing glycoprotein structures on the surface of human buccal epithelial cells . The dependence of this binding on host age was examined . S-fimbriated E . coli adhered in comparable numbers to cells in newborns, infants, children and adults (23.0 +/- 8.6; 23.1 +/- 11.5; 24.7 +/- 7.9; 28.9 +/- 8.8) . Thus, the increased susceptibility of neonates to infections caused by S-fimbriated E . coli cannot be explained by enhanced adhesion to epithelial cells.

Gut, 1992 Sep, 33(9), 1184 - 9
Effects of enteropathogenic Escherichia coli on microvillar membrane proteins during organ culture of rabbit intestinal mucosa; Embaye H et al.; This study examines the effects of an enteropathogenic Escherichia coli on microvillar membrane proteins during organ culture of rabbit ileal explants . Explants maintained with enteropathogenic E coli showed brush border effacement affecting approximately 50% of enterocytes, and where enteropathogenic E coli were closely adherent to the enterocyte surface microvilli were apparently being shed as vesicles . The microvillar membrane enzymes alkaline phosphatase, aminopeptidase N and alpha-glucosidase were released into the culture medium during organ culture, and this process was significantly enhanced by enteropathogenic E coli . This increased loss of microvillar membrane enzymes into the culture medium was associated with decreased tissue activities of microvillar membrane enzymes in enteropathogenic E coli infected ileal explants compared with control . For aminopeptidase N in particular, however, total enzyme activities in the tissue plus culture medium were increased comparing enteropathogenic E coli with control, suggesting that there might be an increase in the rate of synthesis of certain microvillar membrane proteins . Reorientating sucrose density gradient centrifugation of culture medium showed that alkaline phosphatase, aminopeptidase N and alpha-glucosidase were predominantly associated with particles of peak modal density 1.19 g/ml in both groups, confirming that enteropathogenic E coli accelerate release of microvillar membrane enzymes as vesicles . Analytical fractionation of ileal explants showed that enteropathogenic E coli resulted in a loss of microvillar membrane enzyme activities from the main brush border peak of modal density 1.21 g/ml present in controls . The density of the remaining smaller and lighter peak increased from 1.19 g/ml to 1.23 g/ml after homogenisation in digitonin, confirming association of these proteins with cholesterol containing membranes and not endoplasmic reticulum . These findings suggest that enteropathogenic E . coli accelerate the normal shedding of microvillar membrane proteins as vesicles, and may stimulate a compensatory increase in microvillar membrane protein synthesis.

Am J Vet Res, 1992 Sep, 53(9), 1488 - 92
Pathogenicity of porcine enterotoxigenic Escherichia coli that do not express K88, K99, F41, or 987P adhesins; Casey TA et al.; Three-week-old weaned and colostrum-deprived neonatal (less than 1 day old) pigs were inoculated to determine the pathogenicity of 2 enterotoxigenic Escherichia coli isolates that do not express K88, K99, F41, or 987P adhesins (strains 2134 and 2171) . Strains 2134 and 2171 were isolated from pigs that had diarrhea after weaning attributable to enterotoxigenic E coli infection . We found that both strains of E coli adhered in the ileum and caused diarrhea in pigs of both age groups . In control experiments, adherent bacteria were not seen in the ileum of pigs less than 1 day old or 3 weeks old that were noninoculated or inoculated with a nonpathogenic strain of E coli . These control pigs did not develop diarrhea . Antisera raised against strains 2134 and 2171 and absorbed with the autologous strain, grown at 18 C, were used for bacterial-agglutination and colony-immunoblot assays . Both absorbed antisera reacted with strains 2134 and 2171, but not with strains that express K99, F41, or 987P adhesins . A cross-reaction was observed with 2 wild-type K88 strains, but not with a K12 strain that expresses K88 pili . Indirect immunofluorescence with these absorbed antisera revealed adherent bacteria in frozen sections of ileum from pigs infected with either strain . We concluded that these strains are pathogenic and express a common surface antigen that may be a novel adhesin in E coli strains that cause diarrhea in weaned pigs.

Proteins, 1992 Sep, 14(1), 1 - 9
Alkaline phosphatase-somatostatin hybrid proteins as probes for somatostatin-14 receptors; Langen HT et al.; By inserting appropriate peptide ligands into surface loops on globular proteins, we expect to develop probes for the location, accessibility, and steric and electrostatic environment of these ligand-binding sites on their membrane-bound receptors . Three residues in a loop on the surface of E . coli alkaline phosphatase were substituted by an 18-residue peptide containing the receptor-binding segment of somatostatin-14 without significantly affecting the catalytic properties of the enzyme . This hybrid protein was then used to investigate the ligand-binding site of somatostatin receptors . Tryptic cleavage of the hybrid protein within the inserted sequence, and binding of the hybrid protein to antisomatostatin antibodies demonstrated the surface accessibility of the guest peptide . Both the wild-type enzyme and the hormone-enzyme hybrid displaced 125I-labeled somatostatin from rat brain membrane receptors only at high concentrations . However, chemical cationization of the hybrid protein, which again did not disturb the phosphatase activity, enhanced its receptor-binding potency to a level only 23 times lower than that of somatostatin itself and 280 times higher than that of the cationized wild-type protein . This alkaline phosphatase/somatostatin hybrid protein appears, therefore, to be a suitable starting point for the development of probes for the steric and electrostatic environment of the ligand-binding site of somatostatin receptors.

Mol Microbiol, 1992 Sep, 6(17), 2429 - 35
Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap; van der Woude MW et al.; Pyelonephritis-associated pilus (Pap) expression is regulated by a phase variation control mechanism involving PapB, Papl, catabolite activator protein (CAP), leucine-responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam) . Lrp and Papl bind to a specific non-methylated pap regulatory DNA region containing the sequence 'GATC' and facilitate the formation of an active transcriptional complex . Evidence indicates that binding of Lrp and Papl to this region inhibits methylation of the GATC site by Dam . However, if this GATC site is first methylated by Dam, binding of Lrp and Papl is inhibited . These events lead to the formation of two different pap methylation states characteristic of active (ON) and inactive (OFF) pap transcription states . The fae (K88), daa (F1845) and sfa (S) pilus operons share conserved 'GATC-box' domains with pap and may be subject to a similar regulatory control mechanism involving Lrp and DNA methylation.

J Surg Res, 1992 Sep, 53(3), 298 - 305
Mechanisms of increased hepatic glutamine uptake in the endotoxin-treated rat; Pacitti AJ et al.; The mechanisms underlying the accelerated hepatic consumption of glutamine that occurs during endotoxemia were investigated in rats 12 hr after treatment with Escherichia coli lipopolysaccharide . Hepatic glutamine delivery and consumption were calculated from measurements of hepatic blood flow and blood glutamine levels . Hepatic glutaminase activity and glutamine and glutamate content were determined . Hepatocyte plasma membrane transport activity was evaluated employing isolated hepatic plasma membrane vesicles (HPMVs) . Endotoxin treatment resulted in an 11-fold increase in hepatic glutamine consumption and a 2-fold increase in the delivered load of glutamine to the liver . Hepatic glutamate content doubled while glutamine content was unaffected, not withstanding a decrease in the specific activity of glutaminase . Studies employing HPMVs demonstrated that hepatic plasma membrane transport activity was unaffected by endotoxin treatment . The enhanced hepatic consumption of glutamine secondary to endotoxemia appears to be the result of both a mass-action effect and the concurrent activation of intracellular metabolism . Responses at the level of plasma membrane transport do not appear to play an active role in mediating this enhanced hepatic uptake.

Int J Radiat Biol, 1992 Sep, 62(3), 321 - 6
Synergism between electrolysis and methylene blue photodynamic action in Escherichia coli; Capella MA et al.; There is interest in the use of photodynamic therapy for the treatment of certain diseases, including cancer . However, weak penetration of visible light in tissues has restricted its use . In this study the possibility of enhancing photodynamic effects by the use of energies that penetrate more deeply in tissues was investigated . Weak electric currents (1.0 mA) applied to Escherichia coli cells for short periods, producing little or no lethal damage, was found to act synergistically with the photodynamic action of methylene blue, significantly enhancing the effects of this treatment . This synergism exists also between electrolysis and X-rays but not between electrolysis and UV-254 nm . It is suggested that this synergism might eventually be used to improve the results obtained in therapeutic practice based on the utilization of photodynamic action.

J Leukoc Biol, 1992 Sep, 52(3), 343 - 8
Lectinophagocytosis of type 1 fimbriated (mannose-specific) Escherichia coli in the mouse peritoneum; Bernhard W et al.; Bacteria can bind specifically to phagocytic cells via lectin-carbohydrate interactions and such binding is often followed by activation and degranulation of the phagocytes, as well as uptake and killing of the bacteria, a phenomenon designated lectinophagocytosis . Although extensively studied in vitro, no direct evidence for the occurrence of lectinophagocytosis in vivo has been available . To obtain such evidence, we injected type 1 fimbriated (mannose-specific) or nonfimbriated Escherichia coli into the peritoneal cavity of mice (10(7)-10(10) bacteria/animal) in the absence or presence of sugars and quantified the phagocytic activity by assaying the release of lysosomal N-acetyl-beta-D-glucosaminidase into the peritoneal fluid, up to 45 min after injection . Following injection of the type 1 fimbriated bacteria, significant release of the enzyme was observed which was time dependent and increased with the number of bacteria injected, whereas the nonfimbriated bacteria caused only little release . Methyl alpha-D-mannoside (50 mM), but not methyl alpha-D-galactoside or sucrose, inhibited the release by 60 to 100% . No release of N-acetyl-beta-D-glucosaminidase was induced by bacteria injected into a peritoneal cavity from which the macrophages had been removed . Our findings show that lectinophagocytosis can occur in vivo and may contribute to the host's defence against type 1 fimbriated bacteria.

J Bacteriol, 1992 Sep, 174(18), 5923 - 35
Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding; Russell PW et al.; We describe the characterization of two genes, fimF and fimG (also called pilD), that encode two minor components of type 1 pili in Escherichia coli . Defined, in-frame deletion mutations were generated in vitro in each of these two genes . A double mutation that had deletions identical to both single lesions was also constructed . Examination of minicell transcription and translation products of parental and mutant plasmids revealed that, as predicted from the nucleotide sequence and previous reports, the fimF gene product was a protein of ca . 16 kDa and that the fimG gene product was a protein of ca . 14 kDa . Each of the constructions was introduced, via homologous recombination, into the E . coli chromosome . All three of the resulting mutants produced type 1 pili and exhibited hemagglutination of guinea pig erythrocytes . The latter property was also exhibited by partially purified pili isolated from each of the mutants . Electron microscopic examination revealed that the fimF mutant had markedly reduced numbers of pili per cell, whereas the fimG mutant had very long pili . The double mutant displayed the characteristics of both single mutants . However, pili in the double mutant were even longer than those seen in the fimG mutant, and the numbers of pili were even fewer than those displayed by the fimF mutant . All three mutants could be complemented in trans with a single-copy-number plasmid bearing the appropriate parental gene or genes to give near-normal parental piliation . On the basis of the phenotypes exhibited by the single and double mutants, we believe that the fimF gene product may aid in initiating pilus assembly and that the fimG product may act as an inhibitor of pilus polymerization . In contrast to previous studies, we found that neither gene product was required for type 1 pilus receptor binding.

Genes Dev, 1992 Sep, 6(9), 1740 - 51
Concentration-dependent activities of the even-skipped protein in Drosophila embryos; Manoukian AS et al.; The Drosophila pair-rule gene even-skipped (eve) encodes a homeo-domain-containing protein (Eve) that is required for the development of both odd- and even-numbered parasegments . We have used a heat shock-inducible eve transgene to study the regulatory functions of Eve in vivo . Transcripts encoded by eight other segmentation genes were monitored for changes in distribution and abundance following short pulses of ectopic Eve expression . Two tiers of response times appeared to distinguish between genes that were direct {fushi tarazu (ftz), odd-skipped (odd), runt (run), paired, and wingless} and indirect {eve, hairy, and engrailed (en)} targets of Eve . Genes that appeared to be directly regulated by Eve were differentially repressed in a concentration-dependent fashion . Interestingly, the run and ftz genes could also be activated by Eve during a brief 20- to 30-min stage in development . The delayed actions upon the eve and en genes appeared to be mediated by run and odd . As in eve- embryos, these effects on segmentation gene expression patterns caused defects in both odd- and even-numbered parasegments . Four sequential phenotypes could be induced, each of which was attributable to the altered expression of a unique subset of target genes.

