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J Biol Chem, 1994 Mar 18, 269(11), 8366 - 75
Intracellular analysis of in vitro modified HIV Tat protein; Koken SE et al.; Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that specifically activates transcription from the viral long terminal repeat . To characterize the properties of the Tat proteins, we have expressed them in Escherichia coli . The purified Tat protein was biochemically analyzed and tested for activity upon electroporation into human cell lines . This protein electroporation was used for the intracellular analysis of in vitro modified Tat protein . Our results indicate that the transcriptionally active form of the Tat protein is a monomer . Furthermore, we found that Tat activity is dramatically inhibited by preincubation of the protein with strongly reducing agents . In contrast, no inhibitory effect was measured upon incubation with metal-chelating reagents . These results suggest that the cysteine residues of Tat are involved in the formation of intramolecular disulfide bonds.

J Biol Chem, 1994 Mar 18, 269(11), 8334 - 40
A novel 39-kDa phosphoribosylpyrophosphate synthetase-associated protein of rat liver . Cloning, high sequence similarity to the catalytic subunits, and a negative regulatory role; Kita K et al.; The rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of the 34-kDa catalytic subunits (PRS I and II) and other 39- and 41-kDa proteins (Kita, K., Otsuki, T., Ishizuka, T., and Tatibana, M . (1989) J . Biochem . (Tokyo) 105, 736-741), which are termed here PRPP synthetase-associated proteins (PAPs) . We have cloned the cDNA for the major one of 39 kDa (PAP39) from a rat liver cDNA library . Nucleotide sequencing showed that the clone encoded 356 amino acids containing sequences of all five peptides derived from PAP39 . Surprisingly, the deduced amino acid sequence is markedly similar to those of the 34-kDa catalytic subunits . Excluding two regions (about 45 residues in total), PAP39 has a 48% identity with PRS I . Northern analysis detected a major 1.9-kilobase transcript in all 16 rat tissues examined, and the relative amounts of PAP39 mRNA to PRS I mRNA varied with tissues . Covalent cross-linking experiments gave definitive evidence for molecular interaction of PAP39 with the catalytic subunits . Immunoprecipitation experiments revealed that all the catalytic subunits existed as complexes containing PAP39 . When PAPs were eliminated from the rat liver enzyme complex by gel filtration in the presence of 1 m MgCl2, a lyotrope, or by mild tryptic treatment, the enzyme activity of the remaining catalytic subunits increased . Based on these results, we propose that PAP39, the major component of PAPs, plays a negative regulatory role in PRPP synthesis and hence is an important factor controlling nucleotide syntheses in general.

J Biol Chem, 1994 Mar 18, 269(11), 8280 - 6
Transcription factor (TF) IIB and TFIIA can independently increase the affinity of the TATA-binding protein for DNA; Imbalzano AN et al.; The general transcription factors (TF) IIB and TFIIA are the first factors to associate with the TATA-binding protein (TBP) during formation of a transcription initiation complex on RNA polymerase II promoters . DNase I footprint titration was used to measure the effects of TFIIB and TFIIA on binding of TBP to a consensus TATA box . Under reaction conditions optimized for TBP-DNA complex formation, the presence of TFIIB increased affinity of TBP for the TATA box by 2.5-fold, while TFIIA had no effect . When TBP binding conditions were sub-optimal, both TFIIB and TFIIA independently increased TBP affinity by approximately 10-fold . Therefore both TFIIB and TFIIA have the intrinsic ability to directly increase the affinity of TBP for the TATA box . We suggest that this property of TFIIA and TFIIB may increase the range of conditions under which high affinity TBP-DNA interactions can occur and may therefore favor the formation of the preinitiation complex.

J Biol Chem, 1994 Mar 18, 269(11), 8246 - 54
Purification and biochemical characterization of Escherichia coli RecA proteins mutated in the putative DNA binding site; Cazaux C et al.; Escherichia coli RecA protein plays a central role both in DNA repair and in recombination . We report biochemical properties of three new RecA proteins mutated at positions 199 (RecA694), 207 (RecA659), and 211 (RecA611) in the putative DNA binding site . RecA694 had a wild-type phenotype, whereas RecA611 and RecA659 were deficient in promoting both the self-cleavage of LexA repressor and the DNA-strand exchange reaction . In order to determine the origin of this inhibition, we examined the capacity of wild-type and mutant proteins to bind to single-stranded DNA (with and without single-stranded binding protein, SSB), double-stranded DNA, and ATP . DNA strand exchange defects were correlated with the inability of mutant proteins to displace SSB from DNA . For the recA659 mutation this inhibition was reversed by equimolar wild-type protein . In contrast, mixtures of either wild-type/RecA659 or wild-type/RecA611 proteins remained deficient in LexA cleavage, suggesting that the dominant negative phenotype of the mutant proteins may be a consequence of the formation heterologous RecA complexes . Various mutations in the putative DNA binding site of RecA protein altered ATP binding, ATPase activity, displacement of SSB from single-stranded DNA, and protein-protein interactions . These results are consistent with the hypothesis that DNA binding to this site of RecA relays allosteric effects to several functional domains throughout the protein.

J Biol Chem, 1994 Mar 18, 269(11), 8220 - 5
The sulfurtransferase activity and structure of rhodanese are affected by site-directed replacement of Arg-186 or Lys-249; Luo GX et al.; Two mutants of the enzyme rhodanese that replace Arg-186 with Leu (R186L) or Lys-249 with Ala (K249A) were prepared to test suggestions that these residues are involved in catalysis and structure . The predominant effect with R186L was functional, and Km for the sulfur donor, thiosulfate, increased from 3.7 mM to 73 mM with a modest decrease in Vmax (672 IU/mg to 576 IU/mg) . However, K249A was virtually inactive using thiosulfate, but it was active with thiosulfonates such as p-toluene-, 2-aminoethane-, or ethanethiosulfonate, and these compounds could be demonstrated to form persulfide-substituted rhodanese . Compared with wild type enzyme, K249A had (a) reduced stability, (b) comparable secondary structure, (c) more easily exposed hydrophobic surfaces, and (d) a core structure that denatured similarly to the wild type enzyme . Thus, Arg-186 and Lys-249 are important in rhodanese catalysis, and Lys-249 is particularly critical for substrate selectivity and protein stability . Finally, the results suggest that there can be active rhodanese species in vivo that will be undetected using thiosulfate as a sulfur donor.

J Biol Chem, 1994 Mar 18, 269(11), 8182 - 8
The NAD(P)H:flavin oxidoreductase from Escherichia coli as a source of superoxide radicals; Gaudu P et al.; The NAD(P)H:flavin oxidoreductase (encoded by the fre gene) of Escherichia coli is a soluble enzyme which, under aerobic conditions and together with NAD(P)H and flavins, generates superoxide radicals selectively . This was demonstrated from spin trapping experiments and from the ability of the flavin reductase to achieve a superoxide dismutase (SOD)-sensitive reduction of cytochrome c . The participation of the flavin reductase to O2- . generation in E . coli cells has been studied . Superoxide production in dialyzed cytosolic fraction of SOD-deficient E . coli was stimulated by the addition of flavins . There was no stimulation in soluble extracts of flavin reductase-deficient strains . Moreover, using fusions of sodA promoter to lacZ, we showed that sodA transcription was diminished in flavin reductase-deficient E . coli and that the induction of MnSOD by flavin reductase was SoxRS-independent . These results suggest that the flavin reductase might: (i) in vivo, be an important cytosolic site of O2- . generation; (ii) in vitro, serve as a simple, efficient, and selective O2- . generator.

J Biol Chem, 1994 Mar 18, 269(11), 8146 - 52
Anti-metatype antibodies stabilize the fluorescein single-chain antibody 4-4-20 complex against dissociation by hydrostatic pressure; Coelho-Sampaio T et al.; Hydrostatic pressure was used to promote dissociation of fluorescein (Fl) from single-chain antibody 4-4-20 (SCA 4-4-20) . Fl fluorescence intensity was quenched by 97% upon binding to SCA 4-4-20 . Increasing pressure to 2.4 kbar enhanced Fl fluorescence from the remaining 3% to 14-17% . The capacity of anti-metatype antibodies (anti-Met), which specifically recognize liganded anti-Fl antibodies, to protect against pressure-induced Fl dissociation was tested . Both polyclonal and monoclonal anti-Met antibodies protected against Fl dissociation, reducing the fluorescence intensity at 2.4 kbar from 14-17% to 6-8% . Additive effects of anti-Met antibodies in protection against pressure-induced Fl dissociation were suggested by the fact that a 2-fold molar excess polyclonal anti-Met reagent promoted additional protection relative to an equimolar amount . On the other hand, combination of different monoclonal anti-Met antibodies did not promote additive protection, suggesting recognition of overlapping metatopes by these monoclonals . The complex formed by SCA 4-4-20 and the Fl analog HPF was more sensitive to pressure than the Fl-SCA 4-4-20 complex . Addition of both polyclonal and monoclonal anti-Met antibodies reduced the Fl fluorescence recovery at 2.4 kbar from 75% to 40-55% . In order to directly study binding of anti-Met antibodies to mAb 4-4-20, monoclonal anti-Met antibody 3A5-1 was labeled with 2-dimethylaminonaphthalene-5-sulfonyl chloride (2,5-Dns-Cl) and Dns fluorescence anisotropy measured . Unliganded mAb 4-4-20 did not bind to 2,5-Dns-3A5-1 as indicated by the absence of measurable changes in Dns fluorescence anisotropy upon increasing mAb concentration . Addition of mAb 4-4-20 bound to Fl produced a sigmoidal increase in Dns anisotropy, compatible with association of the primary immune complex and 3A5-1 . An affinity constant, K0.5, of 1.5 x 10(-7) M and a cooperativity coefficient (n) of 3.1 were calculated for formation of the Fl-mAb 4-4-20 complex . The HPF-mAb 4-4-20 complex was also recognized by 2,5-Dns-3A5-1 but with lower affinity, indicating that the monoclonal anti-Met 3A5-1 distinguished between mAb 4-4-20 liganded to different haptens.

J Biol Chem, 1994 Mar 18, 269(11), 8091 - 8
Mutations of an active site threonyl residue promote beta elimination and other side reactions of the enediol intermediate of the ribulosebisphosphate carboxylase reaction; Morell MK et al.; The side chain of residue threonine 65 within the active site of ribulosebisphosphate carboxylase participates in a network of hydrogen bonds and ionic interactions involving the phosphate moiety attached to C-1 of the substrate . This residue was replaced with serine, alanine, and valine in the enzyme from Synechococcus PCC 6301 . The mutant enzymes were stable, expressed abundantly by Escherichia coli, and retained the ability to form gel-filterable complexes with the reaction-intermediate analog, 2'-carboxyarabinitol-1,5-bisphosphate . The substitutions reduced the kcat/Km(CO2) (where kcat is the substrate-saturated turnover rate) of the enzyme from 17- to 340-fold with the more radical substitutions causing more severe reductions . The CO2/O2 specificity also deteriorated progressively, the valine replacement causing a 2.3-fold reduction . In concert with these changes, a compound tentatively identified as 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate, the product of beta elimination of the 2,3-enediol(ate) intermediate of the catalytic reaction, appeared among the reaction products in progressively increasing amounts . In the case of the valine substitution, it comprised 13% of the ribulose bisphosphate consumed . The mutant enzymes also partitioned more of their reaction flux to pentulose bisphosphate isomers of ribulose bisphosphate . By contrast, the diversion of carboxylated product to pyruvate, as a result of beta elimination of the three-carbon aci-carbanion intermediate of the carboxylation reaction, was ameliorated by the replacements, the valine mutant showing a 5-fold improvement in this parameter . These observations focus attention on a geometric conflict which exists between the requirements for stabilization of the 5-carbon enediol(ate) and 3-carbon aci-carbanion intermediates . This conflict must be resolved by a change in the angle of the C-1/bridge oxygen bond during each catalytic cycle . The network of hydrogen bonds involving the side chain of threonine 65 must play a crucial role in facilitating reaction of the enediol(ate) with the gaseous substrate and in shepherding this subsequent movement.

J Biol Chem, 1994 Mar 18, 269(11), 8029 - 35
Tn10/IS10 transposase purification, activation, and in vitro reaction; Chalmers RM et al.; We describe a method for the purification of Tn10/IS10 transposase that relies on the aggregation of the protein after overexpression in Escherichia coli . Aggregated transposase was solubilized before the final purification step, a gel-filtration column, using a combination of salt and detergent . This procedure is the first reported for the preparation of concentrated and active transposase from any IS element . The yield is 11 mg of purified protein at a concentration of 1 mg/ml from 2.5 g of cells . The procedure can be scaled up with ease . We also describe a treatment that activates transposase in either a crude or purified state . This involves dilution into a solution of salt plus organic solvent . In transposition reactions using supercoiled substrate plasmid, the activity was directly proportional to the amount of transposase added over a wide range of transposase/DNA ratios (0.2-2.0 molecules/DNA substrate molecule) . In this range 8 transposase molecules were added per transposition event . Maximum conversion of substrate to product (40%) was with 18 transposase molecules/transposition event . At higher levels of transposase with a constant amount of substrate, activity was reduced but could be restored by addition of nonspecific DNA . Both the specific activity of transposase and the type of products generated can be altered by changing in vitro assay conditions . The effects of salts, solvents, and pH value on the reaction are described.

J Biol Chem, 1994 Mar 18, 269(11), 7982 - 8
Lactate monooxygenase . I . Expression of the mycobacterial gene in Escherichia coli and site-directed mutagenesis of lysine 266; Muh U et al.; Lactate monooxygenase utilizes oxygen in the conversion of L-lactate to acetate, CO2, and water . The gene for the enzyme from Mycobacterium smegmatis had been cloned into Escherichia coli (Giegel, D . A., Williams, C . H., Jr., and Massey, V . (1990) J . Biol . Chem . 265, 6626-6632) and the derived amino acid sequence compared to glycolate oxidase and flavocytochrome b2, enzymes of known three-dimensional structure (Lindqvist, Y., and Branden, C . I . (1989) J . Biol . Chem . 264, 3624-3628; Xia, Z . X., and Mathews, S . F . (1990) J . Mol . Biol . 212, 837-863) . There is strong homology, especially around residues in the active site . The mechanism proposed for lactate monooxygenase involves an intermediate having a negative charge at the N(1)-position of the FMN . Based on the homology, lysine 266 is the residue suggested to neutralize that charge . Wild type enzyme and several forms of the enzyme altered at active site residues by site-directed mutagenesis have been expressed in E . coli and purification procedures developed . The properties determined for the recombinant wild type enzyme were, in every case, the same as those previously determined for the enzyme isolated from M . smegmatis . Mutation of lysine 266 to a methionine created K266M . The semiquinone showed spectral features different from those found in the wild type enzyme and was no longer thermodynamically stable . This indicates a redox potential for the enzyme-bound semiquinone/reduced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple . The two-electron redox potential was determined to be -180 mV at 25 degrees C, pH 7.0 . In wild type enzyme, attack of the flavin ring by sulfite creates a negative charge at the FMN N(1)-position . In K266M, the stabilization of the sulfite adduct was 17,000-fold weaker (Kd approximately 10(-3) M) than in the wild type enzyme, with a rate of association that is lowered by 10,000-fold (kon = 1.2 M-1 s-1) . The rate of reduction with L-lactate is significantly decreased in K266M . Unexpectedly, binding of substrate and inhibitors is significantly weaker in K266M than in the wild type enzyme . In all properties involving a negative charge at position N(1) of the FMN, K266M is distinctly different from wild type enzyme . This makes it quite likely that lysine 266 serves the postulated role of interacting with this negative charge.

J Biol Chem, 1994 Mar 18, 269(11), 7970 - 5
A proteolytic fragment of Trypanosoma cruzi trans-sialidase lacking the carboxyl-terminal domain is active, monomeric, and generates antibodies that inhibit enzymatic activity; Schenkman S et al.; trans-Sialidase isolated from trypomastigote forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is multimeric and heterogeneous in size . We show here that limited proteolysis of tans-sialidase with papain yields a single monomeric polypeptide chain of 70 kDa that conserves full enzymatic activity on soluble and membrane-bound substrates . The papain fragment lacks most of the 12-amino acid repeats of the carboxyl-terminal domain that comprises about 50% of the native trans-sialidase . When injected into rabbits, the papain-generated fragment induces antibodies that inhibit trans-sialidase activity and trypomastigote sialylation . The repeats are also not required for the stability of the enzyme or for the correct folding during the biosynthesis in Escherichia coli, but seem essential for trans-sialidase oligomerization . We conclude that trans-sialidase is composed of two structurally and functionally independent domains.

J Biol Chem, 1994 Mar 18, 269(11), 7901 - 7
Expression of mammalian S-adenosylmethionine decarboxylase in Escherichia coli . Determination of sites for putrescine activation of activity and processing; Stanley BA et al.; Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature enzyme activity . To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis . Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B . A., and Pegg, A . E . (1991) J . Biol . Chem . 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine . Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur . Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing . The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography . Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease . Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q . It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity.

J Biol Chem, 1994 Mar 18, 269(11), 7869 - 73
Differential estrogenic regulation of small M(r) heat shock protein expression in osteoblasts; Cooper LF et al.; Polymerase chain reaction amplification and sequencing of heat shock protein (HSP) 27-related transcripts present in cDNAs generated from heat-shocked osteoblast RNA revealed the expression of three related open reading frames that encode proteins of 208, 197, or 175 amino acids . Their expression as recombinant proteins in Escherichia coli demonstrated that they encode 27,000, 25,000, or 22,000 M(r) proteins . Northern blot analysis of heat shock protein 27-related transcript expression revealed that, while HSP 27 expression (850-nucleotide transcript) was induced by heat shock alone, the expression of a smaller transcript was facilitated by estrogen treatment prior to heat shock . This corresponded with the co-induction of 18,000 and 22,000 M(r) proteins by estrogen-pretreated, heat-shocked osteoblasts as revealed by fluorography . The identification of these multiple small M(r) heat shock-induced proteins demonstrated that the stress response of mammalian cells is composed of multiple, related proteins whose expression is differentially regulated . The importance of estrogen-regulation of the small M(r) heat shock protein component of the stress response may be of particular significance to osteoblast physiology.