J Exp Med, 1992 Sep 1, 176(3), 881 - 6
Resolution of Pneumocystis carinii pneumonia in CD4+ lymphocyte-depleted mice given aerosols of heat-treated Escherichia coli; Harmsen AG et al.; Mice were thymectomized and depleted of CD4+ lymphocytes by treatment with monoclonal antibody to induce Pneumocystis carinii (PC) pneumonia (PCP) . These mice were then exposed to aerosols of heat-treated Escherichia coli three times a week . Aerosol treatment for 10 d caused a slight reduction in numbers of PC nuclei in the lungs of mice, and treatment for 22 d resulted in nearly complete resolution of PCP . Large numbers of macrophages, polymorphonuclear leukocytes, and lymphocytes accumulated in lungs of aerosol-treated mice . Depletion of either CD8+ lymphocytes or asialo GM1+ cells that remained in the mice after CD4+ cell depletion had no effect on the ability of the mice to resolve PCP after E . coli aerosol treatments . However, depletion of Thy-1+ lymphocytes in these mice abrogated their ability to resolve PCP and reduced the numbers of macrophages that accumulated in the lungs . In addition, it was found that resolution of PCP induced by heat-treated E . coli aerosol treatments was also abrogated when mice were treated with polyclonal antibodies against tumor necrosis factor alpha (TNF-alpha) . Thus, resolution of PCP in CD4+ lymphocyte-depleted mice by heat-treated E . coli aerosols was not dependent on either CD8+ or asialo GM1+ cells but was dependent on Thy-1+CD4-CD8- lymphocytes and on the participation of TNF . These results indicate that heat-treated E . coli aerosols can act as an immune response modifier by inducing resolution of PCP in mice by a mechanism not dependent on the presence of CD4+ lymphocytes.

J Bacteriol, 1992 Sep, 174(17), 5567 - 74
Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI; Maneewannakul S et al.; The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid . We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141 . Studies of traW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing . Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein . Electron microscopy also showed that F lac traW546 hosts do not express F pili, confirming that TraW is required for F-pilus assembly . Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW . The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product . Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein . Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages . Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency . Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance . The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed.

EMBO J, 1992 Sep, 11(9), 3175 - 83
Common accessory genes for the Bordetella pertussis filamentous hemagglutinin and fimbriae share sequence similarities with the papC and papD gene families; Locht C et al.; The Bordetella pertussis filamentous hemagglutinin (FHA) is a major virulence factor responsible for attachment, one of the early events in bacterial pathogenesis . Deletion of its structural gene, fhaB, or a Tn5 insertion in fhaA, downstream of fhaB, resulted in a FHA- and fimbriae- phenotype, although fhaB and the fim genes are not linked . The fhaB downstream region therefore most likely encodes accessory proteins required for the biosynthesis of FHA and fimbriae, despite the lack of sequence similarities between these two proteins . The nucleotide sequence of this area contains the open reading frames fhaD and fhaA, whose products share sequence similarities with the papD and papC gene products, respectively . PapD is a periplasmic chaperone protein able to bind to the Escherichia coli P pilin subunits and to transport them towards the outer membrane protein PapC which is responsible for pilus membrane translocation . An additional open reading frame, fhaE, is located downstream of fhaA . Its amino acid sequence shares similarities with those of the fimbrial subunits . Deletion analyses suggest that fhaB and the downstream genes can be transcribed as a polycistronic operon, and primer extension analysis revealed the presence of a second promoter between fhaB and fhaD.

Infect Immun, 1992 Sep, 60(9), 3799 - 806
Genetic relationship of putative colonization factor O166 to colonization factor antigen I and coli surface antigen 4 of enterotoxigenic Escherichia coli; Sommerfelt H et al.; Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E . coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon . Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA . Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes . The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression . A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy . The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.

Infect Immun, 1992 Sep, 60(9), 3546 - 51
Presence of functional receptors for the Escherichia coli heat-stable enterotoxin in the gastrointestinal tract of the chicken; Katwa LC et al.; Receptors for the Escherichia coli heat-stable enterotoxin (STa) were shown to be present throughout the digestive tract of the chicken, with binding activity present not only in the intestinal epithelium but also in the intestinal smooth muscle . Brush border membrane vesicles (BBMV) purified from chicken enterocyte homogenates and plasma membranes (SMPM) purified from intestinal smooth muscle homogenates were compared with pig enterocyte BBMV . All had similar 125I-STa binding affinities, but the 50% effective concentration for STa activation of guanylate cyclase was higher in SMPM than in BBMV . Maximal STa-stimulated guanylate cyclase activities were similar in chicken and pig BBMV and were seven- to eightfold higher than in SMPM, and the STa receptor density was five- to sixfold higher . Patterns unique to each membrane were demonstrated after affinity labelling of STa receptors with 125I-STa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography . The results demonstrated STa-stimulated guanylate cyclase activity in birds as well as mammals and suggested that there are different functional STa receptors in chicken BBMV and SMPM.

Rev Inst Med Trop Sao Paulo, 1992 Sep-Oct, 34(5), 395 - 7
{The evaluation of the specific gravity of Giardia duodenalis and Entamoeba coli cysts}; Moitinho Mda L et al.; Cysts of Giardia duodenalis and Entamoeba coli were observed as for floatability in sucrose solutions of different specific gravity, contained in counting-chambers of 0.17 mm height . The cysts that floated and those that sedimented were counted and then calculated the respective percentage . Floatability differences of the cysts of each species were not considerable . Solutions of specific gravity 1,200 kg/m3 made 88.49% of G . duodenalis cysts and 95.71% of E . coli cysts float . The greater values of floatability were associated to specific gravity 1,250 kg/m3 and were 89.15% and 98.59% for cysts of G . duodenalis and E . coli, respectively.

Protein Sci, 1992 Sep, 1(9), 1092 - 9
Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition; Zydowsky LD et al.; Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity . The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA . Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA . The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis . Each of the mutants bound to a CsA affinity matrix . The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not . Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.

Res Microbiol, 1992 Sep, 143(7), 665 - 9
Unusual structure of plasmid DNA formed in transformants of Escherichia coli; Kato M et al.; An unusual structural component of plasmid pUC18 was observed in transformants of Escherichia coli strains . This molecule migrated faster than superhelical topoisomers in agarose gel electrophoresis and was difficult to stain with ethidium bromide . This unusual topology appeared independently of gyrase mutation, and the recA genotype may relate to accumulation of the plasmids conforming to the unusual topology.

J Pharmacol Toxicol Methods, 1992 Sep, 28(2), 67 - 72
Pharmacologic action of Escherichia coli heat-stable (STa) enterotoxin; Knoop FC et al.; Escherichia coli produces a heat-stable (STa) enterotoxin that belongs to a family of peptides that mediate several diarrheal diseases, including traveler's diarrhea and epidemic diarrhea in infants and newborns . The STa enterotoxin consists of 18 or 19 amino acids and is encoded by genes specified on a transposon . Intestinal secretion is induced by specific binding to high affinity receptors that reside on the brush border cell membrane of the small intestine . Receptor activation by STa enterotoxin induces a sequence of events that culminate in the release of fluid and electrolytes into the intestinal lumen . These events include the stimulation of particulate guanylate cyclae and subsequent increase of intracellular cyclic GMP, involvement of particulate protein kinase, elevation of intracellular calcium, and activation of the phosphatidylinositol pathway . The release of archidonic acid and production of prostaglandins and/or leukotrienes have also been implicated in the action of STa . Evidence indicates that the STa enterotoxin receptor may be a single multifunctional membrane protein.

Mol Biol (Mosk), 1992 Sep-Oct, 26(5), 1148 - 59
{Hydrolysis of 5'-phosphonates and nucleoside phosphates by phosphatases of various origin and human and calf serum}; Kukhanova MK et al.; The hydrolysis of 5'-phosphonates of 2'-deoxythymidine and its 3'-modified analogs, inhibiting the HIV reproduction, by the E . coli alkaline, calf intestine and human placenta phosphatases as well as by the Crotalus atrox venom 5'-nucleotidase were studied . Transformations of 5'-phosphonates of adenosine and its analogs during incubation with human and fetal calf blood sera were investigated . The nucleotide derivatives modified at the phosphate residue were not hydrolyzed by any of the phosphatases studied except for the cobra venom 5'-nucleotidase, the effectiveness of the latter depended on the substitutes at both phosphate and sugar residues . 2'-Deoxyadenosine incubation with blood sera resulted in its transformation to 2'-deoxyinosine and then to hypoxanthine . 2'-Deoxyadenosine 5'-phosphonates were stable during incubation with blood sera under the same conditions.

Thromb Res, 1992 Sep 1, 67(5), 589 - 99
Binding of recombinant variants of human tissue-type plasminogen activator (t-PA) to human umbilical vein endothelial cells; Stockinger H et al.; Endothelial cells synthesize and secrete hemostatic components like tissue-type plasminogen activator (t-PA) which is thought to be the major determinant of fibrinolytic activity in the blood . Most recently, a receptor protein for t-PA on human umbilical vein endothelial cells (HUVEC) in culture has been described (1); there are, however, in addition low affinity binding sites for t-PA on HUVEC . The sites of binding are of particular interest, because they are potential regulators of t-PA activity and clearance . We analysed the low affinity binding of recombinant t-PA (rt-PA) to normal diploid HUVEC and to the permanent human cell lines Jurkat, Daudi, HL 60 and K562 by flow cytometry applying t-PA specific monoclonal antibodies . Using this test system binding of both recombinant glycosylated human t-PA produced in Chinese hamster ovary cells (CHO-t-PA) and of nonglycosylated t-PA, produced in E . coli (BM 06.021) was investigated . Analysis of the binding pattern to HUVEC and other cell lines revealed that deglycosylation of full length rt-PA increases non-specific binding . Additionally, we investigated the binding properties of an unglycosylated t-PA deletion variant which comprises the kringle 2 and the protease domains (BM 06.022) . Data obtained show that deletion of these domains most drastically reduces non-specific binding to HUVEC and other human cell lines.

Mol Microbiol, 1992 Sep, 6(18), 2599 - 605
Mapping the cAMP receptor protein contact site on the alpha subunit of Escherichia coli RNA polymerase; Zou C et al.; The C-terminal region (amino acid residues 236-329) of the Escherichia coli RNA polymerase alpha subunit carries the contact site I for positive transcription factors . For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C-terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene . Reconstituted holoenzyme containing the mutant alpha subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP-CRP . DNA sequence determination of several 'Lac-' mutant rpoA genes revealed that all had mutations clustered within a short segment near the C-terminus of alpha, between amino acid residues 265 and 270 . A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s).

Biochem Int, 1992 Sep, 27(6), 1119 - 25
Topoisomerase activity in mitochondrial lysates of a higher plant (Chenopodium album L.); Meissner K et al.; We describe an in vitro system for detection of topoisomerase activity from lysed mitochondria . Mitochondria were isolated from a suspension of cultured Chenopodium album cells . We observed a high activity in relaxation of negatively supercoiled DNA (pBR322) . Addition of ATP had no effect on the activity . Topoisomers obtained from negatively supercoiled DNA were identical with topoisomers produced by the topoisomerase I of E . coli . The mitochondrial activity was dependent on the presence of Mg2+ ions and could be inhibited by novobiocin and N-ethylmaleimide . Nalidixic acid and berenil had no influence on the mitochondrial topoisomerase activity . These features characterize the enzyme as a type I topoisomerase.

Radiat Res, 1992 Sep, 131(3), 332 - 7
Excision of sugar-phosphate products at apurinic/apyrimidinic sites by DNA deoxyribophosphodiesterase of Escherichia coli; Sandigursky M et al.; It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli . A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate . It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E . coli . The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM . It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione . The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases.

Brain Res Mol Brain Res, 1992 Sep, 15(1-2), 99 - 107
Multiple nuclear factors interact with the promoter of the human neurofilament M gene; Elder GA et al.; In order to identify potential regulatory elements of the human mid-sized (M) neurofilament (NF) gene we preformed DNase I footprinting, gel mobility shift assays and methylation interference studies with probes from the NF(M) immediate 5' flanking region . These studies identified multiple sites for DNA-binding proteins including four Sp1 sites, and single sites each for members of the NF-1 and AP-1 families of DNA binding proteins . In addition a binding site within a pyrimidine tract likely binds a novel DNA-binding protein which also interacts with the human NF(H) gene promoter . Factors that bind to these sites are found in both neural and non-neural cells suggesting that the NF(M) promoter may not contain tissue specific regulatory signals . In transient assays, addition of these binding sites to an NF(M) minimal promoter containing only a TATA box lead to a greater than 40-fold activation of transcription over background . Progressive 5' deletions reduced expression in a step wise manner suggesting that all the factors likely act synergistically as positive regulators of transcription.

Biophys J, 1992 Sep, 63(3), 682 - 8
Strong electrostatic loop-helix interactions in bundle motif protein structures; Chou KC et al.; Based on CHARMM potential (Brooks et al., 1983) an energetic analysis has been carried out for four typical 4-alpha-helix bundle proteins, i.e., methemerythrin, cytochrome b-562, cytochrome c', and bovine somatotropin . The bovine somatotropin possesses long loops, but all the other three proteins have short loops . It was found that in all these four 4-alpha-helix bundle motif structures the interaction between loops and helices was much stronger than the interaction among the four helices themselves . Particularly for the electrostatic interaction energy, the loop-helix interaction is overwhelmingly stronger than the interhelix interaction although the latter involves the favorable helix dipole interaction due to the antiparallel arrangement of neighboring alpha-helices . The present study indicates that such a conclusion holds true regardless of what loops, long or short, are in the 4-alpha-helix bundle protein, and also regardless of which empirical potential, ECEPP or CHARMM, is used for calculations although in CHARMM the electrostatic energy is much more heavily emphasized than in ECEPP . Therefore, no appropriate conclusion can be drawn in arguing whether the dipole interaction among the four alpha-helices play a stabilizing role or destabilizing role for a 4-alpha-helix bundle protein without taking into consideration the effect of interaction between helices and loops . The calculated results reported here provide, from a different point of view, insights that might be useful for revealing the essence of the driving forces during the folding of proteins.

Am J Physiol, 1992 Sep, 263(3 Pt 1), C607 - 15
Stimulation of intestinal Cl- transport by heat-stable enterotoxin: activation of cAMP-dependent protein kinase by cGMP; Forte LR et al.; Heat-stable enterotoxins activate guanylate cyclase, whereas heat-labile enterotoxins stimulate adenylate cyclase . Both classes of toxins cause secretory diarrhea at least in part by stimulating Cl- secretion in the intestine . The mechanism for regulation of Cl- secretion by guanosine 3',5'-cyclic monophosphate (cGMP) was investigated using cultured T84 intestinal cells as a model for intestinal crypt cells . Escherichia coli heat-stable enterotoxin (ST) markedly stimulated cGMP production in T84 cells . Cl- secretion across T84 cell monolayers cultured on permeable filters was stimulated by E . coli ST, cholera toxin, or 8-BrcAMP, but 8-BrcGMP was ineffective . cGMP analogues that are known to be potent and specific activators of cGMP-dependent protein kinase (cG-kinase) also had little effect on 36Cl- uptake by T84 cells cultured in plastic dishes . E . coli ST, forskolin, cholera toxin, or membrane-permeant cAMP analogues markedly increased 36Cl- uptake into T84 cells . The general protein kinase inhibitor, staurosporine, inhibited the stimulation of Cl- permeability elicited by E . coli ST, vasoactive intestinal peptide (VIP), or 8-BrcAMP . DEAE-Sephacel chromatography revealed a predominant type II isoform of cAMP-dependent protein kinase (cA-kinase) in T84 cells, whereas little or no cytosolic cG-kinase activity was found . Treatment of T84 cells with E . coli ST or VIP resulted in an increase in the cA-kinase activity ratio (-cAMP/+cAMP) if the cytosolic enzyme was assayed at reduced temperature (on ice).(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1992 Sep, 6(17), 2489 - 97
A new aspect of transcriptional control of the Escherichia coli crp gene: positive autoregulation; Hanamura A et al.; Transcription of the Escherichia coli crp gene is negatively regulated by CRP-cAMP that binds to a specific site located downstream of the crp promoter . A second binding site for CRP-cAMP (CRP site II) exists upstream of the crp promoter . Using an in vitro transcription assay, we have demonstrated that CRP-cAMP activates transcription of crp in certain conditions . A promoter which carries an altered CRP-binding site II is no longer activated by CRP-cAMP, indicating that CRP site II mediates the activation of crp transcription . The concentrations of cAMP that are required for positive autoregulation are higher than those for negative autoregulation . Evidence for positive and negative autoregulation in vivo is presented by a quantitative S1 nuclease analysis.

J Gen Virol, 1992 Sep, 73 ( Pt 9), 2337 - 45
Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera; Stacey SN et al.; Existing assays to detect antibodies to human papillomavirus type 16 (HPV-16) proteins in sera from cervical carcinoma patients rely primarily on bacterially produced recombinant proteins or synthetic peptides for use as target antigens . These methods have had limited success in the detection of antibodies against the E6 protein . To produce more authentic E6 protein for use in serological assays, we have employed a recombinant baculovirus vector to synthesize the protein in insect cells . Cells infected with the vector containing E6 gene sequences expressed a stable protein doublet comprising 18.5K and 19.1K bands . This protein reacted in Western blots with an antiserum raised against a purified E6 fusion protein produced in Escherichia coli . This antiserum, and several others raised against E . coli-derived E6 fusion proteins, were unable to recognize the baculovirus E6 protein in radioimmunoprecipitation assays (RIPAs) . However, serum from a cervical carcinoma patient readily immunoprecipitated the baculovirus E6 protein, suggesting that the baculovirus-derived protein represented a realistic antigenic target . A RIPA was developed for the detection of anti-E6 protein antibodies in human sera . The assay was tested on a selected group of sera from carcinoma patients and controls, in comparison with a Western blotting method using bacterial fusion proteins . The baculovirus E6 protein-based RIPA showed a marked increase in detection rate over the Western blotting method . These findings suggest that serum antibodies to HPV-16 E6 protein may be more prevalent than has previously been shown.

Virology, 1992 Sep, 190(1), 184 - 92
Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion; Overton HA et al.; The UL13 open reading frame of herpes simplex virus type 1 (HSV-1) has been expressed in insect cells by a recombinant baculovirus and in Escherichia coli . In the latter case, the UL13 gene was fused to the gene for glutathione S-transferase (GST) to allow high-level expression of an 80-kDa GST-UL13 fusion protein . Antibody raised against the fusion protein reacted specifically with the 55-kDa UL13 gene product expressed by the recombinant baculovirus . This antibody also recognized a late phosphoprotein in HSV-1-infected cell lysates and a component of purified HSV-1 virions, both with the same electrophoretic mobility as the baculovirus-expressed protein . The virion component was efficiently phosphorylated in vitro by a virion-associated protein kinase . Using the same antibody, the probable homolog of the UL13 gene product was identified in HSV-2-infected cells and purified virions.

Virology, 1992 Sep, 190(1), 106 - 15
Expression of the Epstein-Barr virus latent membrane protein (LMP) in insect cells and detection of antibodies in human sera against this protein; Chen HF et al.; Recombinant baculoviruses containing the complete LMP and truncated LMP genes were generated and high levels of the LMP proteins were expressed in Spadoptera Frugiperda insect cells . A specific rabbit antiserum directed against the N-terminal part of LMP was obtained by immunizing the rabbits with Escherichia coli-expressed trpE-N-terminal part of LMP fusion protein . A total of 127 human sera were studied for their immune response to the recombinant full-length LMP . In immunofluorescence analysis, all sera tested showed no detectable reaction with the recombinant full-length LMP . In immunoprecipitation-immunoblotting analysis, however, sera from patients with nasopharyngeal carcinoma (5/22), patients with Hodgkin's disease (16/27), patients with other diseases exhibiting high EA-IgG titers (3/52), and VCA-IgG-positive healthy individuals (2/26) were shown to contain antibodies against this recombinant LMP . The expressed LMP proteins provided a sufficient and economic source of the proteins for further serological and biological studies.

J Surg Res, 1992 Sep, 53(3), 272 - 9
Ibuprofen intervention in canine septic shock: reduction of pathophysiology without decreased cytokines; Coran AG et al.; This study was undertaken to evaluate the effect of a cyclooxygenase inhibitor, ibuprofen, at various time intervals in a live Escherichia coli model of canine septic shock . Group I (control) animals (n = 5) received a LD100 dose of 10(9) live E . coli per kilogram were given no further treatment . Group II animals (n = 5) received a 10 mg/kg bolus of ibuprofen 10 min prior to bacterial infusion . Group III animals (n = 5) received ibuprofen 15 min after the bacterial infusion . Statistical analysis revealed the following: Group II animals had significantly higher MABP and significantly lower levels of serum fluorescent products (superoxide radical activity), plasma thromboxane B2, prostaglandin E2, and endotoxin levels compared to Group I animals (P less than 0.05) . Plasma levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6) were significantly elevated (P less than 0.05) from baseline in all animals (Groups I, II, and III), but ibuprofen treatment failed to either increase or decrease these levels . This study demonstrates that ibuprofen treatment can significantly reverse the deleterious hemodynamic and metabolic effects commonly seen in live E . coli septic shock without depressing the endogenous production of TNF or IL-6 . These data support the hypothesis that sepsis initiates a cascade of mediators with the cytokines TNF and IL-6 being proximal events which in turn stimulate the next level, with ibuprofen probably exerting its inhibitory effect distal to this point in the cascade.

J Bacteriol, 1992 Sep, 174(18), 5971 - 7
Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase; Talarico TL et al.; The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced . This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine . The protein was overexpressed in E . coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid . The enzyme was purified to homogeneity . Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed . The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry . The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.

J Bacteriol, 1992 Sep, 174(18), 5798 - 802
DNA gyrase, CS7.4, and the cold shock response in Escherichia coli; Jones PG et al.; We identify the A subunit of DNA gyrase as a cold shock protein whose synthesis is sustained following transfer of exponentially growing cultures of Escherichia coli from 37 to 10 degrees C . After a lag period in which its synthetic rate declines, synthesis of the A subunit of DNA gyrase increases relative to that of total protein . The duration of the lag period in synthesis and the synthetic rate of the A subunit appear dependent on the synthesis of a rapidly induced cold shock protein, CS7.4 . The promoter of the gyrA gene contains specific binding sites for the CS7.4 protein, suggesting that CS7.4 acts at the transcriptional level to facilitate continued A-subunit synthesis . As synthesis of the B subunit of DNA gyrase is also sustained during cold shock, we suggest that an increase in the amount of DNA gyrase per cell might occur, which would potentially adapt the cells for growth at reduced temperatures (10 degrees C).