J Biol Chem, 1994 Mar 18, 269(11), 7843 - 6
The secA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane; Knott TG et al.; The presence of secA and secY gene homologues in the plastid genomes of red algae and cyanophytes has raised the possibility that the products of these genes are involved in protein translocation across the thylakoid membrane . Bacterial SecA proteins are effectively inhibited by azide, and we have tested the effects of this compound on the transport of lumenal proteins across the thylakoid membrane in pea chloroplasts . Recent studies have shown that lumenal proteins are transported by two different mechanisms, one dependent on the thylakoidal delta pH and the other requiring the presence of a stromal protein factor and ATP . In this report we show that azide inhibits the transport across the thylakoid membrane of the latter group of proteins, which includes plastocyanin and the lumenal 33-kDa protein of photosystem II; translocation of proteins by the delta pH-dependent pathway is unaffected . Following import into isolated chloroplasts in the presence of azide, a substantial proportion of plastocyanin and the 33-kDa protein is found as the stromal intermediate form; the proportion increases with lower ATP concentrations, suggesting that azide and ATP may compete for a single site . The presence of azide completely inhibits the import of the 33-kDa protein by isolated thylakoids, but import is restored if the azide is removed from the stromal extract or thylakoids, prior to the import incubation . The data thus indicate that azide reversibly inhibits the transport of a subset of proteins across the thylakoid membrane, consistent with the involvement of a SecA homolog . The results also indicate that azide is potentially a valuable tool for the future assignment of novel lumenal proteins to one of the thylakoidal protein transport mechanisms.

Science, 1994 Mar 18, 263(5153), 1625 - 9
Mutation of a mutL homolog in hereditary colon cancer; Papadopoulos N et al.; Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene . A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene . One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds . Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease . These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Nature, 1994 Mar 17, 368(6468), 261 - 5
Destabilization of the complete protein secondary structure on binding to the chaperone GroEL; Zahn R et al.; Protein folding in vivo is mediated by helper proteins, the molecular chaperones, of which Hsp60 and its Escherichia coli variant GroEL are some of the best characterized . GroEL is an oligomeric protein with 14 subunits each of M(r) 60K, which possesses weak, co-operative ATPase activity and high plasticity . GroEL seems to interact with non-native proteins, binding one or two molecules per 14-mer in a 'central cavity', but little is known about the conformational state of the bound polypeptides . Here we use nuclear magnetic resonance techniques to show that the interaction of the small protein cyclophilin with GroEL is reversible by temperature changes, and all amide protons in GroEL-bound cyclophilin are exchanged with the solvent, although this exchange does not occur in free cyclophilin . The complete secondary structure of cyclophilin must be disrupted when bound to GroEL.

Biochim Biophys Acta, 1994 Mar 16, 1205(1), 146 - 50
Identification of tyrosine as a functional residue in the active site of Escherichia coli dUTPase; Vertessy BG et al.; dUTP nucleotidohydrolase (dUTPase, EC 3.6.1.23) from E . coli contains a total of six tyrosine residues per trimer . About half of them were found to be susceptible to acetylation with N-acetylimidazole or to nitration with tetranitromethane with concomitant loss of activity . Deacetylation with N-hydroxylamine leads to full reactivation . Inhibitory products of dUTP hydrolysis, i.e., dUMP and inorganic pyrophosphate together with the cofactor Mg2+ protect significantly against inactivation and chemical modification . In the Cu(2+)-dUTPase complex, charge transfer from Cu2+ to the tyrosinate anion was perturbed by the presence of the substrate dUTP . These results, together with the occurrence of one tyrosine residue in a strictly conserved sequence motif suggest the critical importance of this residue for the function of the enzyme.

Biochim Biophys Acta, 1994 Mar 16, 1205(1), 113 - 22
Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues; Fukuchi T et al.; We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat . To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied . cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli . Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end . The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies . Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity . They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability . Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition . The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis . There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum.

FEMS Microbiol Lett, 1994 Mar 15, 117(1), 79 - 83
Phenylalanine- and tyrosine-dependent production of enterobactin in Escherichia coli; Foster MS et al.; Under low-iron conditions, Escherichia coli synthesizes the siderophore enterobactin . When compared to wild-type cells grown in iron sufficient medium, cells grown under iron limitation, in the absence of tyrosine and phenylalanine or the presence of both, increased catechol production (a measure of enterobactin and its degradation product 2,3-dihydroxybenzoic acid) 5- to 9-fold while cells supplemented with tyrosine alone produced a 10- to 20-fold increase . Mutations in fur, tyrA, pheA, or pheU generally resulted in increased enterobactin production, while a tyrR mutant was unaffected by combinations of tyrosine and phenylalanine.

J Immunol, 1994 Mar 15, 152(6), 3064 - 72
Mast cell and neutrophil expression of dog mast cell protease-3 . A novel tryptase-related serine protease; Yezzi MJ et al.; In previous work the primary structure of a previously unknown protease was deduced from the sequence of a dog mastocytoma cDNA . The predicted preproprotein shares some features with mast cell tryptases but is no more than 49% identical in sequence to known trypsin-like enzymes, including dog tryptase . This study explores the expression of this protein, termed dog mast cell protease-3 (dMCP-3) . A polyclonal Ab was raised to a peptide corresponding to residues 166-181 of the deduced sequence . Anti-dMCP-3(166-181) Ig recognizes dMCP-3 expressed as a CheY fusion protein in Escherichia coli and binds to a approximately 36-kDa protein in extracts of dog mastocytomas . The Ab does not recognize dog tryptase, dMCP-3s closest known relative in mastocytoma cells . When used with fluorescein-conjugated and alkaline phosphatase-conjugated secondary Abs, anti-dMCP-3(166-181) Ig yields punctate cytoplasmic staining in mastocytoma cells, suggesting localization to intracellular granules . Staining is greatly reduced by preincubation with synthetic dMCP-3 peptide, supporting the specificity of the Ab . Immunohistochemical staining of normal dog tissues reveals scattered dMCP-3 reactive cells in skin, intestine, trachea, and lung parenchyma . Double staining with Ab and methylene blue shows that anti-dMCP-3(166-188) Ig recognizes extravascular mononuclear tissue cells with metachromatic granules . In addition, cytoplasmic staining is seen in polymorphonuclear leukocytes within vessels in tissue sections and in leukocytes harvested from blood . Hybridization of dMCP-3 cDNA to dog skin RNA provides further evidence of dMCP-3 gene transcription in normal tissue . Thus, this study provides immunochemical evidence of dMCP-3 expression in dog mast cell tumors, normal tissue mast cells, and neutrophils.

Experientia, 1994 Mar 15, 50(3), 253 - 60
Some features of base pair mismatch repair and its role in the formation of genetic recombinants; Fox MS et al.; For the formation of recombinants involving closely linked markers, two distinct processes play a role . The recombinational interaction between homologous DNA molecules results in the presence of heteroduplex DNA joining the parental components of the recombinant . The presence of markers distinguishing the parents in the region of heteroduplex DNA can result in base pair mismatches . The post recombination repair of such mismatches can contribute to the separation of closely linked markers . The processes responsible for such repair also play roles in mutation avoidance . The specificities, functions and contribution to the formation of recombinants for closely linked markers of the processes in Escherichia coli are described.

Experientia, 1994 Mar 15, 50(3), 216 - 22
Processing of Holliday junctions by the Escherichia coli RuvA, RuvB, RuvC and RecG proteins; Muller B et al.; Recent work has led to significant advances in our understanding of the late steps of genetic recombination and the post-replicational repair of DNA . The RuvA and RuvB proteins have been shown to interact with recombination intermediates and catalyse the branch migration of Holliday junctions . Although both proteins are required for branch migration, each plays a defined role with RuvA acting as a specificity factor that directs RuvB (an ATPase) to the junction . The RuvB ATPase provides the motor for branch migration . The next step is catalysed by RuvC protein which recognises Holliday junctions and promotes their resolution by endonucleolytic cleavage . New data indicates an alternative pathway for Holliday junction processing . This pathway involves RecG, a branch migration protein which is functionally analogous to RuvAB, and a protein (activated by a rus mutation) which works with RecG to process intermediates independently of RuvA, RuvB and RuvC.

Experientia, 1994 Mar 15, 50(3), 204 - 15
In vitro reconstitution of homologous recombination reactions; Kowalczykowski SC; The proteins essential to homologous recombination in E . coli have been purified and their individual activities have been identified, permitting biochemical reconstitution of steps that comprise the cellular recombination process . This review focuses on the biochemical events responsible for the initiation and homologous pairing steps of genetic recombination . The properties of an in vitro recombination reaction that requires the concerted action of recA, recBCD, and SSB proteins and that is stimulated by the recombination hotspot, Chi(chi), are described . The recBCD enzyme serves as the initiator of this reaction; its DNA helicase activity produces single-stranded DNA that is used by the recA protein to promote homologous pairing and DNA strand invasion of supercoiled (recipient) DNA . The SSB protein acts to trap the single-stranded DNA produced by recBCD enzyme and to facilitate pairing by the recA protein . The chi regulatory sequence acts in cis by attenuating the nuclease, but not the helicase, activity of recBCD enzyme . This attenuation assures the preservation of ssDNA produced by the DNA helicase activity and is responsible for the simulation in vitro and, presumably, in vivo . The attenuation of nuclease activity by chi results in the loss or functional inactivation of the recD subunit.

Experientia, 1994 Mar 15, 50(3), 192 - 203
Structure and function of RecA-DNA complexes; Stasiak A et al.; While the E . coli RecA protein has been the most intensively studied enzyme of homologous recombination, the unusual RecA-DNA filament has stood alone until very recently . It now appears that this protein is part of a universal family that spans all of biology, and the filament that is formed by the protein on DNA is a universal structure . With RecA's role in recombination given new and greatly increased significance, we focus in this review on the energetics of the RecA-mediated strand exchange and the relation between the energetics and recombination spanning heterologous inserts.

Eur J Biochem, 1994 Mar 15, 220(3), 739 - 44
Escherichia coli elongation-factor-Tu mutants with decreased affinity for aminoacyl-tRNA; Andersen C et al.; The two evolutionary well-conserved histidine residues, His66 and His118, of Escherichia coli elongation factor Tu have been subjected to mutational analysis . The two histidines have each been replaced by alanines, denoted H66A and H118A, respectively . His118 has also been substituted by glutamate, H118E . The three mutants have been characterized with respect to thermostability, GTPase activity and affinity for aminoacylated tRNA . Most conspicuously, the tRNA affinity is reduced or almost abolished . k-1 for dissociation of the ternary complex increases by factors of 14, 40 and 48 for H66A, H118A and H118E, respectively, when compared to the wild type . The half-lives for the non-enzymic deacylation of aminoacylated tRNA in the ternary complex are 391, 107, 69, 54 and 61 min for wild type, H66A, H118A, H118E and free aminoacylated tRNA, respectively . The Kd is about 20-times higher for H66A compared to wild type . Our results strongly suggest that His66 and His118 play major roles in stabilization of the ternary complex.

Eur J Biochem, 1994 Mar 15, 220(3), 693 - 701
Isolation and expression of a gene specifying a cdc2-like protein kinase from the human malaria parasite Plasmodium falciparum; Ross-Macdonald PB et al.; A partially redundant oligonucleotide based on conserved protein sequences of cdk and cdc2-like proteins was used to isolate from genomic libraries of Plasmodium falciparum fragments of chromosome XIII carrying a 288-residue open-reading frame encoding a protein kinase sharing 57-58% identity with yeast p34cdc2 . Based on sequence data, base composition and the striking similarity with other cdk and related proteins, four intervening sequences were identified . Their removal in vitro allowed expression of the gene, designated PfPK5, in Escherichia coli, the resulting product having kinase activity against casein and histone H1 . Western blotting using a polyclonal antibody raised against the expressed protein showed that the kinase was located in the parasite's cytosol and was present in approximately constant amounts throughout the intra-erythrocytic asexual reproductive stage of the life cycle . The PSTAIRE region of the PfPK5 protein differs at three sites from that of p34cdc2, and the gene failed to complement cdc2/28 yeast mutants . However, Western blotting showed that the gene was not expressed in yeast, so this does not eliminate the possibility that it is the malarial version of cdc2.

EMBO J, 1994 Mar 15, 13(6), 1310 - 7
Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway; Michl D et al.; The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen-targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin . This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase . The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids . Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates . The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism . In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light-harvesting chlorophyll-binding protein of photosystem II . The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH . This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen-evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1994 Mar 15, 13(6), 1263 - 9
The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity; Bode W et al.; Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling . They have been implicated in various disease processes including tumour invasion and joint destruction . These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition . Human neutrophil collagenase (PMNL-CL, MMP-8) represents one of the two 'interstitial' collagenases that cleave triple helical collagens types I, II and III . Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non-covalent complex with the inhibitor Pro-Leu-Gly-hydroxylamine . The 2.0 A crystal structure reveals a spherical molecule with a shallow active-site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, composed of a five-stranded beta-sheet, two alpha-helices, and bridging loops . The inhibitor mimics the unprimed (P1-P3) residues of a substrate; primed (P1'-P3') peptide substrate residues should bind in an extended conformation, with the bulky P1' side-chain fitting into the deep hydrophobic S1' subsite . Modelling experiments with collagen show that the scissile strand of triple-helical collagen must be freed to fit the subsites . The catalytic zinc ion is situated at the bottom of the active-site cleft and is penta-coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor . In addition to the catalytic zinc, the catalytic domain harbours a second, non-exchangeable zinc ion and two calcium ions, which are packed against the top of the beta-sheet and presumably function to stabilize the catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 748 - 54
A novel human homologue of a dead-box RNA helicase family; Kitajima Y et al.; Putative cDNA clones for a nuclear antigen that cross-reacts with anti-human aldolase A monoclonal antibody MAb1A2 were isolated from the HeLa lambda gt11 cDNA library and a candidate clone (clone 3) was analyzed . The cDNA has an open reading frame (ORF) of 1,317 bp encoding a novel RNA helicase belonging to the DEAD RNA helicase family . The ORF also contains a nuclear targeting signal and the epitope for MAb1A2 . The putative RNA helicase has sequence similarity to Escherichia coli RNA helicase DEAD, mouse translation factor eIF-4A, and human p68 and p54.

Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 577 - 81
Site-directed mutagenesis of two highly conserved residues near the active site of phosphofructo-1-kinase; Zheng RL et al.; Mutations of either of two highly conserved residues near the active site of Escherichia coli phosphofructokinase, Ile-126 or Asn-128, produce no changes in the Km for ATP, relatively small changes in kcat, and a large increase in the Km for fructose 6-P, despite the fact that these residues are not directly involved in substrate binding . A computer graphics analysis of the three-dimensional structure suggests that the mutations effect the orientation of Arg-252, a residue important both for fructose 6-P binding and for catalysis.

Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 1041 - 8
Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro; Engel M et al.; We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2) . The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia cells were used . The homologous NDPK's from Yeast and E . coli were also substrates for CK-2 in vitro, but not Drosophila NDPK . Phosphorylation of all NDPK types by the CK-2 holoenzyme was entirely polyamine-dependent . The CK-2 phosphorylation site in human NDPK A, that was about 2.5 times stronger phosphorylated than was the B isotype, was tentatively assigned to Ser-122 . The location of the corresponding residue in the 3D-structure of the 80% homologous Drosophila NDPK suggests that its phosphorylation may directly influence substrate binding and/or catalysis.

Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2265 - 9
Cloning and expression of a distinctive class of self-incompatibility (S) gene from Papaver rhoeas L; Foote HC et al.; We present the identification, cloning, and characterization of a self-incompatibility (S) gene from Papaver rhoeas that has no significant homology to any previously reported gene sequences, including S genes from other species . This result suggests that a different self-incompatibility mechanism may be operating in this species and has important implications for the evolutionary relationships between the S genes . The S1 cDNA was cloned by using an oligonucleotide based upon N-terminal amino acid sequence data from stigmatic proteins that show complete linkage with the S1 gene . The single-copy gene has been expressed in Escherichia coli to test biological activity . Although the recombinant S1 protein (S1e) is not processed in the same way as the protein produced in the plant, it exhibits, in vitro, the specific pollen inhibitory activity expected of an S gene product; pollen carrying the S1 allele is inhibited, whereas pollen not carrying S1 is not inhibited . These results provide definitive demonstration that the product of a cloned S gene has S-specific pollen inhibitory activity.

Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2240 - 4
Cloning of a 3-methyladenine-DNA glycosylase from Arabidopsis thaliana; Santerre A et al.; We have isolated an Arabidopsis thaliana cDNA that complements the methyl methanesulfonate-sensitive phenotype of an Escherichia coli double mutant deficient in 3-methyladenine glycosylases (DNA-3-methyladenine glycosidases I and II, EC 3.2.2.20 and 3.2.2.21, respectively, encoded by tag and alkA) . Expression of the Arabidopsis cDNA enhances the methyl methanesulfonate resistance of the E . coli double mutant by nearly four orders of magnitude . The cDNA corresponds to a single-copy, nuclearly encoded sequence which specifies a predicted 28.1-kDa protein with a charge of +8 at pH 7.0 . Enzymatic analysis of extracts prepared from the transformed mutants indicates that the cDNA encodes a 3-methyladenine glycosylase . The predicted amino acid sequence of the Arabidopsis glycosylase has significant homology to other eukaryotic 3-methyladenine glycosylases.

Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2191 - 5
Role of an Escherichia coli stress-response operon in stationary-phase survival; Weiner L et al.; The phage shock protein operon (pspABCE) of Escherichia coli is strongly expressed in response to stressful environmental conditions, such as heat shock, ethanol treatment, osmotic shock, and filamentous phage infection . We show that bacteria lacking the pspABC genes exhibit a substantial decrease in the ability to survive prolonged incubation in stationary phase under alkaline conditions (pH 9) . The psp mutant bacteria grow approximately as well as wild-type strains in the alkaline medium, and stationary-phase survival of the psp mutants improves substantially at pH values closer to the optimal growth range (pH 6-8) . In late stationary-phase (1- to 2-day-old) cells, the operon can be strongly induced under certain conditions, and PspA can become one of the most highly expressed bacterial proteins . The combination of stationary-phase starvation and alkaline pH is likely to place a severe strain on the maintenance of endogenous energy sources, and, consistent with these effects, we find that psp expression is also induced by uncouplers of oxidative phosphorylation and other agents that interfere with energy production . The death rate of psp mutants in stationary phase is accelerated by the presence of wild-type bacteria in the same culture, suggesting that the psp operon may play a significant role in enabling E . coli to compete for survival under nutrient- or energy-limited conditions.

Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2146 - 50
Folded proteins occur frequently in libraries of random amino acid sequences; Davidson AR et al.; A library of synthetic genes encoding 80- to 100-residue proteins composed mainly of random combinations of glutamine (Q), leucine (L), and arginine (R) has been expressed in Escherichia coli . These genes also encode an epitope tag and six carboxyl-terminal histidines . Screening of this library by immunoblotting showed that 5% of these QLR proteins are expressed at readily detectable levels . Three well-expressed QLR proteins were purified and characterized . Each of these proteins has significant alpha-helical content, is largely resistant to degradation by Pronase, and has a distinct oligomeric structure . In addition, one protein unfolds in a highly cooperative manner . These properties of the QLR proteins demonstrate that they possess folded structures with some native-like properties . The QLR proteins differ from most natural proteins, however, in being remarkably resistant to denaturant-induced and thermal-induced unfolding and in being relatively insoluble in the absence of denaturants.

Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2120 - 4
RNA polymerase binding using a strongly acidic hydrophobic-repeat region of sigma 54; Tintut Y et al.; sigma 54 is a rare bacterial protein that substitutes for sigma 70 in the case of Escherichia coli genes transcribed by certain activators with enhancer protein-like properties . It contains a strongly acidic region of previously unknown function . Gel mobility-shift assays using sigma 54 deletion mutants show that this region is essential for sigma 54 to bind the core RNA polymerase and recruit it to the promoter . Multiple-point mutational analysis shows that the acidic amino acids and overlapping periodic hydrophobic amino acids are necessary for this binding . Related sequences are not found within the core binding determinant of sigma 70, which binds the same core RNA polymerase . This comparison suggests that the core RNA polymerase interacts differently with the two sigma factors, likely contributing to the critical differences in transcription mechanism in the two cases.

Biochemistry, 1994 Mar 15, 33(10), 3063 - 70
Arrangement of messenger RNA on Escherichia coli ribosomes with respect to 10 16S rRNA cross-linking sites; Bhangu R et al.; The arrangement of the mRNA on the Escherichia coli ribosome with respect to ribosomal RNA sites has been investigated by photochemical cross-linking experiments . mRNA analogues 51-54 nucleotides in length contained a Shine-Dalgarno sequence, a single codon for tRNA(Gly), and 4-thiouridine (s4U) in the 5' third of the mRNA (-20 to -12), in the middle third of the mRNA (-3 to +6), or in the 3' third of the mRNA (+20 to +26), where the position numbers are counted from the first nucleotide of the codon . Complexes were formed with these mRNAs and 70S ribosomes in the absence or presence of tRNA(Gly) and were irradiated . The extent of cross-linking and the identity of cross-linked rRNA sites were determined on agarose gels and by primer extension . 16S rRNA nucleotides A412, A532, G693 (weakly), U723, and U1381 (weakly) cross-linked with s4U in the 3' third; A532, G693, U723, A1167 (weakly), U1381, G818 (weakly), and A845 cross-linked with s4U in the middle; A532, G693, U723, A1167, G818 (weakly), and A845 cross-linked with s4U in the 5' third . All of these cross-links occur with tRNA independence . Cross-links at C1395 and A1196 occur for all three mRNAs with tRNA dependence . The pattern of these sites provides information about the order of the rRNA sites along the mRNA track, and they also point out the apparent overlapping neighborhoods for the mRNA track . Models for the track of the mRNA on the 30S subunit are considered to explain this pattern of interactions.

Biochemistry, 1994 Mar 15, 33(10), 2951 - 60
Unfolding studies of the protease domain of urokinase-type plasminogen activator: the existence of partly folded states and stable subdomains; Nowak UK et al.; The domain structure and the stability against thermal and chemical denaturation of urokinase-type plasminogen activator (u-PA) have been investigated by NMR spectroscopy and differential scanning calorimetry (DSC) . At least five structurally autonomous regions of this three-domain protein have been found to exist . Two of these are the EGF-like and the kringle domains; the others are all within the third domain, which is a serine protease . The latter undergoes three unfolding transitions in its enzymatically active form . Reaction with a specific affinity label (L-Glu-L-Gly-L-Arg-chloromethyl ketone) to produce an inactivated protein results in a stabilization of the structure involved in two of these transitions, and an increase in cooperativity to give a domain which unfolds in two, not three, distinct steps . These are attributed to the denaturation of the two major subdomains of the protease structure . One of the subdomains has exceptional stability, being unfolded only under extreme conditions such as 75 degrees C at pH 2.5 or 4 M GuDCl at pH 4.5 and 29 degrees C . This region has been identified by isolation and characterization of a fragment (residues Ile-159 to Thr-277) obtained by limited proteolysis with thermolysin under conditions where the protease domain was partly unfolded . The NMR data are consistent with this stable region being at the N-terminus of the protein and indicate that its structure and stability are similar to those of the corresponding region of the native protein . These results support the idea that the u-PA protease domain has structural resemblance to the digestive serine proteases, but that stabilizing interactions within the structure can differ significantly between a group of homologous proteins.

Biochemistry, 1994 Mar 15, 33(10), 2945 - 50
A molecular wedge for triggering the amidotransferase activity of carbamoyl phosphate synthetase; Mareya SM et al.; The reactive cysteine residue within the small subunit of Escherichia coli carbamoyl phosphate synthetase has been identified using the technique of site-directed mutagenesis . Three cysteine residues have previously been found to react with N-ethylmaleimide (NEM) under controlled reaction conditions . Two of these cysteine residues are found on the large subunit, while the third cysteine is located on the small subunit . In the present investigation, Cys-248 of the small subunit has been identified as the residue that reacts with NEM in the presence of MgATP and bicarbonate . Three cysteine residues of the small subunit at positions 131, 214, and 248 were individually mutated to serine residues . These site-specific changes, in addition to N-ethylmaleimide-labeling studies, demonstrated that Cys-248 is the amino acid that reacts with N-ethylmaleimide . Substitution of Cys-248 of the small subunit with larger residues (Asp, Phe, Arg, and Trp) was conducted in order to more closely mimic the observed properties of the NEM-labeled enzyme . The partial glutaminase activity of the C248D mutant increased 40-fold relative to the wild-type enzyme, while the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished . Similar, but less dramatic, effects were observed for the other mutants, C248S, C248R, C248F, and C248W . There was good correlation between the extent of enhancement of the partial glutaminase activity and an uncoupling of the phosphorylation reactions that occur on the large subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Mar 15, 33(10), 2873 - 9
Cellular retinoid-binding proteins: limited proteolysis reveals a conformational change upon ligand binding; Jamison RS et al.; Intracellular retinoid-binding proteins are small, tightly folded, compact proteins, which appear to be involved in the delivery of retinoids to microsomal metabolic enzymes, among other potential roles . Recently, it has been demonstrated that two of these binding proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein type II {CRBP(II)}, interact with the same microsomal enzyme but in different manners, depending on the absence or presence of ligand {Herr, F.M., & Ong, D.E . (1992) Biochemistry 31, 6748-6755} . The structural components of the binding proteins responsible for these differential interactions are presently unknown . In addition, it is not clear how the ligand is able to gain entry into the solvent-inaccessible interior binding cavity . Limited proteolysis of the apo and holo forms of CRBP and CRBP(II) was used to probe the conformational differences between the different states of these two proteins in solution . It was found that the apo forms of both proteins were significantly more susceptible to proteolysis, and probably adopted a more open conformation, than the holo forms . The initial cleavage site of endoproteinase Arg-C in the apo forms occurred at a conserved arginine residue near a possible site of ligand entry . Similar results were obtained by limited proteolysis of cellular retinoic acid-binding protein and heart fatty acid-binding protein, indicating that a common ligand-induced conformational change may occur for other members of this family of intracellular binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Mar 15, 33(10), 2852 - 8
Determination of the secondary structure and folding topology of an RNA binding domain of mammalian hnRNP A1 protein using three-dimensional heteronuclear magnetic resonance spectroscopy; Garrett DS et al.; The secondary structure and folding topology of the first RNA binding domain of the human hnRNP A1 protein was determined by multidimensional heteronuclear NMR spectroscopy . The 92 amino acid long domain exhibits a beta alpha beta beta alpha beta folding pattern, arranged in a four-stranded antiparallel beta-sheet flanked by two alpha-helices, which is very similar to that found for other members of this family . Regions of marked variation between the structurally characterized RNA binding proteins of this class to date are mainly localized in the loops connecting the secondary structure elements.

Biochemistry, 1994 Mar 15, 33(10), 2838 - 42
1H NMR studies of mouse ribonucleotide reductase: the R2 protein carboxyl-terminal tail, essential for subunit interaction, is highly flexible but becomes rigid in the presence of protein R1; Lycksell PO et al.; Mouse ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone . It has earlier been shown that the carboxyl-terminal part of the R2 protein is essential for subunit association to form the active enzyme complex . We now demonstrate that protein R2 gives rise to a number of sharp 1H NMR resonances, significantly narrower than the major part of the resonances . This line narrowing of certain resonances indicates segmental mobility in the molecule . In two-dimensional 1H TOCSY spectra of protein R2, cross-peak patterns from about 25 amino acid residues are visible . Most of these were assigned to the carboxyl-terminal part of the protein by comparisons with cross-peak patterns of oligopeptides corresponding to the carboxyl terminus of mouse R2 and to the patterns of a seven amino acid residue carboxyl-terminal truncated form of protein R2 . These results and the magnitude of the chemical shifts of the assigned residues demonstrate that the carboxyl-terminal part of mouse R2 protein is highly mobile compared to the rest of the protein and essentially unstructured . When protein R1 is added to a solution of protein R2, the sharp resonances are broadened, suggesting that the mobility of the carboxyl-terminal tail of protein R2 is reduced . The possibility of making direct observations of subunit interaction in native and mutagenized R1/R2 proteins should allow discrimination between effects of amino acid replacements on the catalytic mechanism and effects on subunit interaction.

Biochemistry, 1994 Mar 15, 33(10), 2773 - 81
Solubilizing buried domains of proteins: a self-assembling interface domain from glutathione reductase; Leistler B et al.; In the dimeric glutathione reductase (GR) from Escherichia coli, the interface domain is largely surrounded by the other three domains in each subunit of the protein . Subgenes encoding three forms of the interface domain have been expressed in E . coli and the products purified from inclusion bodies: INT is the excised interface domain, as it is found in native GR; INTN and INTFN are variants carrying exchanges of surface residues in what would have been hydrophobic contact regions with other neighboring domains . The isolated INT domain was found to be a soluble and folded protein, but it was isolated as a mixture of the dimer and at least two species of higher molecular weight . The latter were believed to arise by further association of the dimer via the newly exposed and unsatisfied hydrophobic contact regions . In the variant INTN, three hydrophobic residues normally involved in the contact with the NADPH-binding domain in GR were replaced . This partly suppressed the further aggregation of the dimers . However, continued aggregation at high protein concentrations suggested that at least one further site of unwanted aggregation was still present . After four additional amino acid replacements in the region normally in contact with the FAD-binding domain, the resulting variant INTFN exhibited no unspecific aggregation, even at concentrations as high as 3.2 mg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Mar 15, 33(10), 2761 - 72
Three-dimensional solution structure of Escherichia coli periplasmic cyclophilin; Clubb RT et al.; The solution structure of the periplasmic cyclophilin type cis-trans peptidyl-prolyl isomerase from Escherichia coli (167 residues, MW > 18.200) has been determined using multidimensional heteronuclear NMR spectroscopy and distance geometry calculations . The structure determination is based on a total of 1720 NMR-derived restraints (1566 distance and 101 phi and 53 chi 1 torsion angle restraints) . Twelve distance geometry structures were calculated, and the average root-mean-square (rms) deviation about the mean backbone coordinate positions is 0.84 +/- 0.18 A for the backbone atoms of residues 5-165 of the ensemble . The three-dimensional structure of E . coli cyclophilin consists of an eight-stranded antiparallel beta-sheet barrel capped by alpha-helices . The average coordinates of the backbone atoms of the core residues of E . coli cyclophilin have an rms deviation of 1.44 A, with conserved regions in the crystal structure of unligated human T cell cyclophilin {Ke, H . (1992) J . Mol . Biol . 228, 539-550} . Four regions proximal to the active site differ substantially and may determine protein substrate specificity, sensitivity to cyclosporin A, and the composite drug:protein surface required to inhibit calcineurin . A residue essential for isomerase activity in human T cell cyclophilin (His126) is replaced by Tyr122 in E . coli cyclophilin without affecting enzymatic activity.

Biochemistry, 1994 Mar 15, 33(10), 2757 - 60
Shift in pH-rate profile and enhanced discrimination between dicarboxylic and aromatic substrates in mitochondrial aspartate aminotransferase Y70H; Pan P et al.; Tyr70 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by oligonucleotide-directed mutagenesis . Aspartate aminotransferase Y70H retained at pH 7.5 13% of the activity toward dicarboxylic amino acids, whereas the activity toward aromatic amino acids was only 0.6% of that of the wild-type enzyme, corresponding to a 22-fold increase in the ratio of the activities toward these two types of substrates . In comparison to that of the wild-type enzyme, the low-pH limb of the pH-activity profile of the mutant enzyme was shifted to higher pH values, very likely reflecting the titration curve of the newly introduced histidine residue with a pKa' of 6.3 . Apparently, a positively charged residue at position 70 abolishes enzymic activity . The spectrophotometrically determined pKa' value of the internal aldimine formed between pyridoxal 5'-phosphate and Lys258 in the mutant enzyme was 6.0, similar to that in the wild-type enzyme . The rate constant of the dissociation of pyridoxamine 5'-phosphate from the mutant enzyme was increased only 3 times over that of the wild-type enzyme, in contrast to the 80-fold increase in Escherichia coli aspartate aminotransferase Y70F {Toney, M . D., & Kirsch, J . F . (1987) J . Biol . Chem . 262, 12403-12405}, suggesting that His70 can replace Tyr70 in forming a hydrogen bond to the coenzyme.

Structure, 1994 Mar 15, 2(3), 151 - 8
AB5 ADP-ribosylating toxins: comparative anatomy and physiology; Burnette WN; The crystal structures recently determined for pertussis toxin, cholera toxin, and E . coli heat-labile toxins promise advances in rational vaccine design and improved understanding of G protein-mediated signal transduction.

J Physiol, 1994 Mar 15, 475(3), 531 - 7
Enterotoxin Escherichia coli STa activates a nitric oxide-dependent myenteric plexus secretory reflex in the rat ileum; Rolfe V et al.; 1 . Mucosally added enterotoxin Escherichia coli STa increased the electrogenic Cl- secretion measured as the short-circuit current (Isc) across isolated muscle-stripped and muscle-unstripped rat mid-ilea incubated in vitro . 2 . Pretreatment with serosal L-NAME (N omega-nitro-L-arginine methyl ester) or tetrodotoxin (TTX) significantly reduced the maximum Isc and the duration of action of STa in the unstripped but not stripped ilea . D-NAME (serosal), indomethacin or 5-hydroxy-tryptamine-desensitization was ineffective on STa-induced Isc in either stripped or unstripped ilea . 3 . Serosal capsaicin reduced the maximum Isc of STa and its duration of action in unstripped ilea . 4 . L-Arginine induced a significantly larger increase in the Isc across unstripped ilea than across stripped ilea; this could be significantly reduced by serosal L-NAME or TTX, although these were ineffective in stripped ilea . 5 . Pretreatment of anaesthetized rats with I.P . L-NAME suppressed the fluid secretion induced by luminal STa in ilea in vivo but had no effect on that induced by luminal carbachol . 6 . Mucosal STa increased electrogenic Cl- secretion across intact rat ileum in vitro by activating a capsaicin-sensitive, nitric oxide-dependent myenteric plexus-mediated secretory reflex . The suppression by L-NAME of STa induced ileal fluid secretion in vivo probably involves the inhibition of this reflex.

J Biotechnol, 1994 Mar 15, 33(1), 43 - 53
Application of an alkaline phosphatase fusion protein system suitable for efficient screening and production of Fab-enzyme conjugates in Escherichia coli; Weiss E et al.; We report a novel vector system suitable for the efficient preparation of alkaline phosphatase (PhoA)-labelled antibody Fab fragments in Escherichia coli . The previously described pGE20 vector used for the functional expression of truncated heavy (Fd) and light (L) chains of Fab into the bacterial culture medium was modified by inserting the E . coli PhoA coding region 3' to the Fd cloning sites . The secreted Fd-PhoA fusions and L proteins were found to be disulfide linked and Fab-PhoA complexes, prepared with IgG1 antibodies recognizing specifically human tumor necrosis factor alpha, were shown to be useful for the rapid detection of antigen . When an additional short peptide was interposed between the Fd and PhoA domains, nearly all of the recombinant Fab-PhoA conjugates present in the culture supernatant retained both antigen binding and enzymatic activity . A method for the detection and selection of bacterial colonies expressing bifunctional hybrid molecules of desired antigen specificity was also developed . Taken together, the systems described permit the generation and production of Fab-PhoA conjugates in E . coli, which can replace conventionally prepared PhoA-labelled antibodies in appropriate immunoassays.

Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 969 - 76
Sequence analysis and expression of phospholipase A2 from Taiwan cobra; Pan FM et al.; Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of venom glands in Taiwan cobras to facilitate the cloning and sequencing of phospholipase A2 (PLA2) gene . The PCR product was then subcloned into pUC18 vector and transformed in E . coli strain JM109 . Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method . Sequencing several clones containing about 0.5 kb DNA inserts constructed a complete and unambiguous full-length reading frame of 468 base pairs covering a precursor for phospholipase A2 with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid segment of signal peptide . The sequenced major PLA2 with pI 4.991 shows a high degree of sequence homology to those PLA2 of the same or closely-related genus . The deduced protein sequence allows us to correct and resolve some discrepancy between the sequences determined by conventional protein sequencing (Toxicon, 19, 141(1981)) and X-ray crystallography (Science, 250, 1560(1990)) . Expression of PLA2 in E . coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified PLA2 from the same cobra venom albeit with a much lower activity.

Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 504 - 10
Nitric oxide production depends on preceding tetrahydrobiopterin synthesis by endothelial cells: selective suppression of induced nitric oxide production by sepiapterin reductase inhibitors; Schoedon G et al.; Using murine vascular endothelial cells expressing both constitutive and inducible nitric oxide synthases (cNOS and iNOS), we explored the feasibility of suppressing cytokine-induced nitric oxide (NO) production without affecting constitutive NO production by inhibition of the tetrahydrobiopterin (BH4) biosynthesis . We show in this study that in endothelial cells cytokine/endotoxin-activated BH4 synthesis precedes the induction of NO generation . Using the sepiapterin reductase inhibitors phenprocoumon or dicumarol as BH4 synthesis inhibitors, we achieved a pronounced and selective suppression of induced NO production in cytokine-activated endothelial cells . Addition of exogenous BH4, but not sepiapterin, restored NO production in the presence of the inhibitors . Despite profound inhibition of the BH4 biosynthesis, constitutive NO synthesis was not affected, thereby demonstrating the selectivity and specificity of the inhibitors . Suppression of enhanced NO production by sepiapterin reductase inhibitors such as cumaroles could provide pharmacologic means for therapeutic interventions in NO-mediated pathophysiologic events.

Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2125 - 9
Ribosome binding of DNA analogs of tRNA requires base modifications and supports the "extended anticodon"; Dao V et al.; The efficiency of translation depends on correct tRNA-ribosome interactions . The ability of chemically synthesized yeast tRNA(Phe) anticodon domains to effectively inhibit the binding of native yeast tRNA(Phe) to poly(U)-programmed Escherichia coli 30S ribosomal subunits was dependent on a Mg(2+)-stabilized stem and an open anticodon loop, both facilitated by base modifications . Analysis of tRNA sequences has revealed that base modifications which negate canonical hydrogen bonding are found in 95% of those tRNA anticodon loop sequences with the potential to form two Watson-Crick base pairs across the loop . Therefore, we postulated that a stable anticodon stem and an open loop are prerequisites for ribosome binding . To test this hypothesis, DNA analogs of the yeast tRNA(Phe) anticodon domain were designed to have modification-induced, Mg(2+)-stabilized stems and open loops . The unmodified DNA analog neither bound to poly(U)-programmed 30S ribosomal subunits nor inhibited the binding of native tRNA(Phe) . However, specifically modified DNA analogs did bind to ribosomal subunits and effectively inhibited tRNA(Phe) from binding . Thus, modification-dependent Mg(2+)-stabilized anticodon domain structures with open loops have evolved as the preferred anticodon conformations for ribosome binding.

FEBS Lett, 1994 Mar 14, 341(1), 54 - 8
The thrombospondin-like chains of cartilage oligomeric matrix protein are assembled by a five-stranded alpha-helical bundle between residues 20 and 83; Efimov VP et al.; The N-terminal fragment of rat cartilage oligomeric matrix protein (COMP), comprising residues 20-83, was over-expressed in E . coli and purified under non-denaturing conditions . The fragment forms pentamers similar to the assembly domain of the native protein . Its five chains can be covalently linked in vitro by oxidation of cysteines 68 and 71 . The fragment adopts a predominantly alpha-helical structure as judged by circular dichroism spectroscopy . On the basis of these findings we propose the model of a five-stranded alpha-helical bundle for the assembly domain of COMP . The studied sequence is conserved in thrombospondins 3 and 4 thus raising the possibility that these proteins are also pentamers.

FEBS Lett, 1994 Mar 14, 341(1), 131 - 4
Expression and stability of recombinant RQ-mRNAs in cell-free translation systems; Ugarov VI et al.; Expression of dihydrofolate reductase (DHFR) and chloramphenicol acetyltransferase (CAT) mRNAs in cell-free Escherichia coli translation systems is greatly enhanced as a result of their insertion into RQ135 RNA, a naturally occurring satellite of phage Q beta . The enhancement is due to protection of the recombinant mRNAs against endogenous ribonucleases and to an increased initial rate of translation in the case of the RQ-CAT mRNA.

FEBS Lett, 1994 Mar 14, 341(1), 79 - 85
The crystal structure of human CskSH3: structural diversity near the RT-Src and n-Src loop; Borchert TV et al.; SH3 domains are modules occurring in diverse proteins, ranging from cytoskeletal proteins to signaling proteins, such as tyrosine kinases . The crystal structure of the SH3 domain of Csk (c-Src specific tyrosine kinase) has been refined at a resolution of 2.5 A, with an R-factor of 22.4% . The structure is very similar to the FynSH3 crystal structure . When comparing CskSH3 and FynSH3 it is seen that the structural and charge differences of the RT-Src loop and the n-Src loop, near the conserved Trp47, correlate with different binding properties of these SH3 domains . The structure comparison suggests that those glycines and acid residues which are very well conserved in the SH3 sequences are important for the stability of the SH3 fold.

Nucleic Acids Res, 1994 Mar 11, 22(5), 767 - 72
Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication; Tanaka K et al.; The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV) . If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins . Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above . These were also true for the RSF1010 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site) . Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation . We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication.

Nucleic Acids Res, 1994 Mar 11, 22(5), 711 - 21
The DNA binding domains of the varicella-zoster virus gene 62 and herpes simplex virus type 1 ICP4 transactivator proteins heterodimerize and bind to DNA; Tyler JK et al.; The product of varicella-zoster virus gene 62 (VZV 140k) is the functional counterpart of the major transcriptional regulatory protein of herpes simplex virus type 1 (HSV-1), ICP4 . We have found that the purified bacterially expressed DNA binding domain of VZV 140k (residues 417-647) is a stable dimer in solution . As demonstrated by the appearance of a novel protein--DNA complex of intermediate mobility in gel retardation assays, following in vitro co-translation of a pair of differently sized VZV 140k DNA binding domain peptides, the 140k DNA binding domain peptide binds to DNA as a dimer . In addition, the DNA binding domain peptide of HSV-1 ICP4 readily heterodimerizes with the VZV 140k peptide on co-translation, indicating that HSV-1 ICP4 and VZV 140k possess very similar dimerization interfaces . It appears that only one fully wild type subunit of the dimer is sufficient to mediate sequence specific DNA recognition in certain circumstances . Co-immunoprecipitation analysis of mutant DNA binding domain peptides, co-translated with an epitope-tagged ICP4 DNA binding domain, shows that the sequence requirements for dimerization are lower than those necessary for DNA binding.

Science, 1994 Mar 11, 263(5152), 1440 - 3
Coding of hemolysins within the ribosomal RNA repeat on a plasmid in Entamoeba histolytica; Jansson A et al.; The pathogenesis of amoebic dysentery is a result of cytolysis of the colonic mucosa by the parasitic protozoan Entamoeba histolytica . The cytolysis results in extensive local ulceration and allows the amoeba to penetrate and metastasize to distant sites . Factors involved in this process were defined with three clones that express hemolytic activities in Escherichia coli . These potential amoebic virulence determinants were also toxic to human colonic epithelial cells, the primary cellular targets in amoebal invasion of the large intestine . The coding sequences for the hemolysins were close to each other on a 2.6-kilobase segment of a 25-kilobase extrachromosomal DNA element . The structural genes for the hemolysins were within inverted repeats that encode ribosomal RNAs.

J Mol Biol, 1994 Mar 11, 236(5), 1299 - 309
Partitioning of plasmid R1 . The parA operon is autoregulated by ParR and its transcription is highly stimulated by a downstream activating element; Jensen RB et al.; The parA partitioning system of plasmid R1 mediates efficient stabilization of R1 and F-derived replicons . The parA system is encoded by a continuous DNA segment of approximately 1600 base-pairs and consists of three components . Two adjacent genes, parM and parR, coding for the trans-acting proteins ParM and ParR, and the cis-acting parC site . The centromere-like parC site is located upstream of parM and parR and contains the parA promoter . The parM and parR genes are co-transcribed as an operon from the parA promoter . The 5' end of the parA encoded transcript was mapped to the center of the parC region at +115 . The -10 and -35 core promoter sequences are flanked by the two sets of five direct repeats in parC (the ParR boxes) . The parA promoter was found to be negatively regulated by the parR gene product, whereas the parM gene product seemingly was not involved in the regulation . Surprisingly, a region downstream of the parA promoter enhanced transcription from the promoter many-fold (30 to 50-fold) . The parC site titrated the ParR protein, suggesting that the ParR protein interacts directly with the parC site . Using an engineered parA system we found that the parC site could be complemented in cis by the parM and parR genes . Furthermore, the proper function of the parC site was highly dependent on the expression level of ParM and ParR . The incompatibility associated with the parC site could not be suppressed by overexpression of the ParM and ParR proteins . Based on these results we suggest a novel partition model involving pairing of newly replicated plasmid molecules.

J Mol Biol, 1994 Mar 11, 236(5), 1289 - 98
Partitioning of plasmid R1 . Ten direct repeats flanking the parA promoter constitute a centromere-like partition site parC, that expresses incompatibility; Dam M et al.; The parA partitioning system of plasmid R1 consists of three different components: the cis-acting centromere-like parC site, and the two trans-acting proteins ParM and ParR . These three components are contained within a region of 1.6 kb . The parC site is located upstream of the two genes, parM and parR, which are expressed as an operon from the parA promoter . The parC site contains an array of ten 11 base-pair direct repeats, organized in two sets of five repeats flanking the parA core promoter sequences . Deletions and point mutations were introduced in the parA locus, resulting in partially stable and unstable plasmids . An analysis of these parA- plasmids showed that ParM and ParR are transacting . The 160 bp minimal parC region contained sufficient in cis information for efficient trans-complementation . Both proteins were required for maximal stabilization of a parC+ mini-R1 plasmid, although ParR alone, donated either in cis or in trans, yielded partial stabilization . Plasmids that overexpressed ParR caused destabilization of a co-resident parA+ plasmid, whereas overexpression of ParM had no such effect . The parC site exerted incompatibility (incA) at high but not at low copy number . Likewise, the entire parA system exerted incompatibility in a copy number-dependent fashion, and stronger than the incompatibility expressed by parC alone.

J Mol Biol, 1994 Mar 11, 236(5), 1283 - 8
Asymmetric arrangement of two alpha subunits within Escherichia coli RNA polymerase . Involvement of one alpha subunit in contact with cAMP receptor protein; Zou C et al.; Class I transcription factors of Escherichia coli have been proposed to make contact with contact site I on the alpha subunit, C-terminal region of RNA polymerase with the subunit composition of alpha 2 beta beta ' sigma . Both a reconstituted mutant holoenzyme containing two C-terminally truncated alpha-235 subunits and a hybrid enzyme containing one wild-type alpha (alpha-329) and one C-terminal truncated alpha (alpha-235) subunit were found to be as active in transcription from factor-independent simple promoters as the wild-type holoenzyme . The mutant enzyme was, however, inactive in cAMP receptor protein (CRP)-dependent transcription from lacP1 promoter, but the hybrid enzyme was about 50% as active in lacP1 transcription as the wild-type enzyme . The results indicate that only one specific alpha subunit makes contact with CRP.

J Biol Chem, 1994 Mar 11, 269(10), 7689 - 95
Identification of temperature-sensitive mutants of the human immunodeficiency virus type 1 protease through saturation mutagenesis . Amino acid side chain requirements for temperature sensitivity; Manchester M et al.; Human immunodeficiency virus type 1 encodes a protease whose activity is required for the production of infectious virus . An Escherichia coli expression and processing assay system was used to screen 285 protease mutants for temperature-sensitive activity . Fourteen protease mutants had a temperature-sensitive phenotype, and approximately half resulted from conservative amino acid substitutions . Of the 14 substitutions that conferred a temperature-sensitive phenotype, 11 substitutions occurred at 6 positions that represent 3 pairs of residues in the protease that contact each other in the three-dimensional structure . These mutants assist in pinpointing regions of the protease that are important for enzyme activity and stability.

J Biol Chem, 1994 Mar 11, 269(10), 7674 - 81
The sequence of a second member of the lima bean lectin gene family and the expression and characterization of recombinant lectin in Escherichia coli; Jordan ET et al.; The lima bean lectin recognizes terminal alpha-D-GalNAc groups and agglutinates human type A erythrocytes . We have cloned a portion of the gene encoding the alpha subunit of the lima bean lectin . The clone was obtained using the polymerase chain reaction and verified from a genomic clone encoding the mature protein of 253 amino acids . The deduced amino acid sequence has significant overall homology with other leguminous plant lectins and contains all of the known peptide sequences isolated from lima bean lectin (LBL) . Southern blot analysis reveals the presence of several genes which hybridize to the cloned gene and which we propose are genes included in the lima bean lectin gene family . We report here the sequence, expression, and characterization of LBL 2, the second member of this gene family . Milligram quantities of soluble active recombinant lima bean lectin (rLBL) were obtained from Escherichia coli, using the T7 RNA polymerase expression system . SDS-polyacrylamide gel electrophoresis and Western blot analysis indicate expression of one protein band of about 27 kDa in induced E . coli cells . This protein cross-reacts with polyclonal antibodies raised against seed lectin (sLBL) and gave a reaction of identity with seed lectin by Ouchterlony double diffusion, specifically agglutinates type A blood cells, and is specifically inhibited by D-GalNAc . The isoelectric point of rLBL is 5.86, whereas those of the seed lectin subunits were determined to be 5.86, 5.58, and 5.20 (previously designated alpha, beta, alpha', respectively) . rLBL binds to hydrophobic ligands independent of sugar binding, an observation similar to results obtained with sLBL . However, despite the similar activities described, several significant differences between recombinant and native lima bean lectin were found, including mobility on gel filtration, aggregation in solution, and its CD spectrum . These differences may be due to a number of factors, which will be discussed.

J Biol Chem, 1994 Mar 11, 269(10), 7532 - 7
Arginine 41 of subunit c of Escherichia coli H(+)-ATP synthase is essential in binding and coupling of F1 to F0; Fraga D et al.; Two substitutions were made for Arg41 in the polar loop of subunit c of the Escherichia coli F1F0 H(+)-transporting ATP synthase . The R41K and R41H mutants were initially studied by use of a plasmid carrying the complete c R41K or c R41H unc (F1F0) operon in a chromosomal strain deleted for the unc operon . The extent of F0 incorporation into membranes of these cells was quite variable, and the system was concluded to be unsuitable for biochemical characterization . Ultimately, the mutant genes were recombined into the chromosome using a novel method for the unc system . The biochemical phenotype of the chromosomally expressed mutants proved to be reproducible . The c R41H mutation causes a specific defect in assembly of F0, i.e . subunit a was not incorporated into the membrane despite near normal incorporation of subunits b and c . On the other hand, c R41K mutant F0 assembled normally in one of two background strains studied . (In the second genetic background, subunit a was inefficiently incorporated into the c R41K membrane.) In membranes prepared from a c R41K strain assembling a complete F0, R41K F0 was found to bind F1 with near normal affinity and to transport H+ at near normal rates . Although R41K F0 binds F1, F1-ATPase activity and H+ transport remained uncoupled . The uncoupling was indicated by a lack of ATP-driven H+ translocation and by the high proton permeability of membranes with F1 bound to F0 . The uncoupled phenotype of the R41K mutant closely resembles that previously reported for the c Q42E mutant.

J Biol Chem, 1994 Mar 11, 269(10), 7494 - 500
Analysis of the translational initiation region on the Euglena gracilis chloroplast ribulose-bisphosphate carboxylase/oxygenase (rbcL) messenger RNA; Koo JS et al.; The chloroplast mRNAs from Euglena gracilis fall into two classes . One group of mRNs from this organelle contains a Shine-Dalgarno sequence 5' to the start codon, while the other group of mRNAs does not have a conserved sequence signal in the 5'-untranslated region . To investigate the start signals for E . gracilis chloroplast mRNAs that do not carry a Shine-Dalgarno sequence, 30 S initiation complex formation has been studied using a series of transcripts carrying the wild-type translational start site of ribulose-bisphosphate carboxylase/oxygenase (rbcL) or mutated derivatives of this site . Mutation of the start codon of the rbcL gene indicates that the chloroplast 30 S subunit is recognizing only the correct AUG codon . The analysis of the messages from a series of deletion mutants shows that a minimum of delta 20 residues 5' to the AUG codon is required for activity . Maximal activity requires the full 55-base leader sequence . Surprisingly, a transcript carrying the inverse complement of 48 bases in the leader is delta 60% as active as the wild-type message in promoting initiation complex formation . Introduction of a Shine-Dalgarno sequence in the 5'-leader increases the activity of the mRNA only delta 1.4-2-fold . The presence of an oligodeoxynucleotide containing a strong Shine-Dalgarno sequence does not significantly inhibit the formation of initiation complexes at the rbcL start site . Similar results are obtained when initiation complexes are formed with initiation factors from either E . gracilis chloroplasts or Escherichia coli.

J Biol Chem, 1994 Mar 11, 269(10), 7483 - 8
Site-specific biotinylation of colicin Ia . A probe for protein conformation in the membrane; Qiu XQ et al.; Channel-forming colicins are Escherichia coli proteins that form voltage-dependent channels in lipid bilayer membranes and are lethal to sensitive strains of E . coli . Experiments with colicin E1 have led to a model of voltage dependence based on the insertion of alpha-helical segments of the protein into the membrane in response to cis-positive voltages . This model was tested on the partly homologous colicin Ia protein, which offers certain advantages over colicin E1 as a model channel, it is active at neutral pH and exhibits comparatively well-defined single channel conductance . We describe here the creation of a specific probe for locating a particular amino acid residue on one side or the other of a planar lipid bilayer membrane, by using the biotin-streptavidin system . Site-directed mutagenesis was used to change lysine 544 of colicin Ia to cysteine . This placed a unique cysteine at a site expected, by homology to colicin E1, to cross the membrane from the cis to the trans side in association with the opening of the channel . This unique cysteine was biotinylated chemically, so that it could serve as a target for streptavidin . Incubation of the biotinylated mutant colicin with streptavidin blocked its killing activity, in vivo; incubation of wild-type colicin, which lacks cysteine, with streptavidin, did not affect its activity . Channels formed by the biotinylated mutant protein in planar lipid bilayers were abolished by streptavidin added to the cis side of the membrane, if the channels were closed, but not if they were open . Trans streptavidin had no effect on either open or closed channels . Thus, when the channel is closed, residue 544 of colicin Ia is accessible to cis streptavidin in the closed state, but the opening of the channel eliminates this accessibility.

J Biol Chem, 1994 Mar 11, 269(10), 7297 - 303
Recombinant human retinoic acid receptor beta . Binding of synthetic retinoids and transcriptional activation; Lombardo A et al.; All-trans-retinoic acid mediates cell growth and differentiation by binding to and then activating nuclear retinoid receptor proteins that regulate gene transcription . Recombinant human retinoic acid receptor beta was cloned and expressed in Escherichia coli as a fusion protein rMBP-RAR beta with maltose-binding protein to facilitate purification . After isolation from bacterial lysates, rMBP-RAR beta was used for binding with selected retinoids . Scatchard analysis with {11,12-3H2}all-trans-retinoic acid gave a Kd of 0.34 nM . Competitive binding studies with a series of conformationally restricted aromatic retinoids indicated that the Ki values for binding to rMBP-RAR beta correlated with the logs of the EC50 values for gene transcriptional activation (p < or = 0.05) and with those for the relative activation compared to that of all-trans-retinoic acid (p < or = 0.01) . Inspection of binding-activation correlation diagrams indicates candidate structures for improved retinoid agonists or antagonists.