Eur J Biochem, 1992 Sep 1, 208(2), 419 - 25
In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE . Evidence that SecB prevents the precursor from aggregating by forming a functional complex; Breukink E et al.; We characterised the behaviour of the purified precursor protein prePhoE upon dilution from 8 M urea by CD, fluorescence spectroscopy and gel-filtration techniques . It is demonstrated that prePhoE rapidly adopts beta structure, folds and aggregates upon dilution to urea concentrations below 3 M . These processes are paralleled by a loss of translocation competence . Furthermore the interaction of prePhoE with SecB was investigated . SecB is shown to have a very high content of beta structure, therefore we propose that precursor recognition by SecB is mediated through beta-beta interaction . It is shown that SecB has little effect on the adoption of secondary structure and tertiary folding upon dilution of the precursor from urea . However, SecB prevents the precursor from aggregating by forming a functional and stable complex.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8020 - 4
Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme; McIntosh EM et al.; Two functional human dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) cDNAs were isolated from a cDNA expression library by genetic complementation in Escherichia coli . These cDNAs differed in size but exhibited a common overlapping DNA sequence . Contained within this sequence was a single long open reading frame sufficient to encode a polypeptide of 141 amino acids with a calculated molecular mass of 16.6 kDa . The amino acid sequence of this protein exhibits 35% identity with the E . coli dUTPase and 53% identity with the Saccharomyces cerevisiae enzyme . The human dUTPase was found to contain five characteristics amino acid sequence motifs that are common to the dUTPases of E . coli, yeast, and herpesviruses and to dUTPase-like sequences encoded by some retrovirus gag and pol genes . A high degree of amino acid sequence identity (greater than 60%) was also observed between the human dUTPase and the putative pseudoproteases of two poxviruses, indicating that these virus proteins are dUTPases . Northern hybridization analysis reveals that dUTPase is encoded by at least two species of poly(A)+ mRNA and possibly a third, smaller species . All of these mRNAs are present in a variety of human tissues but their relative levels vary between tissues . Southern analysis indicates that the dUTPase gene has been conserved to some extent throughout vertebrate evolution; however, the gene may be very large, or its organization somewhat complex in some systems . We suggest that dUTPase may generally perform an essential role in DNA replication and therefore could serve as a target enzyme for the development of chemotherapeutic compounds.

Clin Exp Immunol, 1992 Sep, 89(3), 362 - 8
Mononuclear cells from HIV-infected patients produce factors which enhance functional activity of polymorphonuclear neutrophils from healthy subjects; Gabrilovich DI et al.; The influence of mononuclear cell supernatants (MNCS) from nine healthy donors and 35 HIV-infected patients (17 with lymphoadenopathy syndrome (LAS), 15 with ARC and three with AIDS) on functional activity of polymorphonuclear neutrophils (PMN) from healthy donors was investigated . MNC after short-term cultivation (24 h) produced factors which enhanced chemiluminescence (CL) and chemotaxis of PMN . This augmentation did not depend on stimulation of MNC by mitogens (lipopolysaccharide Escherichia coli (LPS) and concanavalin A (Con A)) or on activation of PMN by FMLP . After 48 h of cultivation only MNC stimulated by LPS produced these factors . MNCS from HIV-infected patients provoked a more pronounced augmentation of PMN CL compared with MNCS from healthy subjects . This enhancement was observed in patients at all stages of infection, but was more pronounced in patients with LAS . MNCS impact on PMN CL was not connected with proliferative activity of MNC but was correlated with the level of CD4 cells . It was shown that removal of adherent cells from MNC fraction resulted in decreased MNCS impact . Treatment of MNCS by antibody to IL-1 beta, IL-8, interferon-alpha (IFN-alpha) and tumour necrosis factor-alpha (TNF-alpha) did not decrease MNCS impact on PMN CL.

J Bacteriol, 1992 Sep, 174(17), 5647 - 53
dcd (dCTP deaminase) gene of Escherichia coli: mapping, cloning, sequencing, and identification as a locus of suppressors of lethal dut (dUTPase) mutations; Wang L et al.; In Escherichia coli, most of the dUMP that is used as a substrate for thymidylate synthetase is generated from dCTP through the sequential action of dCTP deaminase and dUTPase . Some mutations of the dut (dUTPase) gene are lethal even when the cells are grown in the presence of thymidine, but their lethality can be suppressed by extragenic mutations that can be produced by transposon insertion . Six suppressor mutations were tested, and all were found to belong to the same complementation group . The affected gene was cloned, it was mapped by hybridization with a library of recombinant DNA, and its nucleotide sequence was determined . The gene is at 2,149 kb on the physical map . Its product, a 21.2-kDa polypeptide, was overproduced 1,000-fold via an expression vector and identified as dCTP deaminase, the enzyme affected in previously described dcd mutants . Null mutations in dcd probably suppress the lethality of dut mutations by reducing the accumulation of dUTP, which would otherwise lead to the excessive incorporation of uracil into DNA.

Biochemistry, 1992 Sep 1, 31(34), 8055 - 8
Assessment of uncoupling by amiloride analogs; Davies K et al.; The amiloride analogs N5-methyl-N5-isobutylamiloride, N5-ethyl-N5-isopropylamiloride, and N5,N5-hexamethyleneamiloride are frequently used to investigate NaH exchange on the premise that they are highly specific inhibitors of the NaH-antiporters . We assessed the relative protonophoric activity of these compounds in reconstituted and native membrane vesicles, using acridine orange fluorescence to measure intravesicular pH . All the compounds tested were found to be potent protonophores at concentrations which are normally used to demonstrate inhibition of NaH exchange . Uncoupling was dependent on both the pH of the assay system and the total amount of lipid present . At the pH optima, which lay in a range from 7.5 to 8.5, these amiloride analogs were more potent uncouplers than the classical protonophore carbonyl cyanide m-chlorophenylhydrazone . Therefore, extreme care must be taken in the interpretation of results obtained using these or similar derivatives of amiloride.

Biochemistry, 1992 Sep 1, 31(34), 7899 - 907
Proton uptake accompanies formation of the ternary complex of citrate synthase, oxaloacetate, and the transition-state analog inhibitor, carboxymethyl-CoA . Evidence that a neutral enol is the activated form of acetyl-CoA in the citrate synthase reaction; Kurz LC et al.; Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CM-CoA), are believed to mimic those with the activated form of acetyl-CoA . The X-ray structure {Karpusas, M., Branchaud, B., & Remington, S.J . (1990) Biochemistry 29, 2213} of the ternary complex of the enzyme, oxaloacetate, and CMCoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form . Since the inhibitor carboxyl has a pKa of 3.90, analogy with an enolic acetyl-CoA intermediate leads to the prediction that a proton should be taken up from solution upon formation of the analog complex so that the transition-state analog carboxyl is protonated when bound . We have obtained evidence in solution for this proposal by comparing the isoelectric points and the pH dependence of the dissociation constants of the ternary complexes of the pig heart enzyme with the neutral ground-state analog inhibitor, acetonyl-CoA (KCoA), and the anionic transition-state analog inhibitor (CMCoA) and by studying the NMR spectra of the transition-state analog complexes of allosteric (Escherichia coli) and nonallosteric (pig heart) enzymes . The pH dependence of the dissociation constant of the ground-state analog indicates no proton uptake, while that for the transition-state analog indicates that 0.55 +/- 0.04 proton is taken up when the analog binds to the citrate synthase-oxaloacetate binary complex . The overall charges of ternary complexes of the pig heart enzyme with the transition-state and ground-state analog inhibitors are the same, as monitored by their isoelectric points.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1992 Sep 1, 149(5), 1744 - 50
Role of complement in endotoxin/platelet-activating factor-induced lung injury; Rabinovici R et al.; C receptor-1 is a protein involved in the regulation of C3 and C5-convertases . Recombinant human soluble C receptor-1 has recently been produced and shown to reduce infarct size in a rat model of myocardial ischemia/reperfusion injury . The present study aimed to investigate whether recombinant human soluble C receptor-1 exerts any protective effect on pulmonary injury produced in a rodent model of adult respiratory distress syndrome . In this model, Escherichia coli endotoxin (LPS, 0.1 microgram/kg) combined with platelet-activating factor (1 pmol/kg/min over 60 min, n = 10) caused microvascular lung injury characterized by elevation of myeloperoxidase activity, deposition of C3 and C5b-9 on the endothelium of pulmonary vessels, and pulmonary edema . Furthermore, bronchoalveolar lavage revealed increased neutrophil count and elevated protein concentration . These pulmonary responses were associated with elevated serum TNF-alpha . Pretreatment (10 min, i.v.) with recombinant human soluble C receptor-1 at 10 mg/kg (n = 13), but not at 1 mg/kg, prevented the LPS/platelet-activating factor-induced pulmonary edema (p less than 0.01) and changes in the bronchoalveolar lavage fluid cell count (p less than 0.01) and protein concentration (p less than 0.05), and attenuated the deposition of C3 and C5b-9 to lung vessels . There was no effect on lung myeloperoxidase activity and serum TNF-alpha . Also, C depletion by cobra venom factor (500 U/kg, i.v.) eliminated the pulmonary edema and elevated leukocyte count in bronchoalveolar lavage fluid, but had no effect on lung myeloperoxidase activity and serum TNF-alpha . These data suggest that C factors may play an important role in the pathophysiology of adult respiratory distress syndrome.

EMBO J, 1992 Sep, 11(9), 3203 - 8
X-ray structure of nucleoside diphosphate kinase; Dumas C et al.; The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution . The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold . Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet . The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position . Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase . Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme . Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.

J Virol, 1992 Sep, 66(9), 5242 - 7
Polyprotein processing in cis and in trans by hepatitis A virus 3C protease cloned and expressed in Escherichia coli; Harmon SA et al.; To determine the P3 region protein-processing sites cleaved by the hepatitis A virus 3C protease, a nested set of constructs containing a portion of 3A (3A* {the asterisk denotes an incomplete protein}), 3B and 3C and various amounts of 3D, fused in frame to Escherichia coli TrpE-coding sequences under control of the tryptophan promoter, was made . Additional plasmids that encoded a portion of 2C (2C*) and the P3 proteins, including complete or incomplete 3D sequences, were constructed . After induction, E . coli containing these recombinant plasmids produced high levels of fusion proteins as insoluble aggregates . 3C-mediated cleavage products were identified by comparison of expression with a matching set of plasmids, containing an engineered mutation in 3C . Cleavage products were detected by immunoblot analyses by using antisera against the TrpE protein, against 3D*, and against 3CD* . Scissile bonds were determined by N-terminal amino acid sequencing of the proteins formed by cleavage . The results showed that when a portion of 2C was present, the primary cleavage by the 3C protease was between 2C and 3A, and the cleavage site was QG, as predicted by J . I . Cohen, J . R . Ticehurst, R . H . Purcell, A . Buckler-White, and B . M . Baroudy, J . Virol . 61:50-59, 1987 . Very little further cleavage of the released P3 protein was detected . When the fusion protein contained no 2C and included only 3A*-to-3D sequences, efficient cleavage occurred between 3B and 3C, at the QS pair, also as predicted by Cohen et al . (J . Virol . 61:50-59, 1987) . The latter proteins were also cleaved between 3C and 3D, but less efficiently than between 3B and 3C . Extracts of bacteria expressing proteins from 3A* to 3D also cleaved a radiolabelled hepatitis A virus substrate containing VP1*2ABC* sequences in trans.

Protein Sci, 1992 Sep, 1(9), 1206 - 14
Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli; Wang CC et al.; The cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane . Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water-soluble 43-kDa and 41-kDa proteolytic fragments has never been achieved . In order to obtain pure cdb3 for crystallization and site-directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N-terminal 379 amino acids of the erythrocyte band 3 gene . Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3 . About 10 mg of recombinant cdb3 can be easily purified from 4 L of E . coli culture in two simple steps . Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N-terminal sequence, secondary structure, and pH-dependent conformational change . The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3 . The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N-terminus exhibits a slightly lower affinity for aldolase.

Protein Sci, 1992 Sep, 1(9), 1185 - 205
Molecular dynamics studies of a DNA-binding protein: 2 . An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor; Guenot J et al.; Although aqueous simulations with periodic boundary conditions more accurately describe protein dynamics than in vacuo simulations, these are computationally intensive for most proteins . Trp repressor dynamic simulations with a small water shell surrounding the starting model yield protein trajectories that are markedly improved over gas phase, yet computationally efficient . Explicit water in molecular dynamics simulations maintains surface exposure of protein hydrophilic atoms and burial of hydrophobic atoms by opposing the otherwise asymmetric protein-protein forces . This properly orients protein surface side chains, reduces protein fluctuations, and lowers the overall root mean square deviation from the crystal structure . For simulations with crystallographic waters only, a linear or sigmoidal distance-dependent dielectric yields a much better trajectory than does a constant dielectric model . As more water is added to the starting model, the differences between using distance-dependent and constant dielectric models becomes smaller, although the linear distance-dependent dielectric yields an average structure closer to the crystal structure than does a constant dielectric model . Multiplicative constants greater than one, for the linear distance-dependent dielectric simulations, produced trajectories that are progressively worse in describing trp repressor dynamics . Simulations of bovine pancreatic trypsin were used to ensure that the trp repressor results were not protein dependent and to explore the effect of the nonbonded cutoff on the distance-dependent and constant dielectric simulation models . The nonbonded cutoff markedly affected the constant but not distance-dependent dielectric bovine pancreatic trypsin inhibitor simulations . As with trp repressor, the distance-dependent dielectric model with a shell of water surrounding the protein produced a trajectory in better agreement with the crystal structure than a constant dielectric model, and the physical properties of the trajectory average structure, both with and without a nonbonded cutoff, were comparable.