J Biol Chem, 1994 Mar 11, 269(10), 7192 - 200
Characterization of the mitochondrial binding and import properties of purified yeast F1-ATPase beta subunit precursor . Import requires external ATP; Hajek P et al.; To better understand the early events of the mitochondrial protein import process, we purified the precursor of the F1-ATPase beta subunit (pre-F1 beta) and examined its import into isolated mitochondria . Import of purified urea-denatured pre-F1 beta did not require cytosolic factors . However, the period of productive import was prolonged by the addition of reticulocyte lysate, suggesting that cytosolic factors such as molecular chaperones were acting to extend the period of import competence of pre-F1 beta . Purified pre-F1 beta bound extensively to both cardiolipin-containing liposomes and to intact mitochondria, indicating that a direct interaction between mitochondrial precursors and the mitochondrial outer membrane surface can occur . The ability to chase this surface-bound pre-F1 beta into mitochondria suggests that precursors bound to the mitochondrial surface can be maintained in an import competent conformation . Finally, our defined mitochondrial import system was used to characterize the ATP requirements of pre-F1 beta import in the absence of cytosol . We found a strong requirement for ATP on both sides of the mitochondrial inner membrane, suggesting that one or more previously undetected mitochondrial proteins outside the inner membrane utilize ATP to promote efficient pre-F1 beta import.

J Biol Chem, 1994 Mar 11, 269(10), 7066 - 9
Effect of DNA cytosine methylation upon deamination-induced mutagenesis in a natural target sequence in duplex DNA; Zhang X et al.; Are 5-methylcytosine residues in DNA hot spots for transition mutagenesis? Numerous studies identify 1) structural changes induced by DNA methylation, 2) high percentages of human mutations that result from GC to AT transition pathways, and 3) differences between G.C and G.mC base pairs in susceptibility to nonenzymatic deamination . However, investigations of chemical stability necessarily involve non-physiological conditions for chemical analysis of deamination . Here we describe an experiment that compares rates of deamination-induced mutagenesis between a G.C and G.mC base pair, when both are present in duplex DNA, incubated at 37 degrees C and pH 7.4, within identical sequence contexts, in a natural mutational target (the Escherichia coli lacZ alpha gene) that selects for mutagenesis at the specific site under investigation . Under these conditions the rate of spontaneous deamination at G.mC exceeds that at G.C by more than 21-fold . Our data implicate differences in chemical stability toward deamination as a major causal factor releasing DNA cytosine methylation to spontaneous mutagenesis.

J Biol Chem, 1994 Mar 11, 269(10), 7062 - 5
Mammalian ferrochelatase, a new addition to the metalloenzyme family; Ferreira GC et al.; A {2Fe-2S} cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway . Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy . In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of {2Fe-2S}+ cluster . Mossbauer spectra of the recombinant ferrochelatase, purified from a strain of E . coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in 57Fe-enriched medium, demonstrated unambiguously that the cluster is a {2Fe-2S} cluster . No change in the cluster oxidation state was observed during catalysis . The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the {2Fe-2S} center in regulation of mammalian ferrochelatases.

Gene, 1994 Mar 11, 140(1), 73 - 7
Newly identified genes involved in the signal transduction of Escherichia coli K-12; Utsumi R et al.; We cloned and sequenced two Escherichia coli genes which are members of a family of an environmentally responsive two-component system . The nucleotide (nt) and deduced amino-acid sequences of these two genes were found to be homologous to those of the Bordetella pertussis bvgA and bvgS genes . They were mapped at 51 min (clones 6B9 to 7G9 of the Kohara miniset library of the E . coli chromosome) . Both proteins, deduced from their nt sequences, were identified in the coupled in vitro transcription-translation system; their molecular masses were consistent with BvgA and BvgS (23 and 135 kDa, respectively) . Furthermore, when these genes were expressed on a multicopy plasmid in an envZ deletion strain, ompC expression was induced . This expression was found to be regulated by low temperature, MgSO4 and nicotinic acid, factors known to control the virulence of B . pertussis via BvgA and BvgS . These results indicate that the newly cloned genes were structurally and functionally similar to bvgA and bvgS, and we designated these genes evgA and evgS.

Gene, 1994 Mar 11, 140(1), 59 - 62
A reliable amplification technique with single-sided specificity for the isolation of 5' gene-regulating regions; Luo M et al.; A simple and efficient method is described for the isolation of extension fragments of known DNA sequences by polymerase chain reaction (PCR) using a single specific primer . With this method, size-selected genomic DNA fragments are ligated to a plasmid vector (pGEM-4Z) which contains sequencing primers and the population of chimeric plasmids is used for transforming Escherichia coli . DNA is extracted from an aliquot of the resulting mini-library and PCR performed using a sequence-specific primer and either of the standard sequencing primers of the plasmid vector . This method appears to be more versatile than inverse PCR (IPCR), since: (i) the DNA sequence needed as the specific primer can be as short as about 20 nucleotides (nt); (ii) the DNA templates to be used in PCR are available in high amount, thus facilitating all manipulations; and (iii) if relinearization of the DNA by restriction enzyme digestion is desired before the PCR reaction, many restriction sites can be chosen from the vector polylinker . Using this method, we have isolated the genomic 5' region of the carrot bifunctional dihydrofolate reductase-thymidylate synthase-encoding gene dhfr-ts using a 21-nt sequence of the 5' region of the dhfr-ts cDNA clone as the specific primer.

Gene, 1994 Mar 11, 140(1), 137 - 8
Sequences of the rplJL operon containing the L10 and L7/L12 genes from Brucella abortus; Oliveira SC et al.; The rplJL operon encodes the L10 and L7/L12 proteins, essential for ribosomal function and protein synthesis . In this study, we report the nucleotide sequence of the rplJ and rplL genes from Brucella abortus . The deduced amino-acid sequences show 37 and 67% identity to Escherichia coli L10 and L7/L12, respectively.

Cell, 1994 Mar 11, 76(5), 925 - 32
Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport; Noueiry AO et al.; Plant viruses establish a systemic infection by moving through plasmodesmata, but little is known of the mechanism(s) involved . The roles of two movement-associated proteins of a single-stranded DNA virus were investigated in vivo, using functional proteins expressed in E . coli and microinjection into plant cells . We report here that the BL1 protein of bean dwarf mosaic geminivirus moves extensively from cell to cell, increases mesophyll plasmodesmal size exclusion limit, and potentiates the movement of double-stranded DNA from cell to cell . Movement of single- and double-stranded DNA out of the nucleus is mediated by the BR1 protein . These results provide direct experimental evidence for intercellular macro-molecular transport in plants, and suggest that the BR1 and BL1 proteins coordinate the movement of viral DNA across both nuclear and plasmodesmal boundaries.

J Biol Chem, 1994 Mar 11, 269(10), 7450 - 7
Glutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B . Searching for the catalytic triad; Boehlein SK et al.; The mechanism of nitrogen transfer in glutamine-dependent amidotransferases remains to be unambiguously established . We now report the overexpression, purification, and kinetic characterization of both the glutamine- and ammonia-dependent activities of Escherichia coli asparagine synthetase B (AS-B) and a series of mutants . In common with other members of the purF family of amidotransferases, the recombinant enzyme possesses an NH2-terminal cysteine residue . Replacement of Cys-1 by either alanine or serine results in a loss of glutaminase and glutamine-dependent activity, without out any significant effect upon ammonia-dependent asparagine synthesis . As previously observed for human AS (Sheng, S., Moraga-Amador, D., Van Heeke, G., Allison, R . D., Richards, N . G . J., and Schuster, S . M . (1993) J . Biol . Chem . 268, 16771-16780), glutamine is an inhibitor of the ammonia-dependent reaction catalyzed by both the Cys-1-->Ala (C1A) and Cys-1-->Ser (C1S) mutants of AS-B . In the case of C1A, the inhibition pattern suggests that an abortive complex is formed . This is consistent with a recent proposal implicating the formation of an imide intermediate in the nitrogen transfer reaction (Richards, N . G . J., and Schuster, S . M . (1992) FEBS Lett . 313, 98-102) . In contrast, glutamine appears to be only a competitive inhibitor of the ammonia-dependent activity of C1S . Cys-1 does not appear to be required for glutamine binding . Replacement of Asp-33 by either asparagine or glutamic acid has little effect on the kinetic properties of the mutant enzymes when compared to wild-type AS-B . Cys-1 and Asp-33 are cognate to residues Cys-1 and Asp-29 in glutamine phosphoribosylpyrophosphate amidotransferase which have been proposed to be members of a catalytic triad responsible for mediating nitrogen transfer in this enzyme (Mei, B., and Zalkin, H . (1989) J . Biol . Chem . 264, 16613-16619) . In the case of AS-B, although Cys-1 is essential for glutamine-dependent activity, Asp-33 does not appear to participate in mediating nitrogen transfer . In an effort to locate other residues which might form part of a "catalytic triad" in the glutamine amidotransferase domain of AS-B, we have expressed and characterized mutant proteins in which His-29 and His-80, which are conserved within the glutamine amidotransferase domain of purF amidotransferases, are replaced by alanine (H29A and H80A).(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1994 Mar 11, 269(10), 7379 - 86
Functional roles of Glu-269 and Glu-325 within the lactose permease of Escherichia coli; Franco PJ et al.; Acidic residues which are found on transmembrane segments within the lactose permease may play an important role in H+ and/or sugar recognition . To examine the functional roles of Glu-269 and Glu-325, we have constructed a variety of amino acid substitutions (e.g . aspartate, glycine, alanine, serine, or glutamine) via site-directed mutagenesis . At position 269, all mutations appear to have a detrimental effect on sugar affinity, downhill transport, and counterflow . The Asp-269 mutant was able to accumulate lactose against a concentration gradient, whereas all of the nonionizable substitutions at position 269 were completely defective . Nevertheless, in spite of their inability to actively accumulate sugars, Gly-269, Ala-269, and Gln-269 mutants were observed to transport H+ upon the addition of galactosides . Mutations at position 325 had a markedly different phenotype . For example, the Asp-325, Gly-325, and Gln-325 mutants exhibited an apparent Km for lactose transport (e.g . 0.21, 0.47, and 0.50 mM, respectively), which was actually lower than that of the wild-type strain (1.44 mM) . In counterflow assays, all position 325 mutants also appear to catalyze lactose exchange . Similar to the results obtained at position 269, the Asp-325 mutant exhibited moderate levels of accumulation, whereas none of the nonionizable mutations at position 325 were able to accumulate galactosides against a concentration gradient . However, unlike the position 269 mutants, no H+ transport was observed in the Gly-325, Ala-325, Ser-325, or Gln-325 strains upon the addition of lactose, S-beta-D-galactopyranosyl-(1,1)-beta-thiogalactopyranoside, 1-O-methyl-beta-D-galactopyranoside, or melibiose . Furthermore, in these mutants, the efflux of lactose during counterflow assays became insensitive to delta pH . Overall, these results are consistent with the notion that an acidic residue at position 325 is required for H+ transport via the lactose permease . Alternative hypotheses are also discussed.

Biochim Biophys Acta, 1994 Mar 8, 1184(2-3), 284 - 90
Mutagenesis of the b'-subunit of Synechocystis sp . PCC 6803 ATP-synthase; Lill H et al.; We investigated the F0F1 ATP synthase of the cyanobacterium, Synechocystis sp . PCC 6803 . The gene for the F0-subunit b', a peptide probably located at the interface between F0 and F1, has been partially or completely evicted from the bacterial genome . We found that the complete deletion of the subunit was lethal to the cells . However, the subunit could be truncated down to its hydrophobic N-terminal stretch without much harm . Since the gene for b' probably shares a common ancestor with the gene for subunit b and emerged by gene duplication, we propose that b' gathered a new role during evolution, perhaps in the regulation of photophosphorylation.

Biochemistry, 1994 Mar 8, 33(9), 2688 - 95
Evidence for participation of aspartate-84 as a catalytic group at the active site of porphobilinogen deaminase obtained by site-directed mutagenesis of the hemC gene from Escherichia coli; Woodcock SC et al.; The role of aspartate-84, an invariant residue in the active site cleft of Escherichia coli porphobilinogen deaminase, has been investigated by site-directed mutagenesis . Substitution of aspartate-84 by glutamate results in an enzyme that retains less than 1% of its activity and which can form highly stable enzyme-intermediate complexes . Substitution of aspartate-84 by either alanine or asparagine, however, results in proteins unable to catalyze the formation of preuroporphyrinogen but which, nevertheless, appear able to assemble the dipyrromethane cofactor . The mechanisms of the tetramerization reaction and cofactor assembly are discussed.

Biochemistry, 1994 Mar 8, 33(9), 2644 - 50
Influence of substrates and MgADP on the time-resolved intrinsic fluorescence of phosphofructokinase from Escherichia coli . Correlation of tryptophan dynamics to coupling entropy; Johnson JL et al.; The influence of that MgADP and the substrate ligands MgATP and fructose 6-phosphate (Fru-6-P) have on the structure of E . coli phosphofructokinase (PFK) in the vicinity of the single tryptophan that exists in each subunit has been examined by employing both steady-state and time-resolved measurements of the tryptophan fluorescence . The accessibility of the tryptophan to iodide quenching is over 1 order of magnitude less than experienced by N-acetyltryptophanamide in solution but varies nonetheless with the state of ligation . Most, but not all, of these changes correlate with changes in the degree of local motion available to the tryptophan side chain as determined by steady-state and time-resolved polarization measurements . When the data obtained from differential polarization experiments are fit to a model in which the motion of the tryptophan side chain is able to move with high frequency within a cone of limited amplitude as part of an otherwise slowly tumbling spherical protein, it was found that ligands primarily affect the amplitude of the available local motion . By interpreting these effects with reference to the disproportionation equilibria which define the negative coupling free energy between MgADP and Fru-6-P and the positive coupling free energy between MgADP and MgATP, it is apparent that changes in the local motion amplitudes correlate with the sign of the component coupling entropy previously determined from van't Hoff analyses (Johnson & Reinhart, 1994).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Mar 8, 33(9), 2635 - 43
Influence of MgADP on phosphofructokinase from Escherichia coli . Elucidation of coupling interactions with both substrates; Johnson JL et al.; A comprehensive assessment is presented of the mutual influence that MgADP, MgATP, and fructose 6-phosphate (Fru-6-P) have on each other's binding to phosphofructokinase (PFK) from E . coli . When virtually any combination of these ligands binds to PFK it produces a significant perturbation in the intrinsic tryptophan fluorescence intensity and/or polarization which not only provides a means to follow binding in titration experiments but which also underscores the fact that more than two different enzyme conformations result from the binding of these ligands . When MgATP is saturating, the binding of MgADP to the allosteric site increases the affinity the enzyme subsequently displays for Fru-6-P . However, in the absence of MgATP, MgADP can bind to both the allosteric site and the nucleotide portion of the active site, with the latter antagonizing the binding of Fru-6-P to an extent that leads to an overall inhibition of Fru-6-P binding by MgADP . MgADP binding at the allosteric site also inhibits the binding of MgATP, indicating that under many circumstances MgADP should be more properly viewed as an inhibitor rather than an activator of E . coli PFK . After quantifying all of the 20 dissociation constants and 11 coupling parameters between ligand pairs pertinent to this three-ligand system, the more significant coupling parameters have been further characterized by examining their variation with temperature to establish the apparent enthalpy and entropy contributions to the corresponding coupling free energies . For both activating and inhibitory couplings, the enthalpy and entropy terms have the same sign as the coupling free energy.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Mar 8, 33(9), 2628 - 34
Nativelike secondary structure in interleukin-1 beta inclusion bodies by attenuated total reflectance FTIR; Oberg K et al.; Attenuated total reflectance FTIR has been used to study the structure of human interleukin-1 beta in inclusion bodies (IBs) and other aggregated forms . The secondary structure composition of native wild-type IL-1 beta determined by FTIR is in excellent agreement with that previously determined by crystallography and NMR: 52% beta-sheet, 25% loop/irregular structure, and 23% turn . Remarkably, IL-1 beta inclusion bodies exhibit secondary structural composition very similar to that of the native protein . The results indicate that the IBs form from a folding intermediate that has nativelike secondary structure . The secondary structure content of aggregated IL-1 beta, formed either in refolding or by thermal denaturation, was identical within experimental error to that of the IB, indicating that these aggregates were formed from intermediates with structures similar to that of the inclusion body.

FEBS Lett, 1994 Mar 7, 340(3), 281 - 6
Formation of sulphmyoglobin during expression of horse heart myoglobin in Escherichia coli; Lloyd E et al.; Expression of recombinant horse heart myoglobin in Escherichia coli has been found to result in the production of both native and variable amounts (approximately 16-17% total) of two sulphmyoglobin isomers . The recombinant sulphmyoglobin produced consists primarily of the A and B isomers as identified by 1H NMR spectroscopy with no evidence for production of the C isomer . Conversion of recombinant sulphmyoglobin to the native protein can be achieved by reconstitution with protohaem IX . The possible relationship of this observation to recombinant expression of other heme proteins is discussed.

FEBS Lett, 1994 Mar 7, 340(3), 202 - 6
Predicted topology of the N-terminal domain of the hydrophilic subunit of the mannose transporter of Escherichia coli; Markovic-Housley Z et al.; A folding topology for the homodimeric N-terminal domain (IIA, 2 x 14 kDa) of the hydrophilic subunit (IIABman) of the mannose transporter of E . coli is proposed . The prediction is based on (i) tertiary structure prediction methods, and (ii) functional properties of site-directed mutants in correlation with NMR-derived alpha/beta secondary structure data . The 3D structure profile suggested that the overall fold of IIA is similar to that of the unrelated protein, flavodoxin, which is an open-stranded parallel beta-sheet with a strand order of 5 4 3 1 2 . The 3D model of IIA, constructed using the known atomic structure of flavodoxin, is consistent with the results from site-directed mutagenesis . Recently NMR results confirmed the open parallel beta-sheet with a strand order of 4 3 1 2 (residues 1-120) of our model whereas beta-strand 5 (residues 127-130) was shown to be antiparallel to beta-strand 4 . The correctly predicted fold includes 90% of the monomeric subunit sequence and contains all functional sites of the IIA domain.

J Mol Biol, 1994 Mar 4, 236(4), 1227 - 40
Unfolding-refolding kinetics of the tryptophan synthase alpha subunit by CD and fluorescence measurements; Ogasahara K et al.; To elucidate the folding mechanism of the tryptophan synthase alpha subunit from Escherichia coli, the kinetics of the unfolding-refolding were studied by peptidyl circular dichroism (CD) and aromatic fluorescence measurement at pH 7 and 25 degrees C . The reactions were induced by concentration jumps of guanidine hydrochloride (GuHCl) . The results can be summarized as follows . (1) The kinetic properties of the unfolding-refolding monitored by CD at 222 nm and aromatic fluorescence coincided with each other, indicating that the changes in the secondary and tertiary structures proceed simultaneously . (2) The unfolding kinetics showed two phases in the range of final GuHCl concentration above 1.8 M . The total amplitudes in the unfolding kinetics accounted for about 100% of the total change . (3) The refolding kinetics also showed two phases in the native condition . The total amplitudes observed in the two phases accounted for only 41% of the total change in maximum, indicating the presence of an undetectable early folding intermediate in the folding process . (4) The fast phases in both the unfolding and refolding were major phases as judged by the magnitudes of the amplitudes . (5) The amplitudes in terms of the CD values at 222 nm for the undetectable early folding intermediate in the refolding kinetics showed little dependence on final GuHCl concentration in the native condition, but depended on final GuHCl concentration in the transition zone, resulting in a similar equilibrium GuHCl unfolding curve . (6) The CD spectrum in the far-UV region for the early folding intermediate was similar to that for the equilibrium unfolding intermediate . (7) It is concluded that the early folding intermediate of the alpha subunit is equivalent to the equilibrium unfolding intermediate, which is assumed to be a molten globule.

J Mol Biol, 1994 Mar 4, 236(4), 1079 - 92
Crystal structure of the tenth type III cell adhesion module of human fibronectin; Dickinson CD et al.; The crystal structure of the cell adhesion module of fibronectin (FNIII10) has been determined at 1.8 A resolution . A recombinant fragment corresponding to the tenth type III module of human fibronectin was crystallized in space group P2(1) with a = 30.7, b = 35.1 and c = 37.7 A and beta = 107 degrees . The structure was determined by molecular replacement and refined by least squares methods . The crystallographic R-factor for the final model of the 91 amino acid module plus 56 solvent atoms is 0.18 for 10 to 1.8 A data . The module consists of two layers of beta-sheet, one with three antiparallel strands and the other with four antiparallel strands . The beta-sheets enclose a hydrophobic core of 24 amino acid side-chains . The module contains the RGD cell recognition sequence in a flexible loop connecting two beta-strands . The tertiary structure of the FNIII10 module has been used to develop a structure-based sequence alignment of 17 type III modules in fibronectin based on the striking conservation of homologous hydrophobic residues . A similar pattern of homologous alternating hydrophobic residues is also evident in a comparison of type III modules in proteins unrelated to fibronectin such as cytokine receptors and muscle proteins.

J Mol Biol, 1994 Mar 4, 236(4), 1011 - 21
The domains of a type I DNA methyltransferase . Interactions and role in recognition of DNA methylation; Cooper LP et al.; The DNA methyltransferases of type I restriction-modification systems are trimeric enzymes composed of one DNA specificity (S) subunit and two modification (M) subunits . The S subunit contains two large regions, each of which recognizes one part of the split, asymmetrical DNA target sequence . Each M subunit contains an amino acid motif for binding the methyl group donor and cofactor, S-adenosyl methionine . The EcoKI methyltransferase has a strong preference for methylating a hemimethylated DNA target rather than an unmodified target . We have used partial proteolytic digestion of EcoKI methyltransferase to generate polypeptide domains that we have identified by amino acid sequencing . The S subunit was cut into two large, folded domains each containing one DNA binding region . Binding of DNA partially protected the S subunit from digestion . The M subunit was also cut into two large domains joined together by a short flexible loop, and a C-terminal tail region . The short loop contained part of the S-adenosyl methionine binding motif, and cofactor binding protected the loop and the two large domains from proteolysis . The C-terminal domain of M remained associated with the N-terminal domain of the S subunit even after the rest of the protein had been digested . The conformation of the tail region of the M subunit was sensitive to the methylation state of DNA in ternary complexes also containing S-adenosyl methionine, and could differentiate between unmethylated and hemimethylated DNA substrates.

J Biol Chem, 1994 Mar 4, 269(9), 6908 - 17
Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme; Li N et al.; 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is a key enzyme regulating biosynthesis of the plant hormone ethylene . The expression of an enzymatically active, wound-inducible tomato (Lycopersicon esculentum L . cv Pik-Red) ACC synthase (485 amino acids long) in a heterologous Escherichia coli system allowed us to study the importance of hypervariable COOH terminus in enzymatic activity and protein conformation . We constructed several deletion mutants of the gene, expressed these in E . coli, purified the protein products to apparent homogeneity, and analyzed both conformation and enzyme kinetic parameters of the wild-type and truncated ACC syntheses . Deletion of the COOH terminus through Arg429 results in complete inactivation of the enzyme . Deletion of 46-52 amino acids from the COOH terminus results in an enzyme that has nine times higher affinity for the substrate S-adenosylmethionine than the wild-type enzyme . The highly efficient, truncated ACC synthase was found to be a monomer of 52 +/- 1.8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar conditions, is a dimer . These results demonstrate that the non-conserved COOH terminus of ACC synthase affects its enzymatic function as well as dimerization.

J Biol Chem, 1994 Mar 4, 269(9), 6810 - 4
Modulation of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase activity by phosphorylation . Role of serine 871; Omkumar RV et al.; Attenuation of Syrian hamster 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) activity by in vitro phosphorylation was studied using AMP-activated protein kinase and wild-type and mutant forms of HMG-CoA reductase . The only residue of the wild-type enzyme phosphorylated was Ser871 . Substrates protected against kinase-mediated attenuation of activity, consistent with substrate-induced conformational changes at the C-terminal region . Although close to the catalytic histidine His865, Ser871 appears to play no direct role in catalysis or substrate recognition . Mutant enzymes S871A, S871H, S871N, and S871Q exhibited from 62-106% of wild-type activity and had wild-type Km values for HMG-CoA and NADPH . Replacement of Ser871 by aspartate or glutamate, but not by glutamine, asparagine, histidine, or tyrosine, severely attenuated activity . Attenuation of catalytic activity that accompanies phosphorylation thus appears to result primarily from the introduction of negative charge, not merely steric hindrance . Other than the wild-type enzyme, only mutant enzyme S871T was phosphorylated, and phosphorylation was accompanied by attenuation of activity . The AMP-activated kinase thus can also phosphorylate threonyl residues.

J Biol Chem, 1994 Mar 4, 269(9), 6784 - 9
Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase; Zhou G et al.; Glutamine 5-phosphoribosylpyrophosphate amidotransferase from Escherichia coli is subject to synergistic feedback regulation by adenine and guanine nucleotides . Inhibition assays and equilibrium binding measurements have established that synergistic inhibition by AMP and GMP results from synergistic binding to two sites/enzyme subunit in the homotetramer . Although each nucleotide can bind to both sites, analyses of the wild type and mutant enzymes indicate that binding of GMP to an A (allosteric) site and AMP to a proximal C (catalytic) site are necessary for synergistic inhibition . K326Q and P410W amino acid replacements result in decreased binding affinity for GMP and AMP and lead to corresponding reductions in feedback inhibition . The K326Q A site mutation results not only in decreased affinity of GMP for the mutant A site but also has an adverse effect on AMP affinity for the C site . Similarly, the P410W C site mutation has a detrimental effect on binding of AMP to the mutant C site and also on affinity of GMP to the A site . The fact that a mutation in one site affects binding of nucleotides to both sites provides further evidence for synergistic binding of nucleotides.

J Biol Chem, 1994 Mar 4, 269(9), 6645 - 50
Non-sterol compounds that regulate cholesterogenesis . Analogues of farnesyl pyrophosphate reduce 3-hydroxy-3-methylglutaryl-coenzyme A reductase levels; Bradfute DL et al.; Farnesyl acetate and ethyl farnesyl ether, two analogues of farnesyl pyrophosphate, stimulate post-transcriptional down-regulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids . Farnesyl acetate and ethyl farnesyl ether reduce translation of HMG-CoA reductase mRNA and enhance degradation of the enzyme, the same regulatory effects attributed to the putative non-sterol regulatory metabolite (Goldstein, J.L., and Brown, M.S . (1990) Nature 343, 425-430) . HMGal, a fusion protein consisting of the membrane domain of HMG-CoA reductase linked to Escherichia coli beta-galactosidase, is subject to the same regulated degradation as HMG-CoA reductase (Skalnik, D . G., Narita, H., Kent, C., and Simoni, R . D . (1988) J . Biol . Chem . 263, 6836-6841) . At 10 micrograms/ml (37.8 microM), farnesyl acetate and ethyl farnesyl ether trigger a 50-80% reduction in HMGal activity . Farnesyl acetate reduces the synthesis of HMG-CoA reductase and HM-Gal by 60-80%, but neither farnesyl compound affects HMG-CoA reductase mRNA levels . Farnesyl acetate and ethyl farnesyl ether stimulated the degradation of HMG-CoA reductase and HMGal, reducing the half-lives of the enzymes by 40-70% . In addition to their regulatory effects on HMG-CoA reductase, these farnesyl compounds also directly disrupt sterol synthesis.

J Biol Chem, 1994 Mar 4, 269(9), 6578 - 8
Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli; Lucht JM et al.; The Escherichia coli hns gene encodes the abundant nucleoid-associated DNA-binding protein H-NS . Mutations in hns alter the expression of many genes with unrelated functions and result in a derepression of the proU operon (proVWX) without abolishing the osmotic control of its transcription . We have investigated the interactions of H-NS with the proU regulatory region by deletion analysis of cis-acting sequences, competitive gel retardation assays, and DNase I footprinting . The negative effect of H-NS on proU transcription was mediated by cis-acting sequences within proV but did not depend on the presence of a curved DNA segment upstream of the proU-35 region previously characterized as a target for H-NS binding in vitro . We detected a 46-base pair high affinity H-NS binding region downstream of the proU promoter at the 5' end of the proV gene and a complex array of additional H-NS binding sites which suggest the presence of an extended H-NS nucleoprotein complex . Most of the H-NS binding sites were highly A+T-rich and carried stretches of 5 or more consecutive A-T base pairs . The implications of our results for the osmotic regulation of proU transcription are discussed.

J Biol Chem, 1994 Mar 4, 269(9), 6511 - 6
Activation of the skeletal muscle calcium release channel by a cytoplasmic loop of the dihydropyridine receptor; Lu X et al.; Expression studies with skeletal and cardiac muscle cDNAs have suggested that the putative cytoplasmic loop region of the dihydropyridine receptor (DHPR) alpha 1 subunit between transmembrane repeats II and III (DCL) is a major determinant of the type of excitation-contraction coupling (skeletal or cardiac) in rescued dysgenic muscle cells (Tanabe, T., Beam, K . G., Adams, B . A., Niidome, T., and Numa, S . (1990) Nature 346, 567-569) . In this study, the possibility of a direct functional interaction with the sarcoplasmic reticulum ryanodine receptor/Ca2+ release channel has been tested by expressing the DCLs of the mammalian skeletal and cardiac muscle DHPR alpha 1 subunit in Escherichia coli . The purified peptides activated the skeletal muscle ryanodine receptor/Ca2+ release channel in single channel and {3H}ryanodine binding measurements, by increasing channel open probability and the affinity of {3H}ryanodine binding, respectively . The two peptides did not activate the cardiac muscle Ca2+ release channel . Other proteins (polylysine, serum albumin) also increased {3H}ryanodine binding and Ca2+ release channel activity, but their activation mechanisms were distinguishable from DCLs . These results show that the II-III cytoplasmic loop of the skeletal and cardiac DHPR alpha 1 subunit functionally interacts with the skeletal, but not cardiac, muscle Ca2+ release channel . Furthermore, our studies suggest that in addition to the DHPR, the sarcoplasmic reticulum Ca2+ release channel may determine the type of E-C coupling that exists in muscle.

J Biol Chem, 1994 Mar 4, 269(9), 6506 - 10
Relief of YY1-induced transcriptional repression by protein-protein interaction with the nucleolar phosphoprotein B23; Inouye CJ et al.; Previous studies have shown that the transcription factor YY1 can both activate and repress transcription of many mammalian genes (reviewed by Hahn (Hahn, S . (1992) Curr . Biol . 2, 152-154)) . Given the diverse effects of the YY1 protein, it seems likely that its function depends on interaction with other cellular factors . We have used the yeast two-hybrid system to isolate mouse cDNAs encoding proteins capable of directly binding to YY1 . Sequence analysis of one clone revealed it had an open reading frame with the potential to code for a protein nearly identical to the previously published mouse nucleolar phosphoprotein B23 . The YY1.B23 complex is specific, and occurs in vivo and in vitro . Overexpression of the B23 protein can reverse the transcriptional repression exerted by YY1 . These results suggest a role for a nucleolar protein as a component in transcription and provide a possible mechanism for transcriptional regulation by YY1.

J Biol Chem, 1994 Mar 4, 269(9), 6458 - 70
Tracing the path of messenger RNA on the Escherichia coli small ribosomal subunit . Immune electron microscopy using defined oligodeoxynucleotide analogs of mRNA; Montesano-Roditis L et al.; Oligodeoxynucleotide models of mRNA were used to determine the ribosomal site of specific nucleotides 3' to the initiation codon . Each mRNA analog had a 5'-terminal 9-base Shine-Dalgarno sequence, a 7-nucleotide spacer, and an ATG initiation signal, followed by up to 31 nucleotides, one of which carried an antibody-recognizable marker . All probes bound efficiently to activated Escherichia coli 30 S ribosomal subunits . Complexes were formed using ribosomal subunits, initiator tRNA, an oligodeoxynucleotide probe, and antibodies . Electron microscopy was then used to place specific positions in the mRNA analog on the subunit and thus to trace the pathway of the messenger . As viewed from the cytoplasmic side of the subunit, the 5' segment of the mRNA lies on the left, along the inner surface of the platform . The initiation codon and the next 9 nucleotides are located in the cleft between the subunit platform and body; within this segment the mRNA makes a U turn and emerges from the cleft at the left of the neck that separates the subunit head and body . The mRNA then loops around the neck to the right, along the cytoplasmic surface of the subunit and toward the site of the 7-methylguanosine residue of the 16 S ribosomal RNA.

J Biol Chem, 1994 Mar 4, 269(9), 6450 - 7
The NAD-glycohydrolase activity of the pertussis toxin S1 subunit . Involvement of the catalytic HIS-35 residue; Antoine R et al.; Pertussis toxin is a member of ADP-ribosylating bacterial toxins that are capable of catalyzing the cleavage of the N-glycosidic bond of NAD+ and the transfer of its ADP-ribose moiety to G proteins . The catalytic S1 subunit of pertussis toxin uses signal transducing G proteins as acceptor substrates but can also catalyze the transfer of the ADP-ribose moiety to water in the absence of G proteins . Site-directed mutagenesis followed by kinetic analyses of truncated soluble mutant proteins revealed that His-35 of S1 is a catalytic residue because alterations of this residue affect the turnover rate of NAD-glycohydrolysis by approximately two orders of magnitude without significantly affecting substrate binding . Replacement of the imidazole of His-35 by the side chain of glutamine maintained the highest residual activity . The pH dependence of the enzyme activity showed only slight variations over the experimental range with an optimum at pH 7.5 and an approximate pKa of 6.5 to 7 . This pH dependence was abolished by the Gln substitution, which still retained significant activity, suggesting that His-35 probably does not act as a true base but rather as a proton acceptor . Direct catalytic roles for several other residues were ruled out . Ser-52 substitutions resulted in slight alterations of both kcat and Km for NAD+ suggesting an involvement in maintaining the local geometry of the active site rather than a direct role in catalysis for this residue . Kinetic studies on mutants with substitutions of Ser-40 indicate a role in NAD+ binding for this residue . In conjunction with previous findings, these studies suggest that the NAD-glycohydrolase activity of S1 utilizes 2 catalytic residues, His-35 and the previously identified Glu-129 . The enzyme mechanism could therefore proceed through an activation by polarization of the acceptor substrate water or G protein by His-35, and the stabilization of an oxocarbonium-like transition state intermediate by Glu-129.

J Biol Chem, 1994 Mar 4, 269(9), 6355 - 61
The conserved serine 211 is essential for reduction of the dinuclear iron center in protein R2 of Escherichia coli ribonucleotide reductase; Regnstrom K et al.; The R2 protein family of class I ribonucleotide reductases contains a highly conserved serine residue close to the essential tyrosyl radical and the dinuclear iron center . In order to test its physiological importance, we have engineered the Ser-211 of Escherichia coli R2 to an alanine and a cysteine residue . The three-dimensional structure of R2 S211A solved to 2.4-A resolution is virtually identical to the wild-type structure apart from the substituted residue . Both mutant proteins contain oxidized dinuclear iron and tyrosyl radical, and their specific enzyme activity per radical are comparable to that of the wild-type protein . In R2 S211A the stability of the tyrosyl radical is substantially decreased, probably caused by movement of Gln-80 into hydrogen bonding distance of Tyr-122 . The major defect in R2 S211A, however, is the inability of its iron center to be reduced by enzymic or chemical means, a characteristic not found in R2 S211C . We propose that Ser-211 is needed as a proton donor/transporter during reduction of the iron center of R2, a reaction which in vivo precedes reconstitution of the tyrosyl radical . This offers a physiological explanation for the high conservation of a serine residue at this position in the R2 family.

J Biol Chem, 1994 Mar 4, 269(9), 6340 - 6
Characterization and oxidoreduction properties of cytochrome c3 after heme axial ligand replacements; Dolla A et al.; Cytochrome c3 (M(r) 13,000) is a tetrahemic cytochrome in which the four heme iron atoms are coordinated by 2 histidine residues at the axial positions . The presence of several oxidoreduction centers in the same molecule raises the question of their coupling . To investigate this mechanism, four single mutations were introduced in cytochrome c3 by site-directed mutagenesis, leading to the replacement of each histidine, the sixth axial ligand of the heme iron atom, by a methionine residue . Characterization of the new set of molecules using biochemical and biophysical techniques was carried out . The novel methionine was correctly coordinated to the iron atom of hemes 3 and 4 in H25M and H70M cytochromes c3, respectively, and this coordination induced a large increase in the oxidoreduction potential of the mutated heme . In contrast, in the case of H22M and H35M cytochromes c3, in which the corresponding methionine is in an oxidized form, only slight changes in redox potential values were observed . In H22M, H25M, and H35M cytochromes c3, two conformations of the molecule were possible, in which the methionine is either free or coordinated to the iron atom . The rate constants for the electron exchange reactions between the cytochrome mutants and the hydrogenase were measured using electrochemical techniques . Distinct behaviors were revealed depending on the mutation . The values of the rate constants for the electron exchange reactions are interpreted in terms of intramolecular electron exchange among the four hemes of the cytochrome.