Protein Sci, 1992 Sep, 1(9), 1173 - 84
Molecular dynamics studies of a DNA-binding protein: 1 . A comparison of the trp repressor and trp aporepressor aqueous simulations; Howard AE et al.; The results of two 30-ps molecular dynamics simulations of the trp repressor and trp aporepressor proteins are presented in this paper . The simulations were obtained using the AMBER molecular mechanical force field and in both simulations a 6-A shell of TIP3P waters surrounded the proteins . The trp repressor protein is a DNA-binding regulatory protein and it utilizes a helix-turn-helix (D helix-turn-E helix) motif to interact with DNA . The trp aporepressor, lacking two molecules of the L-tryptophan corepressor, cannot bind specifically to DNA . Our simulations show that the N- and C-termini and the residues in and near the helix-turn-helix motifs are the most mobile regions of the proteins, in agreement with the X-ray crystallographic studies . Our simulations also find increased mobility of the residues in the turn-D helix-turn regions of the proteins . We find the average distance separating the DNA-binding motifs to be larger in the repressor as compared to the aporepressor . In addition to examining the protein residue fluctuations and deviations with respect to X-ray structures, we have also focused on backbone dihedral angles and corepressor hydrogen-bonding patterns in this paper.

Plant J, 1992 Sep, 2(5), 815 - 20
A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator; Brandt J et al.; A full length cDNA of a barley leaf messenger, found to increase in amount during infection attempts by the powdery mildew fungus (Erysiphe graminis), is characterized . The messenger encodes a polypeptide of 261 amino acid residues with a calculated mass of 29.2 kDa and a pI of 4.6 . Sequence comparisons as well as serological studies demonstrate that the encoded protein is closely related to a family of mammalian proteins believed to have functions associated with the multifunctional Ca(2+)-dependent protein kinases.

Mol Gen Mikrobiol Virusol, 1992 Sep-Oct, (9-10), 19 - 21
{Cloning the gene for vaccinia virus strain L-IVP growth factor in Escherichia coli}; Pak VN et al.; The growth factor gene of the vaccinia virus LIVP strain has been primarily cloned in a 4.3 kbp long BamHI-EcoRI fragment and then subcloned in a 440 bp fragment . It was shown that clone 4 of the LIVP strain contains a single copy of this gene while the WR strain contains a repeat . The gene is located on a 4.3 kbp BamHI-EcoRI fragment but not on a 2.2 kbp fragment and has four nucleotide changes, three of which result in amino acid substitutions.

Mol Gen Mikrobiol Virusol, 1992 Sep-Oct, (9-10), 11 - 3
{Determination of thermostable and thermolabile enterotoxins in Escherichia coli strains by genetic, biological, and immunoserological methods}; Domaradskaia TI et al.; The Escherichia coli strains (75) isolated from patients suffering from diarrhea were screened for ability to produce the temperature-labile or stable toxins (ST or LT) by the different techniques (the hybridization with DNA probes, biological, enzyme immunoassay) . The majority of tested strains was shown to harbor the tox-genes controlling the synthesis of ST, LT or both enterotoxins . However, the phenotypic expression of the genes was registered in only some of the strains . The hybridization with the DNA probes is noted to be most perspective in the mass screening of toxigenic strains . The DNA probe used contained the fused estA-eltB genes that makes one able to detect the genes for both enterotoxins.

Hua Xi Yi Ke Da Xue Xue Bao, 1992 Sep, 23(3), 293 - 6
{Mutagenesis study of ethylmethane sulfonate with shuttle vector plasmid pZ189}; Fan Q et al.; Shuttle vector plasmid pZ189 was used as a molecular tool and the SupF inserted in the plasmid was worked as a target gene for mutagenesis study . The host cells (E . coli MBM 7070) with pZ189 were treated with ethylmethane sulfonate (EMS) and plated on the selective media containing X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) and IPTG (isopropyl-beta-D-thiogalactoside) . The SupF and the LacZ amber mutant carried by the host cells complemented each other and thus made the colonies blue on the selective media . However the colonies derived from the SupF mutants changed the colour from blue to white . The mutant frequencies in a series of experiments with different concentrations of EMS were estimated . Furthermore, the DNA isolated from 5 SupF mutants was digested with restruiction enzyme Hha I . It suggests that the 214 bp Hha I fragments containing mutant SupF could be distinguished from their wild type counterparts by temperature-gradient gel electrophoresis under optimal conditions.

Hua Xi Yi Ke Da Xue Xue Bao, 1992 Sep, 23(3), 245 - 7
{Colony hybridization with digoxigenin labelled DNA probe}; Jiang S et al.; A simple, rapid method for colony hybridization has been developed . The DNA probes were labelled by digoxigenin . Hybridization signal was detected by enzyme-linked immunoassay reaction . The results showed that this method is sensitive and reproducible, and it can be used for colony hybridization instead of isotopic methods.

Zhonghua Yu Fang Yi Xue Za Zhi, 1992 Sep, 26(5), 291 - 3
{A study of vitamin inhibition on the mutagenicity of the antineoplastic drugs}; Zhao ZZ et al.; The inhibitory effects on the mutational specificity of antineoplastic drugs of 14 kinds of vitamin were tested with the method of mutational and anti-mutational synchoronous test, add S9 and no S9 . Vit C, Vit B6, and nicotinic acid had distinct inhibitory effects on the mutational specificity of 6 antineoplastic drugs, namely, mitomycin C, bleomycin, fluorouracil, cis-Diaminodichloroplatinum, arabinosylcytosine and mustargen Vit K3 showed inhibitory effect to mitomycin C, fluorouracil, cis-diaminodichloroplatinum, and arabinosylcytosine but Vit AD, Vit B1, Vit B2, Vit Bco, Vit D3, Vit E, Rutin, Vit K1, Vit K4 and folic acid did not . The fact that Vit C, Vit B6, nicotinic acid and Vit K3 showed anti-mutational effects is of some significance with reference to clinical therapeutics and prevention of tumours.

J Struct Biol, 1992 Sep-Oct, 109(2), 87 - 96
Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli: structure in projection at 40 A resolution; Weaver AJ et al.; SecA is a single-chain, membrane-associated polypeptide (102 kDa) which functions as an essential component of the protein export machinery of Escherichia coli . SecA has been crystallized from ammonium sulfate as small, three-dimensional bipyramidal crystals (0.1 x 0.1 x 0.05 mm) . These crystals did not demonstrate detectable diffraction of X-rays from rotating anode sources . For study by electron microscopy, individual crystals were cross-linked in glutaraldehyde and OsO4 solutions, dehydrated, embedded in epoxy resin, and sectioned normal to crystallographic axial directions inferred from the external morphology of the crystals . Fourier transformation of processed images of untilted thin sections stained with uranyl acetate and lead citrate show reflections extending to 31 A resolution . Diffraction data and reconstructed images of the projected density of the unit cell contents indicate that the bipyramidal SecA crystals belong to orthorhombic space group C222(1) with unit cell dimensions a = 414 A, b = 381 A, and c = 243 A . Filtered images and density maps of mutually orthogonal projections of the unit cell contents are consistent with a three-dimensional model in which the asymmetric unit contains eight SecA monomers . The large unit cell dimensions and packing of protein monomers suggest that SecA is crystallizing as an oligomer of either dimers or tetramers.

J Struct Biol, 1992 Sep-Oct, 109(2), 109 - 15
Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy; Tumminia SJ et al.; A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM) . From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data . Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles . Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the 80S monosomes and the large (60S) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular . These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the "native" eukaryotic ribosome 3-D model.

Chin Med Sci J, 1992 Sep, 7(3), 161 - 5
The role of endotoxin in the pathogenesis of experimental multiple system organ failure: a preliminary report; Yao Y et al.; In this study, an animal model of multiple system organ failure (MSOF) in rabbits, engendered by feeding E . coli prior to severe hemorrhagic shock, was used for the purpose of investigating 1) the relationship between lipopolysaccharide (LPS) and MSOF, and 2) the effectiveness of Re-LPS antiserum in preventing MSOF . The results showed that endotoxemia occurred very early, and its degree correlated well with that of organ dysfunction . Re-LPS antiserum administration abated the toxic effects and lowered the incidence of MSOF . These results suggest that sequential analysis of circulating LPS levels may be useful for the early diagnosis of MSOF, and that gut-derived endotoxin might play an important role in the pathogenesis of experimental MSOF.

Int J Lepr Other Mycobact Dis, 1992 Sep, 60(3), 382 - 9
Enzyme linked immunosorbent assay of a 12-kDa protein of Mycobacterium leprae with sera from leprosy patients; Navalkar RG et al.; A low molecular weight protein was obtained from a sonicate of armadillo-derived Mycobacterium leprae cells and from a lambda gt11 phage lysate of Escherichia coli (specifying the M . leprae 12-kDa protein) by a single step of ultrafiltration . Both proteins had an approximate molecular weight of about 12,000 (by SDS-PAGE) and were recognized by the M . leprae 12-kDa-specific monoclonal antibody ML06 by immunoblotting . Sera from 79 leprosy patients across the clinical spectrum, 17 contacts, and 12 normal healthy individuals were screened in an enzyme-linked immunosorbent assay (ELISA) using the 12-kDa proteins as the antigens . Antibodies to the 12-kDa protein (from lysate as well as sonicate) were detected in patients' sera across the clinical spectrum (44%-100% positivity), while no detectable reactivity was observed with control or contact sera . Sera from patients who had undergone a year or more of chemotherapy exhibited no reactivity compared to those from patients with only 3-6 months of chemotherapy . The 12-kDa proteins were also recognized by rabbit hyper-immune M . leprae antiserum.

Int J Lepr Other Mycobact Dis, 1992 Sep, 60(3), 368 - 75
Recognition of Mycobacterium leprae recombinant 18-kDa proteins in leprosy; Hussain R et al.; Three different, purified, Escherichia coli-derived, recombinant preparations of the Mycobacterium leprae 18K protein were compared for their immunological recognition in leprosy . The preparations tested were 18K fusion proteins containing 70% (amino acids 38-148) of the full 18K protein fused to either a short leader sequence containing six asparagine residues or to beta-galactosidase, and the full length 18K protein . All three recombinant antigens were recognized by IgG antibodies which were restricted mostly to lepromatous leprosy patients . The 18K antigen with the asparagine leader sequence showed better reactivity with IgG antibodies compared with the other two 18K preparations . In lymphocyte proliferation assays, the truncated 18K and the full-length 18K showed equivalent responses in the same donors with strongest recognition in donors who were also strongly responsive to the M . leprae soluble sonicate . These results indicate that the major human B- and T-cell epitopes are located within the segment 38-148, although some individuals may recognize additional epitopes at the NH2-terminal end.

Trop Med Parasitol, 1992 Sep, 43(3), 139 - 45
Specific and sensitive IgG4 immunodiagnosis of onchocerciasis with a recombinant 33 kD Onchocerca volvulus protein (Ov33); Lucius R et al.; The full length cDNA of the immunodominant Ov33 protein of Onchocerca volvulus was expressed in E . coli using various vector constructs . Expression was best with the vectors pGEX2T and pCG808fx, yielding fusion protein Ov33-GST and Ov33-MBP, respectively . Purified fusion protein Ov33-GST and O . volvulus antigen extracts (OvAg) were used to compare antibody responses (IgM and IgG-subclasses) of patients infected with O . volvulus, Brugia malayi, Wuchereria bancrofti, Mansonella perstans/Loa loa and of Sudanese control sera . Sera of all groups contained IgM reacting with Ov33-GST and with OvAg . There was no IgG1 response to Ov33-GST . IgG1 responses to OvAg were only detected in filariasis sera . IgG2 and IgG3 responses were not detectable or marginal in all groups . The IgG4 reaction of onchocerciasis patients to Ov33-GST and to OvAg was high, whereas few other filariasis sera contained IgG4 antibodies to Ov33-GST and to OvAg . A serodiagnostic test for onchocerciasis based on detection of IgG4 to Ov33-GST had a sensitivity of 93.3% and a specificity of 96% . An epitope common to Ov33 and to the homologous proteins of other filarial species was demonstrated with a monoclonal antibody . Purified Ov33-MBP fusion protein was used to follow the development of the antibody response of four chimpanzees experimentally infected with O . volvulus . The data indicates that antibodies to Ov33 are induced by developing worms and later parasite stages.

Ukr Biokhim Zh, 1992 Sep-Oct, 64(5), 23 - 30
{Escherichia coli ribosomes having peptidyl-tRNA and deacylated tRNA at the A- and P-sites, respectively, may not be competent in translocation}; Koval'chuk O; Ribosomes can have two states at 0 degree C: competent and noncompetent in translocation . In both states poly(U)-programmed ribosomes bind phenylalanyl-tRNA to A and P sites and form peptide bond . Elongation factor G promotes fast translocation in competent ribosomes and makes them noncompetent ones . Initial correlation between competent and noncompetent ribosomes is 2:1 . Addition of deacylated tRNA does not influence phenomenon described as well as thermal reactivation of the ribosomes before beginning of the experiments . The possibility of deacylated tRNA translocation is shown . The translocation does not occurred provided that at least one of the ribosome sites is filled with shortened tRNA analog (tRNA with truncated CCA-end or tRNA anticodon arm).

Cell, 1992 Aug 21, 70(4), 671 - 9
A new mechanism of transcriptional regulation: release of an activator triggered by small molecule binding; Henry MF et al.; The FadR protein of E . coli activates transcription of the fabA gene, a key enzyme of fatty acid synthesis . We report that FadR binds to a DNA sequence positioned at -40 relative to the start site of the FadR-regulated fabA transcript (the location favored by positive activators) . This binding was found to be specifically antagonized by long chain acyl-CoAs . The chain length specificity of the disassociation of the FadR-DNA complex by acyl-CoAs observed in vitro reflects that seen in the repression of fabA transcription observed upon addition of fatty acids to bacterial cultures . Acyl-CoA antagonism of FadR-DNA interactions is readily reversible . These data indicate that repression of fabA transcription by fatty acids is the first reported example of a repression system mediated by positive control.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 544 - 51
Expression, purification and biochemical comparison of natural and recombinant human non-pancreatic phospholipase A2; Johansen B et al.; The gene coding for human non-pancreatic phospholipase A2 (npPLA2) was cloned in a eukaryotic expression vector and transfected into chinese hamster ovary (CHO) cells . A number of cell lines stably expressing npPLA2 were obtained . Northern analysis of these cell lines showed an abundant transcript of expected size 1200 nt . The recombinant enzyme was efficiently secreted in quantities up to 400 micrograms npPLA2 per liter culture medium in the most productive cell lines . npPLA2 was purified to homogeneity from conditioned medium as previously described (1) . The recombinant npPLA2 migrated by SDS--PAGE as a single band with an apparent mass of 14,000 . The recombinant enzyme displayed the pH-optimum, calcium dependence and substrate preference that were characteristic of the human platelet and synovial fluid enzymes.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 375 - 80
Molecular cloning of nucleobindin, a novel DNA-binding protein that contains both a signal peptide and a leucine zipper structure; Miura K et al.; We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mouse produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier . We purified this factor, a 55 kD protein that we termed nucleobindin (Nuc), and obtained its cDNA clone . Although the gene for Nuc encodes a signal peptide and, in fact, Nuc was identified as a secreted protein, Nuc had a DNA-binding property . The putative polypeptide predicted from the cDNA sequence featured a signal peptide, a leucine zipper structure and a basic amino acid-rich region . The DNA-binding property of Nuc was destroyed by deletion of either the leucine zipper structure or the basic amino acid-rich region . The amino acid sequences of Nuc are highly conserved between mouse and human . We discuss the possible role of Nuc in autoimmunity.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 209 - 16
Changes in bioactive lipids, alkylacylglycerol and ceramide, occur in HIV-infected cells; Van Veldhoven PP et al.; The mass levels of bioactive lipids known to modulate signal transduction or to possess other biological activities were measured in HIV-infected CEM cells . The levels of diacylglycerol, an activator of protein kinase C, as well as of alkylacylglycerol were elevated . A more drastic increase was observed in the ceramide levels after HIV-infection, whereas sphingosine levels were hardly influenced . Interestingly, the magnitude of the changes was related to the infection time, being higher at 8 days after infection then at 4 days . The possible role of these lipids in the cytopathic effects of HIV-infection is discussed . In addition, an improved methodology to quantitate simultaneously diacylglycerol and alkylacylglycerol in crude lipid extracts, based upon their phosphorylation by E . coli diacylglycerol kinase, is presented.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 18 - 25
Effect of semi-random mutagenesis at the C-terminal 4 amino acids of human interleukin-6 on its biological activity; Yasueda H et al.; The carboxyl(C)-terminus of human interleukin-6 (hIL-6) has a critical role in the expression of the biological activity of this cytokine . To define the structure-function relationships of this region, semi-random mutagenesis of the C-terminal Leu181-Arg182-Qln183-Met184 sequence of hIL-6 was performed . The mutants were produced in Escherichia coli, renatured, and purified . Alterations of the C-terminal 4 amino acids caused a significant reduction of the proliferative effect of the mutants on MH60.BSF2 and KT-3 cells, and also led to a drastic decrease in receptor binding affinity . These results suggest the importance of a positively charged residue at position 182 or 183 and an alpha-helix at position 181 for the biological activity of hIL-6.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 101 - 7
Identification of an amino acid residue involved in the substrate-binding site of rat liver uricase by site-directed mutagenesis; Ito M et al.; Computer analysis has shown that a conserved amino acid sequence (Leu 160 to Lys 164) of rat liver uricase is also present in other enzymes with purine substrates . The significances of the amino acids in this sequence were studied by site-directed mutagenesis . Replacement of Lys 164 by Glu or Ile resulted in loss of uricase activity and decrease in binding of the competitive inhibitor xanthine . The far ultraviolet circular dichroic spectra of the mutant uricases were identical to that of the wild type protein, indicating that the replacement of Lys 164 by other amino acids did not result in serious modification of the conformation of uricase . These findings suggest that this amino acid is involved in the substrate-binding site of the enzyme.

FEBS Lett, 1992 Aug 31, 309(1), 5 - 9
Distinct and specific GAP activities in rat pancreas act on the yeast GTP-binding proteins Ypt1 and Sec4; Jena BP et al.; Previous studies have demonstrated that Sec4, a 23.5 kDa guanine nucleotide-binding protein of the ras superfamily is required for exocytosis in the budding yeast Saccharomyces cerevisiae . Ypt1, another ras-like 23 kDa guanine nucleotide-binding protein in yeast has been found to be involved in ER-Golgi transport . A mammalian homologue of Ypt1 called rab1 has also been identified . Recent studies using purified Sec4 protein have identified a component of yeast lystate that specifically stimulates the hydrolysis of GTP bound to Sec4 . In the present study, purified recombinant Sec4 and Ypt1 proteins expressed in E . coli have been used as substrates to determine if GTPase activating proteins (GAPs) directed toward these proteins are present in rat pancreas . Our studies showed that 65% of Sec4-GAP activity was associated with the 150,000 x g pancreatic particulate fraction with approximately 35% being found in the cytosol . On the other hand, more than 95% of Ypt1-GAP activity was found to associate with the particulate fraction . Sec4 and Ypt1 competition assays further demonstrated the specificity of the Sec4 and Ypt1 GAPs . The results from the present study suggest the presence of a distinct GAP in the pancreas that interacts with Sec4, and another that interacts with Ypt1.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 94 - 100
The oxygenated flavohaemoglobin from Escherichia coli: evidence from photodissociation and rapid-scan studies for two kinetic and spectral forms; Orii Y et al.; The kinetics of dissociation and reassociation of the oxygenated species of Escherichia coli flavohaemoglobin (Hmp) were studied using stopped-flow rapid-scan and flash photolysis spectrophotometry at 25 degrees C . The oxygenated compound(s) form rapidly on mixing oxygen with the NADH-reduced flavohaemoglobin . On exhaustion of NADH, with residual oxygen, decay occurs in two phases to give a form in which haem b and flavin are oxidized . Spectral changes during this process suggest a direct release of O2- from the oxy form . Photodissociation of the oxygenated species generates the unliganded protein, which recombines with oxygen to give two spectrally and kinetically distinct forms . The reversibility of the oxygen reaction and the rapid reassociation kinetics after photodissociation confirm the haemoglobin-like features of this protein.

Science, 1992 Aug 28, 257(5074), 1264 - 7
Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors; Pitcher JA et al.; The rate and extent of the agonist-dependent phosphorylation of beta 2-adrenergic receptors and rhodopsin by beta-adrenergic receptor kinase (beta ARK) are markedly enhanced on addition of G protein beta gamma subunits . With a model peptide substrate it was demonstrated that direct activation of the kinase could not account for this effect . G protein beta gamma subunits were shown to interact directly with the COOH-terminal region of beta ARK, and formation of this beta ARK-beta gamma complex resulted in receptor-facilitated membrane localization of the enzyme . The beta gamma subunits of transducin were less effective at both enhancing the rate of receptor phosphorylation and binding to the COOH-terminus of beta ARK, suggesting that the enzyme preferentially binds specific beta gamma complexes . The beta gamma-mediated membrane localization of beta ARK serves to intimately link receptor activation to beta ARK-mediated desensitization.

Biochim Biophys Acta, 1992 Aug 17, 1132(1), 11 - 6
Direct expression of active spinach glycolate oxidase in Escherichia coli; Macheroux P et al.; Spinach glycolate oxidase (GAO) was expressed in Escherichia coli using the T7 RNA polymerase promotor . The enzyme accounts for approx . 1% of the soluble protein fraction and is expressed as a soluble and active enzyme . Comparison with GAO expressed in Saccharomyces cerevisiae (Macheroux, P., Massey, V., Thiele, D.J . and Volokita, M . (1991) Biochemistry 30, 4612-4619) showed that the GAO expressed in E . coli has identical physico-chemical features to the wild-type enzyme, but is expressed at a level approx . 15-fold higher than in the yeast system.

J Biol Chem, 1992 Aug 25, 267(24), 17339 - 46
Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus; Lustigman S et al.; A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein . OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors . In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin . It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations . Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B . Neither fusion polypeptide inhibits serine or metalloproteinases activity . The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively . The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs . This represents the complete coding sequence of the mature onchocystatin (130 amino acids) . A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.

J Biol Chem, 1992 Aug 25, 267(24), 17257 - 63
Enzymic activities of two-chain pepsinogen, two-chain pepsin, and the amino-terminal lobe of pepsinogen; Lin XL et al.; In order to study the relationships of aspartic proteases, we have modified pepsin, a single-chain eukaryotic enzyme, to a two-chain heterodimer, which resembles aspartic proteases from retrovirus, including human immunodeficiency virus . Two fragments of pepsinogen, residues 1P-172 and 173-326, were expressed separately in Escherichia coli . Mixtures of chains were refolded from urea solutions to generate an active two-chain pepsinogen, which was converted to two-chain pepsin in acid solutions . The intramolecular and bimolecular activation constants (k1 and k2) of two-chain pepsinogen are about 1.5-fold and one-sixth, respectively, of those for pepsinogen . Structural evidence suggests that the faster k1 of two-chain pepsinogen is due to decreased interaction of the propeptide with the pepsin moiety, implying that the rate-limiting step in the intramolecular activation of pepsinogen is the "conformational dissociation" of its propeptide . Two-chain pepsin has the same Km but only one-sixth of the kcat of pepsin . Both pepsinogen chains are capable of independent refolding . The refolding of the NH2-terminal chain, which contains the propeptide and the NH2-terminal lobe, generated a small amount of proteolytic activity which is likely derived from the homodimer of the NH2-terminal lobe . It has been postulated that mammalian aspartic proteases, which contain two structurally homologous lobes, are derived in evolution from a homodimer enzyme by gene duplication and fusion (Tang, J., James, M . N . G., Hsu, I.-N., Jenkins, J . A., and Blundell, T . L . (1978) Nature 271, 618-621) . The observation of the homodimer activity of the NH2-terminal lobe of pepsinogen suggests that the interface of the lobes is conservative in evolution.