J Biol Chem, 1994 Mar 4, 269(9), 6332 - 9
Uncoupled steps of the colicin A pore formation demonstrated by disulfide bond engineering; Duche D et al.; Four disulfide bonds were engineered into the pore-forming domain of colicin A to probe the conformational changes associated with its membrane insertion and channel formation . The soluble pore-forming domain consists of 10 alpha-helices with two outer layers (helices 1, 2, and 3-7, respectively) sandwiching a middle layer of three helices (8-10) . Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only . This helical hairpin has been hypothesized to be the membrane anchor . Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), respectively, is unable to promote K+ efflux from sensitive Escherichia coli cells . Activity can be restored by addition of a reducing agent . In vitro studies with brominated lipid vesicles and planar lipid bilayers show that the disulfide bond which connects the helices 1 to 9 prevents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles . All of the engineered bridges prevented the formation of a conducting channel in the presence of a membrane potential . This novel approach indicates that membrane insertion and channel formation are two separate steps . Moreover, the effects of the distance constraints introduced by the different disulfide bonds on colicin A activity indicate that the helical pair 1 and 2 moves away from the other helices upon membrane insertion . Helices 3-10 remain associated together . As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface . In contrast, induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association . These results are discussed in light of several proposed models of the membrane-bound colicin and channel structures.

J Biol Chem, 1994 Mar 4, 269(9), 6313 - 9
Mutagenic investigation of conserved functional amino acids in Escherichia coli L-aspartase; Saribas AS et al.; The potential importance of several functional amino acids in the activity of L-aspartase from Escherichia coli has been examined by site-directed mutagenesis . Amino acids whose importance in enzyme activity was suggested by chemical modification and pH dependence studies were chosen as candidates for investigation . The selection of the particular amino acid targets was guided by homology comparisons among the other sequenced bacterial L-aspartases and by the broader comparison among the fumarase-aspartase enzyme family . Substitution of the most highly conserved cysteine with either serine or alanine, or the most highly conserved histidine with leucine, had no significant effect on the activity of L-aspartase or on the sensitivity of these mutated L-aspartases to cysteine and histidine specific modifying reagents . However, alteration of each of the two conserved lysines to arginine did cause dramatic changes in the catalytic properties of the enzyme . Modification of lysine 54 results in the complete loss of enzyme activity . However, this activity loss appears to be related to changes in the subunit association properties of the arginine 54 mutant . Lysine 326 appears to be involved in substrate binding . Modification of this residue causes a 5-fold increase in the Km for aspartic acid, a drastic decrease in kcat/Km, and a change in the divalent metal ion requirements of the enzyme.

J Biol Chem, 1994 Mar 4, 269(9), 6290 - 5
Genetically altered levels of inorganic polyphosphate in Escherichia coli; Crooke E et al.; The ppk gene encoding polyphosphate kinase (PPK), the enzyme in Escherichia coli that makes long chains of polyphosphate (polyP) reversibly from ATP, was disrupted by insertion of a kanamycin resistance gene . Expression of the exopolyphosphatase gene (ppx) immediately downstream of ppk in the operon was likewise disrupted . Cells were also transformed with a high-copy-number plasmid bearing ppk . Genetically altered polyP levels were estimated in cell extracts by the PPK conversion of ADP to ATP . PolyP levels (microgram/10(11) cells) near 2.0 were reduced in the ppk(-)-ppx- mutants to 0.16 and increased more than 100-fold (e.g . 220) in cells transformed with multiple copies of ppk . Mutant cells, lacking the long polyP chains, showed a growth lag following dilution of a stationary-phase culture . PolyP-deficient cells exhibit a striking phenotype in their failure to survive in stationary phase and loss of resistance to heat (55 degrees C) and to oxidants (42 mM H2O2) . High polyP levels are also associated with reduced survival.

J Biol Chem, 1994 Mar 4, 269(9), 6286 - 9
Fluorescence detection of conformational changes in GroEL induced by thermal switching and nucleotide binding; Hansen JE et al.; GroEL assists other proteins to fold in vivo, usually requiring Mg2+ and ATP to function . Electron micrographs reveal that ATP binding induces a conformational change in this protein . Previously we have suggested that a thermally induced conformational change in GroEL results in the switching between enhanced and arrested reactivation of bacterial glucose-6-phosphate dehydrogenase . This thermal switching occurs over a narrow temperature range (25-30 degrees C) . Recently it has aslo been reported that the binding affinity of the P22 tail spike polypeptide to GroEL changes abruptly over this same temperature range . To determine whether these conformational changes occur in GroEL in solution in absence of folding intermediates, the protein was stoichiometrically labeled with AEDANS (N-(acetylaminoacyl)-5-naphthalene-1-sulfonic acid), a fluorescent label whose emission is environment-sensitive . By measuring changes in the fluorescence Stokes shift we have detected two thermally induced conformational changes between 25 and 30 degrees C . The first change occurs between 25 and 26 degrees C, resulting in less than a 1-nm shift in the fluorescence . The second conformational change occurs between 27 and 30 degrees C, resulting in a 3-nm fluorescence shift . The conformational change induced by ATP binding also results in a 3-nm shift in fluorescence . Our data provide a correlation between functional change and structural change in GroEL.

J Biol Chem, 1994 Mar 4, 269(9), 6962 - 71
Rabbit interleukin-1 receptor antagonist . Cloning, expression, functional characterization, and regulation during intestinal inflammation; Cominelli F et al.; Genomic and cDNA clones for rabbit interleukin-1 receptor antagonist (IL-1ra) were isolate based on homology with the human, mouse, and rat IL-1ra gene . A partial genomic clone, obtained by screening a rabbit genomic library, contained coding sequences for the carboxyl-terminal 108 amino acids of rabbit IL-1ra . Two classes of cDNA for rabbit IL-1ra were obtained using RNA from inflamed rabbit colon tissue . One class of cDNA coded for a secreted form of IL-1ra, whereas the other coded for a putative intracellular form of rabbit IL-1ra . The latter form is similar to that isolated from human epithelial cells . A partially synthetic rabbit IL-1ra gene was constructed and expressed in Escherichia coli . The recombinant rabbit IL-1ra was purified to homogeneity by ion exchange chromatography . Its affinity was similar to that of human IL-1ra for the human and mouse type I IL-1 receptor . From the cDNA clone and the purified recombinant protein, specific probes were developed for measuring levels of rabbit IL-1ra mRNA and protein in normal and inflamed rabbit tissues . Unlike IL-1 alpha and IL-1 beta, IL-1RA mRNA and protein were present at detectable levels in normal rabbit colon . During the development of an experimental formalin-immune complex colitis, rabbit IL-1 alpha showed a dramatic increase in tissue levels, consistent with previous results; IL-1ra also increased 3-4-fold . Treatment of colitis rabbits with corticosteroids significantly suppressed neutrophil infiltration, corticosteroid treatment suppressed IL-1ra but not IL-1 alpha mRNA steady-state levels . Our observations demonstrate that IL-1 and IL-1ra synthesis is differentially regulated in healthy and inflamed intestinal tissue.

Biochim Biophys Acta, 1994 Mar 3, 1211(2), 221 - 8
Arachidonate 12-lipoxygenase of rat pineal glands: catalytic properties and primary structure deduced from its cDNA; Hada T et al.; When a crude extract of rat pineal glands (the 1000 x g supernatant of a homogenate) was incubated with arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was found as a major product . The 12-lipoxygenase of rat pineal gland also reacted with linoleic and alpha-linolenic acids at 35% and 101% the rate of arachidonate 12-oxygenation, respectively . Upon Western blot analysis using polyclonal antibody against porcine leukocyte 12-lipoxygenase, the cytosol fraction of rat pineal gland showed a positive band with a molecular weight of approx . 74 kDa . A full-length cDNA for this enzyme was cloned from a cDNA library of rat pineal gland and the identity of the 12-lipoxygenase cDNA was confirmed by its expression in E . coli . The amino acid sequence of the enzyme was deduced from the nucleotide sequence of the cDNA, encoding 663 amino acids with a calculated molecular weight of 75,305 . The enzyme showed 72% identity of amino acid sequence with porcine leukocyte 12-lipoxygenase and 73% with bovine tracheal 12-lipoxygenase, but only 59% with human platelet 12-lipoxygenase . Taken together, the high reactivity with C-18 fatty acids, the immunoreactivity and the amino acid homology data indicate that the rat pineal 12-lipoxygenase is more closely related to leukocyte 12-lipoxygenase than to platelet 12-lipoxygenase . Upon RNA blot analysis, by far the highest content of 12-lipoxygenase mRNA was observed in the pineal gland and negligible amounts of mRNA were detected in other parts of the brain . The predominant presence of 12-lipoxygenase mRNA in pineal gland was confirmed by in situ hybridization of rat brain . Significant amounts of 12-lipoxygenase mRNA were also detected in rat spleen, aorta, lung and leukocytes.

Biochim Biophys Acta, 1994 Mar 2, 1199(2), 143 - 8
DNA topoisomerases from Streptomyces noursei: influence of coumarins and quinolones on the enzymic activity; Storl K et al.; DNA topoisomerases have been purified from Streptomyces noursei . DNA gyrase and topoisomerase I show a differential sensitivity against quinolones and coumarins compared to the E . coli enzymes . Streptomyces gyrase is resistant to much higher levels of various drugs than is the E . coli enzyme . The observed differences between the gyrases from streptomycetes and E . coli are discussed in the light of present literature data.

J Urol, 1994 Mar, 151(3), 750 - 3
Detection of interleukin-1 activity in human bladder cancer cell lines; Hayashi O et al.; Detection of interleukin (IL)-1 activity was studied in two human bladder cancer cell lines, T24 and EJ1, and one rat bladder carcinoma cell line, 804G . Significantly high proliferation of mouse thymocytes in the assay of IL-1 activity was observed in the conditioned medium (CM) of T24 cells, indicating that the cells released IL-1-like activity . Enzyme-linked immunosorbent assay (ELISA) and Northern blot analysis showed the presence of both IL-1 alpha and IL-1 beta in the CM of T24 cells and expression of mRNAs of both cytokines in the cells . Interleukin-1 activity in EJ1 cells, which produced a little activity, was induced by E . coli lipopolysaccharide (LPS) while it was not induced in T24 by either LPS or other test substances . Conditioned medium of T24 increased proliferation of both T24 and EJ1 in cell-growth assay . Further investigation of the mode of action and role of cytokines, especially those from tumor cells themselves, is necessary in relation to BCG or photodynamic therapy.

Mol Cell Endocrinol, 1994 Mar, 99(2), 183 - 92
DNA topology regulates rat prolactin gene transcription; Ying C et al.; A cell-free transcription system was used to examine the effects of DNA negative supercoiling on transcription from rat prolactin promoters . The mRNA was faithfully transcribed from prolactin promoters that contained the DNA fragment spanning from -1960 to -10 base pairs upstream of the prolactin transcription start site . Prolactin promoters in supercoiled form, prepared from Escherichia coli, have a higher transcriptional efficiency than the promoter in the relaxed form . Deletion of DNA sequences containing the estrogen response element and putative non-B form DNA elements from the upstream region of the prolactin gene did not alter this effect of DNA topology on prolactin transcription . We further separated transcription into preinitiation and elongation steps by using sarkosyl and found that more preinitiation complexes were assembled on the supercoiled prolactin promoters than on the relaxed promoters . DNA supercoiling therefore plays an important role in controlling prolactin gene expression by facilitating the formation of preinitiation complexes on the promoters.

Alcohol, 1994 Mar-Apr, 11(2), 85 - 90
Selective effect of alcohol on cellular immune responses of lymphocytes from AIDS patients; Nair MP et al.; In this study we examined the in vitro effects of alcohol on the proliferative responses of lymphocytes from healthy donors and AIDS patients to a recombinant fusion peptide, env-gag, corresponding to portions of the gp41 envelope (env) and internal core (gag) proteins of HIV . The effects of alcohol (ETOH) on the natural killer (NK) cell activities of lymphocytes from healthy donors and patients with AIDS were also investigated . Peripheral blood mononuclear cells from both normal donors and AIDS patients produced significant levels of lymphocyte proliferative responses to the HIV env-gag peptide; however, these responses were significantly higher in patients with AIDS, showing the specificity of the response . The env-gag-induced proliferative responses of lymphocytes from normal subjects were significantly suppressed when cultures contained only higher levels of ETOH (0.2% and 0.3%), whereas ETOH even at a lower level (0.1%) produced significant suppression of the env-gag-induced proliferation of lymphocytes only from AIDS patients . Direct addition of ETOH at concentrations of 0.1%, 0.2%, and 0.3% to cultures of lymphocytes from normal donors and NK target cells did not produce significant suppression of NK cell activities . However, ETOH at concentrations of 0.2% and 0.3% significantly suppressed the NK activities of lymphocytes from AIDS patients, and the suppressive effect was observed at all E:T cell ratios examined . Control peptide from the Escherichia coli expression vector did not produce any significant effect on lymphocyte proliferative responses or NK activity of both normal donors and AIDS patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis, 1994 Mar, 9(2), 133 - 9
Effect of distamycin on chlorambucil-induced mutagenesis in pZ189: evidence of a role for minor groove alkylation at adenine N-3; Wang P et al.; Previous work has shown that the bifunctional alkylating agent chlorambucil induces thermolabile adenine adducts and that the predominant chlorambucil-induced mutations in shuttle vector pZ189 are transversions at AT base pairs . In order to assess the role of thermolabile adducts in generating these transversions, pZ189 was treated with chlorambucil in the presence of distamycin, which specifically blocks formation of thermolabile adenine adducts . Analysis of the mutations resulting from replication of the damaged vector in human 293 cells showed that base substitutions at AT base pairs were specifically suppressed in concert with suppression of thermolabile adducts at specific sites in the supF target gene, strongly supporting a role for these adducts in mutagenesis . Since there is considerable evidence that these adducts are N-3 alkylations, a computer graphics model of such an adduct was constructed . Modeling studies indicated that the adduct could be formed with little distortion of the DNA helix . Analysis of the adduct using the HINT (Hydropathic INTeractions) program was consistent with the proposal that favorable hydrophobic interactions of the phenyl ring of chlorambucil with the wall of the minor groove may promote adenine N-3 alkylation by this drug.

J Cell Biochem, 1994 Mar, 54(3), 299 - 308
Apolipoprotein E: a potent inhibitor of endothelial and tumor cell proliferation; Vogel T et al.; Recombinant human apolipoprotein E3 (apoE), purified from E . coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose- and time-dependent manner . ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number . Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin-binding growth factors . Thus, at low serum concentrations (0-2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold . The bFGF-dependent proliferation was dramatically inhibited by apoE with an IC50 approximately 50 nM . Under conditions where cell proliferation was mainly serum-dependent, apoE also suppressed growth but required higher concentrations to be effective (IC50 approximately 500 nM) . ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells . The IC50 values obtained with these cells were generally 3-5 times higher than with BAE cells . Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture . In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF . Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE . The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growth, and metastasis.

Arch Histol Cytol, 1994 Mar, 57(1), 87 - 105
A morphological study of acute respiratory tract lesions in a lipopolysaccharide instilled rat model; Ooi H et al.; Acute inflammatory reactions in different respiratory regions were studied in a lipopolysaccharide (LPS) instilled rat model . Rats were sacrificed at 1, 2, 4, 8, 12, 24 and 48 h after administration of Escherichia coli LPS to the lumen of the trachea, and changes in the trachea, bronchioles and alveoli were observed by light and electron microscopy . In the trachea, neutrophils were markedly increased in subepithelial connective tissue and between tracheal epithelial cells, from 2 h on, showing a peak at 8 h . The number of tracheal mast cells increased at 4 and 8 h . Different structural features of secretory granules between mast cells in the tracheal epithelium and subepithelium were noticed, and the possible involvement of mast cells in airway acute inflammation is discussed . In the bronchioles, Clara cells showed characteristic morphological alterations . At 8 to 12 h, Clara cells revealed well-developed smooth and rough endoplasmic reticula and many free ribosomes, possibly for enhanced synthesis of secretory substances . Many Clara cells possessed a large apocrine-like protrusion filled with an amorphous substance, likely indicating an apocrine secretion of the cells . At 24 h, Clara cells were observed to take on a pseudostratified arrangement, suggesting enhanced proliferation of progenitor cells and their differentiation into Clara cells . In the alveoli, neutrophils infiltrated alveolar walls and pulmonary venules at 4 h and disappeared at 12 h . Prior to morphological changes in bronchioles and alveolar walls at 2 h, the macrophages with well-developed filopodia and free-ribosomes appeared in alveolar sacs and ducts . They may contribute to histological changes especially at the initial stage of acute inflammation in alveoli.

Leber Magen Darm, 1994 Mar, 24(2), 81 - 3
{Biliary pseudocyst as a rare complication of choledocholithiasis}; Wirbel RJ et al.; Biliary pseudocysts, so-called biliomas are reported to be an extreme rarity and mostly arising posttraumatic . We report on a case of symptomatic choledocholithiasis 7 years after cholecystectomy . The course was complicated by formation of a biliary pseudocyst . The 71-year-old, septic patient could not be operated because of pulmonal and cardiac high risks . He could be treated successfully by conservative procedure via endoscopic papillotomy, stone extraction and percutaneous, sonographic controlled drainage of the biliary pseudocyst.

J Clin Immunol, 1994 Mar, 14(2), 141 - 8
Endotoxin administration to humans primes alveolar macrophages for increased production of inflammatory mediators; Smith PD et al.; To elucidate potential mechanisms of the acute lung injury associated with endotoxemia, we evaluated the effect of intravenously administered endotoxin on the ability of alveolar macrophages isolated by bronchoalveolar lavage from normal subjects to produce inflammatory mediators . Within 1 hr of endotoxin (4 ng/kg body weight) administration, all 12 study subjects developed constitutional symptoms and leukopenia, and within 3 hr, low-grade fever . Resolution of symptoms and fever by 6 hr was accompanied by systemic granulocytosis . Although intravenously administered endotoxin appeared to activate a subset of circulating monocytes, it did not alter the bronchoalveolar lavage cell number, phenotype (95% macrophages), or constitutively expressed high levels of surface HLA-DR and O2- . In contrast, intravenous endotoxin primed the alveolar macrophages for enhanced lipopolysaccharide-induced secretion of interleukin-1 (11.8 to 25.8 U/ml; P = 0.04), tumor necrosis factor-alpha (titer, 6.8 to 13.6; P = 0.20), and prostaglandin E2 (38.4 to 116.3 ng/ml; P = 0.035) . These results demonstrate that low-dose intravenous endotoxin primes human alveolar macrophages, which are already differentiated in situ, for enhanced secretion of inflammatory mediators . Such mediators may contribute to the pulmonary changes associated with endotoxemia and acute lung injury.