J Biol Chem, 1992 Aug 25, 267(24), 17153 - 8
Characterization of Escherichia coli RNase PH; Kelly KO et al.; We have previously shown that the orfE gene of Escherichia coli encodes RNase PH . Here we show that the OrfE protein (purified as described in the accompanying paper) (Jensen, K . F., Andersen, J . T., and Poulsen, P . (1992) J . Biol . Chem . 267, 17147-17152) has both the degradative and synthetic activities of RNase PH . This highly purified protein was used to characterize the enzymatic and structural properties of RNase PH . The enzyme requires a divalent cation and phosphate for activity, the latter property indicating that RNase PH is exclusively a phosphorolytic enzyme . Among tRNA-type substrates, the enzyme is most active against synthetic tRNA precursors containing extra residues following the -CCA sequence, and it can act on these molecules to generate mature tRNA with amino acid acceptor activity; 3'-phosphoryl-terminated molecules are not active as substrates . The equilibrium constant for RNase PH is near unity, suggesting that at the phosphate concentration present in vivo, the enzyme would participate in RNA degradation . The synthetic reaction of RNase PH displays a nonlinear response to increasing enzyme concentrations, and this may be due to self-aggregation of the protein . Higher order multimers of RNase PH could be detected by gel filtration at higher protein concentrations and by protein cross-linking . The possible role of RNase PH in tRNA processing is discussed.

J Biol Chem, 1992 Aug 25, 267(24), 17147 - 52
Overexpression and rapid purification of the orfE/rph gene product, RNase PH of Escherichia coli; Jensen KF et al.; The pyrE gene, encoding the pyrimidine biosynthetic enzyme orotate phosphoribosyltransferase, is the promoter distal gene of the dicistronic orfE-pyrE operon . The promoter proximal orfE gene, whose transcription and translation is important for regulation of the pyrE attenuator, encodes a 238-amino acid residue protein which was recently identified as the phosphorolytic ribonuclease, RNase PH, that removes nucleotides from the 3' ends of tRNA precursors . In this paper we report the construction of a plasmid, which overexpresses the orfE and pyrE gene products substantially, as well as the purification of the OrfE protein by ammonium sulfate precipitation and chromatography on phosphocellulose . The highly purified protein catalyzes the phosphorolytic cleavage of poly(A) at a rate of 1.6 mumol/min/mg and the formation of CDP from tRNA-CCA-Cn and orthophosphate at a rate equal to 0.14 mumol/min/mg, as characteristic for RNase PH . OrfE/RNase PH contains helix-turn-helix motifs resembling those in DNA-binding proteins, and it binds nonspecifically to DNA . On SDS gels, OrfE/RNase PH migrates as two distinct protein bands . This heterogeneity might be caused by post-translational modification other than proteolysis, or may be an electrophoretic artifact . The native protein is composed of two or more subunits.

J Biol Chem, 1992 Aug 25, 267(24), 17095 - 101
Photoaffinity labeling of fatty acid-binding proteins involved in long chain fatty acid transport in Escherichia coli; Mangroo D et al.; The photoreactive fatty acid 11-m-diazirinophenoxy-{11-3H}undecanoate was shown to be taken up specifically by the fatty acid transport system expressed in Escherichia coli grown on oleate . This photoreactive fatty acid analogue was therefore used to identify proteins involved in fatty acid uptake in E . coli . The fadL protein was labeled by the probe, confirmed to be exclusively in the outer membrane and to exhibit the heat modifiable behavior typical of outer membrane proteins . The apparent pI of the incompletely denatured form of the protein having the mobility of a 33-kDa protein was 4.6 while that of the fully denatured form was consistent with the calculated value of 5.2 . The denaturation was reversible depending upon the protein to detergent ratios . The photoreactive fatty acid partitions into the outer membrane, resulting in extensive photolabeling of the lipid; a high affinity fatty acid-binding site is not apparent in total membranes labeled using free fatty acids due to this large binding capacity of the outer membrane . However, when the free fatty acid concentration was controlled by supplying it as a bovine serum albumin complex, the fadL protein exhibited saturable high affinity fatty acid binding, having an apparent Kd for the probe of 63 nM . The methods described very readily identify fatty acid-binding proteins: the fact that even when the sensitivity was increased 500-fold, no evidence was found for the presence of a fatty acid-binding protein in the inner membrane is consistent with the proposal that fatty acid permeation across the plasma membrane is not protein mediated but occurs by a simple diffusive mechanism.

J Biol Chem, 1992 Aug 25, 267(24), 16816 - 23
Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins . Characterization of AKAP 79; Carr DW et al.; Postsynaptic densities (PSD) are a network of proteins located on the internal surface of excitatory synapses just inside the postsynaptic membrane . Enzymes associated with the PSD are optimally positioned to respond to signals transduced across the postsynaptic membrane resulting from excitatory synaptic transmission or neurotransmitter release . We present evidence suggesting that type II cAMP-dependent protein kinase (PKA) is anchored to the PSD through interaction of its regulatory subunit (RII) with an A-Kinase Anchor Protein (AKAPs) . A cDNA for the human RII-anchoring protein, AKAP 79, was isolated by screening an expression library with radiolabeled RII . This cDNA (2621 base pairs) encodes a protein of 427 amino acids with 76% identity to bovine brain AKAP 75 and 93% identity to a carboxyl-terminal RII-binding fragment of murine brain AKAP 150 . A bacterially expressed 92-amino acid fragment, AKAP 79 (335-427) was able to bind RII alpha . Disruption of secondary structure by site-directed mutagenesis at selected residues within a putative acidic amphipathic helix located between residues 392 and 408 prevented RII binding . Immunological studies demonstrate that AKAP 79 is predominantly expressed in the cerebral cortex and is a component of fractions enriched for postsynaptic densities . AKAP antisera strongly cross-react with a 150-kDa protein in murine PSD believed to be AKAP 150 . Co-localization of the type II PKA in purified PSD fractions was confirmed immunologically by detection of RII and enzymologically by measuring cAMP-stimulated phosphorylation of the heptapeptide substrate Kemptide . Approximately 30% of the PSD kinase activity was specifically inhibited by PKI 5-24 peptide, a highly specific inhibitor of PKA . We propose that AKAP 79 and AKAP 150 function to anchor the type II PKA to the PSD, presumably for a role in the regulation of postsynaptic events.

J Biol Chem, 1992 Aug 25, 267(24), 16783 - 9
trp repressor/trp operator interaction . Equilibrium and kinetic analysis of complex formation and stability; Hurlburt BK et al.; The trp repressor of Escherichia coli regulates transcription initiation in the trp operon by binding at an operator located within the trp promoter region . We have used a filter binding assay to analyze the interaction between purified trp repressor and a synthetic 43-base pair DNA fragment containing the natural trp promoter-operator region . In equilibrium binding experiments, the KD of high affinity binding of trp repressor to this DNA fragment was determined to be 2 x 10(-10) M . Low affinity binding was observed at repressor concentrations above 10 nM . In kinetic experiments with various input ratios of repressor to operator, trp repressor-operator complexes dissociated with equivalent, first-order kinetics . Instantaneous reduction of the tryptophan concentration resulted in increased rates of complex dissociation, indicating that loss of one or both tryptophan molecules from the repressor-operator complex destabilizes the complex . A heterodimeric repressor with a single tryptophan binding site was constructed and its affinity for operator was compared with that of ligand free aporepressor and tryptophan saturated repressor . The heterodimeric repressor had a 20-25-fold higher affinity for operator than did the aporepressor, and it had a 20-25-fold lower affinity for operator than did the tryptophan-saturated repressor.

J Biol Chem, 1992 Aug 25, 267(24), 16779 - 82
The chromosome origin of Escherichia coli stabilizes DnaA protein during rejuvenation by phospholipids; Crooke E et al.; DnaA protein (the initiator protein) binds and clusters at the four DnaA boxes of the Escherichia coli chromosomal origin (oriC) to promote the strand opening for DNA replication . DnaA protein activity depends on the tight binding of ATP; the ADP form of DnaA protein, generated by hydrolysis of the bound ATP, is inactive . Rejuvenation of ADP-DnaA protein, by replacement with ATP, is catalyzed by acidic phospholipids in a highly fluid bilayer . We find that interaction of DnaA protein with oriC DNA is needed to stabilize DnaA protein during this rejuvenation process . Whereas DnaA protein bound to oriC DNA responds to phospholipids, free DnaA protein is inactivated by phospholipids and then fails to bind oriC . Furthermore, oriC DNA facilitates the high affinity binding of ATP to DnaA protein during treatment with phospholipids . A significant portion of the DnaA protein associated with oriC DNA can be replaced by the ADP form of the protein, suggesting that all of the DnaA protein bound to oriC DNA need not be rejuvenated between rounds of replication.

J Biol Chem, 1992 Aug 25, 267(24), 16767 - 70
Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245; Abo A et al.; Activation of the NADPH oxidase of phagocytes in the cell-free system requires the association of several cytosolic components with membrane-bound cytochrome b . In this study we were able to fully reconstitute NADPH oxidase activity in the cell-free system with three recombinant proteins: p67-phox, p47-phox, p21rac1, and pure cytochrome b-245 . Activity was dependent upon the concentration of the proteins, with maximal activity observed with roughly equimolar ratios of the cytochrome b and p67-phox (133 and 163 mol/s/mol, respectively) and concentrations of the other two proteins approximately 1 order of magnitude greater . No activity was observed in the absence of any one of these components . In addition, activation was dependent upon p21rac1 being preloaded with GTP, the cytochrome b being reconstituted with lipid, and the presence of FAD during activation . Half-maximal activity was observed at a concentration of NADPH of approximately 50 microM . These findings confirm our recent description of the membrane-bound cytochrome b as a FAD-containing flavocytochrome b containing the NADPH binding site, and implicate the three cytosolic proteins in its activation.

Biochemistry, 1992 Aug 25, 31(33), 7741 - 4
Secondary structure of human interleukin 2 from 3D heteronuclear NMR experiments; Mott HR et al.; Recombinant 15N-labeled human interleukin 2 (IL-2) has been studied by 2D and 3D NMR using uniformly 15N-labeled protein . Assignment of the backbone resonances has enabled the secondary structure of the protein to be defined . The secondary structure was found to consist of four alpha-helical regions and a short section of antiparallel beta-sheet . This structure is more similar to recent published structures of interleukin 4 and granulocyte-macrophage colony-stimulating factor than to a structure of IL-2 previously obtained from low-resolution X-ray diffraction data.

Biochemistry, 1992 Aug 25, 31(33), 7629 - 37
G1401: a keystone nucleotide at the decoding site of Escherichia coli 30S ribosomes; Cunningham PR et al.; 16S ribosomal RNA contains three highly conserved single-stranded regions . Centrally located in one of these regions is the C1400 residue . Zero-length cross-linking of this residue to the anticodon of ribosome-bound tRNA showed that it was at or near the ribosomal decoding site {Ehresmann, C., Ehresmann, B., Millon, R., Ebel, J-P., Nurse, K., & Ofengand, J . (1984) Biochemistry 23, 429-437} . To assess the functional significance of sequence conservation of rRNA in the vicinity of this functionally important site, a series of site-directed mutations in this region were constructed and the effects of these mutations on the partial reactions of protein synthesis determined . Mutation of C1400 or C1402 to any other base only moderately affected a set of in vitro protein synthesis partial reactions . However, any base change from the normal G1401 residue blocked all of the tested ribosomal functions . This was also true for the deletion of G1401 . Deletion of C1400 or C1402 had more complex effects . Whereas subunit association was hardly affected, 30S initiation complex formation was blocked by deletion of C1400 but much less so by deletion of C1402 . Alternatively, tRNA binding to the ribosomal A site was more strongly affected by deletion of C1402 than by deletion of C1400 . P site binding was inhibited by either deletion . HPLC analysis of the in vitro reconstituted mutant ribosomes showed that none of the functional effects were due to the absence or gross reduction in amount of any ribosomal protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Aug 25, 31(33), 7519 - 26
Proximity relationship between the active site of Escherichia coli RNA polymerase and rifampicin binding domain: a resonance energy-transfer study; Kumar KP et al.; Escherichia coli RNA polymerase has two subsites, i and i + 1, for the binding of the first two substrates, and the first phosphodiester bond is formed between them during the initiation of transcription . Various studies have shown earlier that the inhibitor rifampicin has little effect, if any, on the formation of this phosphodiester bond . On an earlier occasion, we measured the distance of the i nucleotide from the rifampicin binding site on RNA polymerase using Forster's energy-transfer mechanism {Kumar & Chatterji (1990) Biochemistry 29,317} . In this paper, the 1-aminonaphthalene-5-sulfonic acid (AmNS) derivative of UTP in the presence of 10 mM MgCl2 was used as an energy donor, and its distance from rifampicin was estimated . The modified nucleotide (gamma-AmNS)-UTP binds to RNA polymerase with a Kd of 3 microM and has one binding site in the presence of Mg(II) ion . Fluorescence titration studies performed with or without an initiator indicated that (gamma-AmNS)-UTP exclusively binds to RNA polymerase at the (i + 1) site in the presence of Mg(II) . Rifampicin was found to form a 1:1 complex with RNA polymerase bound to labeled UTP . Rifampicin and (gamma-AmNS)-UTP have a substantial spectral overlap with an energy-transfer efficiency close to 50% . Labeled UTP shows a decrease in its excited-state lifetime when bound to the enzyme; the transfer efficiency calculated from lifetime measurements was found to be lower than that estimated from steady-state spectral analysis . Time-resolved emission spectral analysis was carried out to differentiate between the free and bound UTP over the enzyme surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1992 Aug 25, 20(16), 4347 - 53
RecA protein promotes rapid RNA-DNA hybridization in heterogeneous RNA mixtures; Kirkpatrick DP et al.; The nucleoprotein filament formed by the RecA protein of Escherichia coli on single-stranded DNA catalyzes the hybridization of RNA transcripts with single-stranded DNA sequences at 37 degrees C, in vitro . RecA protein rapidly promotes hybridization, even when noncomplementary RNA is in a millionfold nucleotide excess over hybridizing RNA, and in a thousandfold nucleotide excess over hybridizing single-stranded DNA . Heterologous double-stranded DNA and RecA-coated noncomplementary single-stranded DNA are also poor competitors of RNA transcripts produced in vitro . Since large excesses of noncomplementary RNA fail to inhibit sharply the hybridization reaction by RecA protein under mild, non-degradative conditions, the reaction may be useful in the identification and isolation of transcripts produced in vivo.

Nucleic Acids Res, 1992 Aug 25, 20(16), 4213 - 20
Blockage of polymerase-catalyzed DNA chain elongation by chemically modified cytosine residues in templates and the release of blockage for readthrough; Bessho T et al.; The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and -methoxy)-5,6-dihydrocytosine-6-sulfonate residues . We have studied the mechanism of the blockage, using as templates bisulfite-hydrazine (and -methoxyamine)- modified single strand phage-M13mp2 DNA and synthetic oligonucleotides . Both N4-amino-5,6-dihydrocytosine-6-sulfonate and N4-methoxy-5,6-dihydrocytosine-6-sulfonate residues blocked the elongation at one nucleotide before these sites . In this blockage, the idling of polymerase at the lesion site due to its 3'-5' exonuclease action appears not to play a major role, because Sequenase that lacks the 3'-5' exonuclease activity still could not readthrough these sites . It seems possible that conformational distortion of the template near these sites is responsible for the blockage, because on conversion of this 5,6-dihydropyrimidine-6-sulfonate structure into a planar pyrimidine, a complete restoration of polymerase-readthrough resulted . In the presence of RecA and SSB proteins, the Klenow fragment was able to partially readthrough these sites . Since there was no decrease in the 3'-5' exonuclease activity during this readthrough, it seems that the binding of these proteins relaxes the distortion in the modified template to allow the polymerase to readthrough the lesion site . These sites on phage DNA can be lethal but also are capable of inducing C-to-T transitions . This observation suggests that these sites can be read by E . coli DNA polymerases in vivo with accompanying errors.

Nucleic Acids Res, 1992 Aug 25, 20(16), 4193 - 8
The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site; Slany RK et al.; The tgt/sec operon in E . coli consists of five genes: queA, tgt, ORF12, secD, and secF . QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion . Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons . An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found . Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58 . Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis . Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength . An approximately two-fold activation of the promoter by the UAS element was observed.

Nucleic Acids Res, 1992 Aug 25, 20(16), 4137 - 44
Zinc finger-DNA recognition: analysis of base specificity by site-directed mutagenesis; Nardelli J et al.; Zinc fingers of the Cys2/His2 class are conserved 28-30 amino acid motifs that constitute an important and widespread family of eukaryotic DNA-binding domains . It is therefore of great interest to understand the rules that govern specific recognition of DNA by zinc fingers . The DNA-binding domain of the transcription factor Krox-20 consists of three zinc fingers, each of them making its primary contacts with a three-base pair subsite . We have performed a data base-guided site-directed mutagenesis analysis of Krox-20: nine derivatives were generated, in which one to three amino acid changes had been introduced within finger 2, at positions which were likely to affect the specificity of DNA recognition . The affinities of the different proteins for a panel of potential DNA binding sites were estimated by gel retardation assay . Six of the derivatives bound specific targets with affinities comparable to that of wild type Krox-20 for its consensus binding site . However, the specificity of recognition was dramatically modified at the expected bases, in a manner that could be explained by examining the newly introduced amino acids within the context of the overall finger/triplet interaction . These data provide new insights into the details of zinc finger-DNA interactions and, combined with the modular nature of zinc fingers, illustrate both the potential and the difficulties of utilising these motifs for designing DNA-binding proteins with novel specificities.

J Biol Chem, 1992 Aug 25, 267(24), 16829 - 33
Interaction of GroE with an all-beta-protein; Schmidt M et al.; Molecular chaperones are involved in protein folding both in vivo and in vitro . The Escherichia coli chaperone GroEL interacts with a number of nonnative proteins . A common structural motif of nonnative proteins, which is recognized by GroEL, has not yet been identified . In order to study the role of beta-sheet secondary structure on the interaction of nonnative proteins with GroEL, we used the F(ab) fragment of a monoclonal antibody as a model substrate protein . Here we show that GroEL interacts functionally with this all-beta-protein during reactivation . Antibody fragments refold spontaneously in good yield from the guanidine-denatured state . Functional refolding to the native state is inhibited transiently by GroEL, but there is no complete folding arrest in the absence of Mg-ATP and GroES . The yield of these unspecifically released GroEL-bound F(ab) fragments corresponds to that of the spontaneous reactivation in the absence of chaperones . However, the refolding kinetics in the presence of GroEL are considerably slower . The addition of Mg-ATP to the GroEL.F(ab) complex results in an immediate release of bound substrate protein and a significant increase in the amount of reconstituted antibody fragments compared to spontaneous reactivation . GroES is not essential for functional GroEL-mediated refolding of the F(ab) fragment but affects the reactivation yield to a small extent . Interestingly, stimulation of the GroEL-mediated F(ab) refolding depends primarily on the binding and not on hydrolysis of adenosine triphosphates . Previous results indicate the binding of alpha-helices to GroEL . The results presented in this paper suggest that beta-sheet secondary structural elements are recognized by GroEL . We therefore conclude that the interaction of a nonnative protein with GroEL depends mainly on the nature of the early folding intermediate but not on a specific element of secondary structure.

Nucleic Acids Res, 1992 Aug 25, 20(16), 4159 - 65
Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function; Podust VN et al.; By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa . The protein is very likely replication factor C (Tsurimoto, T . and Stillman, B . (1989) Mol . Cell . Biol . 9, 609-619) . This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA . Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA . Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP . Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way . Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon . Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed.

J Biol Chem, 1992 Aug 25, 267(24), 17424 - 9
Structural elements required for the cooperative binding of the herpes simplex virus origin binding protein to oriS reside in the N-terminal part of the protein; Elias P et al.; The origin binding protein (OBP) of herpes simplex virus type 1 is required to activate a viral origin of replication in vivo . We have used intact OBP as well as a truncated form of the protein expressed in Escherichia coli to investigate the protein-protein interactions, as well as the protein-DNA interactions involved in the formation of a nucleoprotein complex at a viral origin of replication (oriS) in vitro . The salient findings demonstrate that the N-terminal part of OBP is required for the cooperative binding of OBP to three sites (boxes I, II, and III) within oriS . A detailed model for the interaction of OBP with the viral origins of replication oriS and oriL is presented.

J Biol Chem, 1992 Aug 25, 267(24), 17279 - 86
TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin; Ziegelin G et al.; Conjugative transfer of the self-transmissible IncP plasmid RP4 requires the product of the RP4 traK gene . By using the phage T7 expression system, the traK gene product was efficiently overproduced and purified to near homogeneity . traK encodes a basic protein (pI = 10.7) of 14.6 kDa that, as shown by DNA fragment retention assay, interacts exclusively with its cognate transfer origin . The apparent equilibrium constant K(app) for the complex of TraK and oriT-DNA was estimated to be 4 nM . Footprinting experiments using DNase I or hydroxyl radicals indicate that several TraK molecules interact specifically with an intrinsically bent region of oriT, covering a range of almost 200 base pairs . The TraK target sequence maps in the leading region adjacent to the relaxation nick site and recognition sequences involved in relaxosome formation but does not overlap them . Specific interactions between TraK and the DNA occur only on one side of the double helix . Electron microscopy of TraK-oriT complexes demonstrates that binding of TraK to its recognition region apparently shrinks the length of the target DNA, suggesting that the nucleic acid becomes wrapped around a core of TraK molecules . Formation of this structure could be favored by the presence of the sequence-directed bend in the TraK recognition region.

J Biol Chem, 1992 Aug 25, 267(24), 17141 - 6
Nucleotide binding by the poliovirus RNA polymerase; Richards OC et al.; Cross-linking of ribonucleoside triphosphates (NTPs) to specific binding sites on the poliovirus RNA-dependent RNA polymerase has been performed by ultraviolet irradiation and by reduction of oxidized nucleotide-protein complexes . The latter method approached a cross-linking efficiency of 1 NTP/molecule of enzyme . Nucleotide competition experiments suggested that the same binding site is occupied by all NTPs . Analysis of peptides produced by proteinase Glu-C and trypsin digestion and labeled with {32P}GTP indicated that a lysine residue between Met-189 and Lys-228 in the polymerase was cross-linked to NTP . Nucleotide binding was exploited for rapid purification of the enzyme by GTP-agarose affinity chromatography . In addition, a set of cloned, modified polymerase molecules with reduced or absent polymerization activity was analyzed for binding efficiency to a GTP-agarose column . Some mutations eliminated GTP binding, whereas others generated proteins with varying affinities for GTP . Incubation of the poliovirus polymerase with high concentrations of NTP, particularly GTP, resulted in a dramatic protection against heat denaturation and activity loss . These data suggest that nucleotide binding results in an alteration of the enzyme conformation or the stabilization of an ordered conformation.

J Biol Chem, 1992 Aug 25, 267(24), 16939 - 42
NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction; Weber DJ et al.; The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate . The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product . This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group . No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate . No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP . Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates . The MutT protein is the first hydrolase shown to do so.

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