Arzneimittelforschung, 1994 Mar, 44(3), 317 - 22
Biological effects of the new platelet-activating factor receptor antagonist (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride; Imanishi N et al.; SM-12502 ((+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl, CAS 119383-00-5) inhibited platelet-activating factor (PAF)-induced aggregation of rabbit and human platelets with IC50 values of 2.3 mumol/l and 4.7 mumol/l, respectively, but did not inhibit platelet aggregation induced by adenosine diphosphate, collagen, thrombin, arachidonic acid, U46619 (a thromboxane A2 agonist) or Ca2+ ionophore A23187 at concentrations up to 400 mumol/l . SM-12502 competitively antagonized 3H-PAF binding to rabbit platelets with an IC50 of 1.0 mumol/l . In contrast, the anti-PAF activity of the optical isomer SM-12501 ((-)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl) was much weaker and its IC50 was more than 100 mumol/l . SM-12502 prevented PAF-induced death in mice with ID50 values of 4.8 mg/kg (i.v.) or 68.6 mg/kg (p.o.) . In guinea pigs, SM-12502 inhibited PAF (0.1 micrograms/kg)-induced hemoconcentration with ID50 values of 1.9 mg/kg (i.v.) or 40.2 mg/kg (p.o.) . In addition, SM-12502 inhibited PAF (10 ng/kg)-induced hypotension in rats with ID50 values of 2.0 mg/kg (i.v.) or 6.5 mg/kg (p.o.) . The in vivo effects of SM-12501 were much weaker . Orally administered SM-12502 showed rapid absorption and a long duration of pharmacological activity in rats . SM-12502 afforded dose-dependent protection against anaphylactic death in mice with ID50 values of 18.4 mg/kg (i.v.) and 136 mg/kg (p.o.) . It also inhibited endotoxin (E . coli 0.55:B5, 60 mg/kg)-induced death in mice, with ID50 values of 119 mg/kg (i.v.) and 182 mg/kg (p.o.).(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Vet Res, 1994 Mar, 55(3), 333 - 8
Resistance of Chinese Meishan, Fengjing, and Minzhu pigs to the K88ac+ strain of Escherichia coli; Michaels RD et al.; The microscopic brush border membrane adherence assay was used to determine resistance (nonadherence) and susceptibility (adherence) of Chinese pigs (n = 289) to the K88ac+ strain of Escherichia coli-mediated disease . This study estimates prevalence of resistance to diarrheal disease in multiple family lines (no common ancestry for a minimum of 3 generations) for the Chinese Meishan, Fengjing, and Minzhu breeds . Results of in vitro assays indicate that pigs of the Meishan breed are highly resistant (nonadherent) to K88ac+ E coli-mediated disease . The gene conferring susceptibility to K88ac+ E coli-mediated disease exists at low frequency in pigs of the Minzhu breed . Minzhu-type (crossbred) pigs of both phenotypes (susceptible and resistant) were identified in ratios consistent with a 1-locus gene model . Given that all susceptible pigs were from 1 site, frequency of susceptibility within this Minzhu population is estimated at 8% . Inheritance within the Fengjing breed is still unclear because a weakly adherent phenotype, as well as the resistant phenotype, was identified . The weakly adherent phenotype was observed in pigs derived from multiple family lines . Expression of the weakly adherent phenotype in terms of susceptibility to disease is not known at this time.

Int J Biochem, 1994 Mar, 26(3), 387 - 96
Phosphorylation of Escherichia coli proteins during the SOS response; Marcandier S et al.; 1 . The phosphorylation of Escherichia coli proteins was analyzed comparatively before and after induction of the SOS response in a temperature-sensitive mutant strain . 2 . The presence of phosphorylated proteins was evidenced by gel electrophoresis and autoradiography after labelling with radioactive orthophosphate in vivo or radioactive adenosine triphosphate in vitro . 3 . Significant changes in the intensity of protein labelling were observed upon induction of the SOS functions: six proteins were found to be more phosphorylated while two others were less phosphorylated . Moreover, five additional proteins appeared to become phosphorylated exclusively during the SOS response . The molecular mass and isoelectric point of these various proteins were determined . 4 . For most proteins, the changes in the pattern of protein phosphorylation were concomitant with variations in the amount of protein synthesized . 5 . The changes in the pattern of phosphoproteins observed during the SOS response were not due to the temperature shift required experimentally for expressing the SOS phenotype . 6 . Phosphorylation was found to be catalyzed by protein kinases that modify amino acid residues at hydroxyl groups in protein substrates . 7 . Both in vivo and in vitro studies brought evidence that neither RecA nor LexA, the two key regulatory proteins of the SOS functions, were capable of undergoing phosphorylation.

Microsc Res Tech, 1994 Mar 1, 27(4), 294 - 306
Electron microscopy of the F1F0 ATP synthase: from structure to function; Gogol EP; The F1F0 ATP synthase is the large multisubunit complex which uses the proton gradient of energetically active membranes to synthesize ATP . While biochemical and genetic approaches have characterized the composition of the enzyme and elucidated many details of its mechanism and assembly, electron microscopy has been the tool of primary importance in determining the arrangement of the many subunits which comprise the F1F0 . The highly cooperative catalytic mechanism is tightly coupled to transmembrane proton translocation in a separate and rather distant sector of the complex . An understanding of this intricate process and its control requires an appreciation of subunit interactions, starting with their locations relative to one another . Electron microscopy has provided most of the available structural information on the F1F0, and recent applications of cryo-electron microscopy have captured different functionally relevant configurations which may finally address longstanding questions about subunit rearrangements during the catalytic cycle.

Int Immunol, 1994 Mar, 6(3), 369 - 76
LacZ inducible, antigen/MHC-specific T cell hybrids; Sanderson S et al.; The nuclear factor of activated T cells (NFAT) enhancer element of the IL-2 gene can regulate expression of the Escherichia coli lacZ reporter gene in activated T cells . Based upon this observation, we showed that this inducible NFAT--lacZ construct could be used to measure TCR mediated, ligand-specific activation in single T cells . Here we describe a general approach to obtaining lacZ inducible, T cell hybrids by generating two new fusion partners BWZ.36 and BWZ.36 CD8 alpha derived by transfecting alpha-beta-BW5147 cells with the NFAT--lacZ construct . Using these fusion partners and normal T cells from immunized mice, we obtained T cell hybrids in which lacZ activity is specifically induced in response to antigen/MHC class II or MHC class I complexes . We show that measuring ligand induced T cell activation by the non-radioactive lacZ assay is simpler, faster, and most cost-effective relative to conventional IL-2 assays . Most importantly, the unique ability to detect activation of single T cells by the lacZ assays permits detection of rare antigen presenting cells and thus provides the basis for developing expression cloning strategies for identifying unknown T cell antigens.

Biotechniques, 1994 Mar, 16(3), 514 - 9
Two-minute miniprep method for plasmid DNA isolation; Tarczynski MC et al.; An extremely rapid method, INSTA-PREP, has been developed to prepare plasmid DNA from 1 to 3 mL miniprep Escherichia coli bacterial cultures . Direct extraction of plasmid DNA from E . coli bacterial cells is achieved by a two-phase solution consisting of phenol-chloroform-isoamyl alcohol and water or buffer with efficient separation of the phases by centrifugation in the presence of the INSTA-PREP gel barrier material . Processing time, from E . coli culture to usable plasmid DNA, is two minutes or less per sample . Supercoiled plasmid DNA yields ranged from 3 to 10 micrograms per mL of culture depending on plasmid copy number . Plasmid DNAs prepared by INSTA-PREP were analyzed and are suitable for use in molecular biology procedures including restriction digestion, ligation with T4 DNA ligase, bacterial transformation, PCR, cultured cell transfection and T7 DNA polymerase or thermostable DNA polymerase-mediated dideoxynucleotide sequencing.

Biotechniques, 1994 Mar, 16(3), 463 - 9
Mapping by insertion mutagenesis without cloning; Sussman R; This paper describes a new strategy for positioning specific loci on known genomic maps or for generating high-resolution physical maps of organisms that are susceptible to transposable elements . The strategy does not require cloning and thus saves time and effort . It is based on isolating cell lines containing appropriate insertions of a DNA element (transposon) carrying a selectable marker and one or more restriction sites . DNA from independent cell lines is digested to completion with a restriction enzyme that cuts within the transposon and the adjacent genomic DNA . The fragments thus produced are analyzed by partial digestions with a panel of restriction enzymes, separated and probed sequentially with oligonucleotides complementary to the ends of the transposon . Algorithms that compare and order the different restriction fingerprints are used to either place the unknown locus on an existing restriction map or, in the case of a new genome, to form contigs to generate a map . The usefulness of this strategy was demonstrated by mapping an Escherichia coli insertion mutation that was difficult to map by more standard procedures.

Biotechniques, 1994 Mar, 16(3), 460 - 3
Large-scale supercoiled plasmid preparation by acidic phenol extraction; Wang Z et al.; A novel method for large-scale plasmid preparation is described . Crude extracts are subjected to acidic phenol extraction to remove any contaminants present in the aqueous phase . The supercoiled plasmid DNA, which preferentially remains in the organic phase and inter-phase, is extracted back into the aqueous phase with 1.5 M TRIZMA base, from which it is precipitated . The resultant plasmid DNA is highly pure and satisfactory for any subsequent procedures . The method is extremely economical and takes only 3-4 h.

Mol Gen Mikrobiol Virusol, 1994 Mar-Apr, (2), 21 - 4
{Expression in Escherichia coli of hepatitis B virus polymerase and its functional domains}; Gazina EV et al.; The plasmids have been constructed permitting expression of hepatitis B viral polymerase and its functional domains, the catalytic and end ones, as hybrid proteins containing 12 alien amino acids in the N-terminal . Immunoblotting with the rabbit antisera to N- and C-terminals of hepatitis B polymerase amino acid sequence has demonstrated that constructed plasmids determine the synthesis of the corresponding proteins in bacterial cells . HBV polymerase is processed in Escherichia coli cells . The successful expression of HBV polymerase and its functional domains makes possible the detailed study of this protein structure and function.

Mol Gen Mikrobiol Virusol, 1994 Mar-Apr, (2), 14 - 6
{Cloning and expression of certain herpes simplex virus type 1 genes in Escherichia coli}; Shatalin KIu et al.; Complete genes IE12, IE63, IE68, IE175, UL19, UL29 of herpes simplex virus type I or their fragments have been cloned in Escherichia coli cells . The peptides expressed were shown to be fused with cro-beta-galactosidase proteins . The recombinant proteins containing amino acid sequences of ICP4, ICP27 and ICP47, major capsid protein, and major DNA-binding protein react in immunoblotting with the anti-HSVI serum from hyperimmune rabbit . The recombinant proteins can be used for creation of diagnosticums and other scientific and practical purposes . The immunological properties of the recombinant proteins are being investigated.

Mol Gen Mikrobiol Virusol, 1994 Mar-Apr, (2), 11 - 4
{Biochemical bases for the effect of combining kanamycin and nitrofuran resistance genes in Escherichia coli cells}; Morozov GI et al.; The fact of a significant increase in resistance to aminoglycosides when nfr genes with chromosomal or plasmid localization are combined with the plasmid genes coding for kanamycin-transferase in E . coli cells is confirmed . Gel-filtration of homogenates of the cells with and without pLD105 plasmid carrying nfr gene and of the cells with a chromosomal nfr gene revealed a 10 kD polypeptide when the plasmid is present . Relying on these results, it is concluded that the discovered polypeptide fulfils two roles: inhibiting of specific nitrofuran-reductase, which leads to nitrofurans resistance and a drop of transmembrane electric potential contributing to the increase of resistance to aminoglycosides (kanamycin) in strains with the plasmid nfr gene . Absence of the 10 kD polypeptide in the cells with a chromosomal nfr gene and other data are indicative of a possible existence of a different mechanism of resistance to nitrofurans and an increase of resistance to aminoglycosides in the strains with a chromosomal nfr mutation.

Mol Biol (Mosk), 1994 Mar-Apr, 28(2), 362 - 73
{Biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli . II . Effect of replacing amino acids at the processing site and N-terminal domain of the mature polypeptide chain of alkaline phosphatase on its biogenesis}; Karamyshev AL et al.; The effect of amino acid substitutions in E . coli alkaline phosphatase on its biogenesis has been studied . The substitution of Val for Ala(-1) in the signal peptide cleavage site completely inhibits all stages of posttranslational modification: processing and formation of isozymes . The absence of processing does not prevent translocation of the precursor across the cytoplasmic membrane and formation of an active enzyme macromolecule . The precursor of the above mutant protein was found in the periplasm and in the cytoplasmic membrane . The substitution of Gln for Glu(+4), as well as the double substitution of Ala for Arg(+1) and Gln for Glu(+4), in the N-terminus of mature polypeptide chain result in the change in the isozyme spectrum . Differences in the rates of processing in vivo of both mutant proteins were not revealed . However, the double amino acid substitution significantly increases the efficiency of in vitro processing . All amino acid substitutions studied have no effect on the peculiarities of biogenesis which are conditioned by oversynthesis of the enzyme encoded by the phoA gene in the plasmid: secretion into the culture medium and accumulation of precursor as insoluble aggregates in the cytoplasm . However, extracellular activities of mutant proteins differ from that of the wild-type protein, which may result from the change either in the efficiency of their secretion or in their catalytic properties.

FEMS Microbiol Lett, 1994 Mar 1, 116(3), 293 - 9
CEL1: a novel cellulose binding protein secreted by Agaricus bisporus during growth on crystalline cellulose; Armesilla AL et al.; The cel1 gene of Agaricus bisporus encodes a protein (CEL1) that has an architecture resembling the multi-domain fungal cellulases, although the sequence of its putative catalytic core is not matched by any other in the protein and nucleic acid data bases . The N-terminal half of the putative catalytic domain of CEL1 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase . The fusion protein was used to raise a CEL1-specific antibody . CEL1 was detected as an extracellular 49.8 kDa protein in A . bisporus cellulose-grown cultures, where it bound strongly to cellulose . CEL1 was neither an endoglucanase, a cellobiohydrolase able to hydrolyze fluorogenic cellobiosides, a beta-glucosidase, a xylanase, nor a cellobiose: quinone oxidoreductase . CEL1 was present in some fractions of culture fluid separated by electrophoresis which released soluble sugars from crystalline cellulose.

FEMS Microbiol Lett, 1994 Mar 1, 116(3), 287 - 91
A possible osmodependent protease in Escherichia coli; Meury J; When a strain of Escherichia coli, expressing a hybrid protein GalK-beta-Gal, is shifted to high osmolarity, the beta-galactosidase activity strongly decreases within 20 min of shock . The loss of beta-galactosidase activity results from degradation of the hybrid protein under osmotic stress . The results raise the possibility that osmotic stress induces a specific osmodependent protease.

Plant J, 1994 Mar, 5(3), 353 - 61
Purification, characterization, and cDNA sequence of glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.); Graeve K et al.; Glucose-6-phosphate dehydrogenase (G6PDH, E.C . 1.1.1.49) has been purified from potato tuber at least 850-fold to apparent homogeneity as judged by SDS-PAGE . The enzyme was characterized by Km values of 260 microM for glucose-6-phosphate and 6 microM for NADP and a broad pH optimum between pH 7.5 and 9 . NADPH, GTP, ATP, acetyl CoA and CoA inhibited G6PDH activity . Dithiothreitol (DTT) did not inactivate the enzyme . A highly specific antiserum was produced in a rabbit and used for immunodetection of G6PDH in Western blots . A cDNA library from potato leaves was screened with DNA probes produced by the polymerase chain reaction (PCR) in the presence of g6pdh-specific primers . A full-length cDNA clone was analyzed and the derived amino acid sequence compared with known G6PDH sequences from various sources . The homology of the plant sequence with G6PDH sequences from animals and yeast was found to be rather high (52%), whereas there was significantly lower homology with sequences of bacterial origin (37%) . The lack of a plastidic signal sequence as well as the insensitivity of the recombinant enzyme towards reduced DTT, support the view that the cDNA sequence of a redox-independent cytosolic isoform was obtained.

Vaccine, 1994 Mar, 12(4), 291 - 8
Protection of mice against experimental murine mycoplasmosis by a Mycoplasma pulmonis immunogen in lysogenized Escherichia coli; Lai WC et al.; A construct of the Mycoplasma pulmonis (MP) genomic library, using randomly sheared DNA, was cloned in lambda gt11 and transfected into C600 Escherichia coli organisms . Clones of E . coli expressing a fusion protein reactive with anti-MP and monospecific serum were transferred orally or intravenously into Balb/c mice . Expression of the fusion protein was induced by adding isopropyl-beta-D-thiogalactopyranoside to the drinking water . This vaccination protocol led to local and systemic antibody formation, to generation of immune lymphocytes and to protection against large numbers of virulent MP organisms . This approach might be generally successful in preventing infectious disease.

Protein Eng, 1994 Mar, 7(3), 413 - 24
Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine . Evidence for an extremely slow conformational change; Onuffer JJ et al.; The strictly conserved active site residue, Asp222, which forms a hydrogen-bonded salt bridge with the pyridine nitrogen atom of the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase (AATase), was replaced with alanine (D222A) in the Escherichia coli enzyme . The D222A mutant exhibits non-hyberbolic saturation behavior with amino acid substrates which appear as apparent negative cooperativity in steady-state kinetic analyses . Single turnover progress curves for D222A are well described by the sum of two exponentials, contrasting with the monophasic kinetics of the wild-type enzyme . An active/inactive heterodimer containing the D222A mutation retains this biphasic kinetic response, proving that the observed cooperativity is not the result of induced allostery . The anomalous behavior is explained by a hysteretic kinetic model involving two slowly interconverting enzyme forms, only one of which is catalytically competent . The slow functional transition between the two forms has a half-life of approximately 10 mins . Preincubation of the mutant with the dicarboxylic inhibitor maleate shifts the equilibrium population of the enzyme towards the catalytically active form, suggesting that the slow transition is related to the domain closure known to occur upon association of this inhibitor with the wild-type enzyme . The importance of Asp222 in the chemical steps of transamination is confirmed by the approximately 10(5)-fold decrease in catalytic competence in the D222A mutant, and by the large primary C alpha-deuterium kinetic isotope effect (6.7 versus 2.2 for the wild-type) . The transamination activity of the D222A mutant is enhanced 4- to 20-fold by reconstitution with the co-factor analog N-methylpyridoxal-5'-phosphate (N-MePLP), and the C alpha-proton abstraction step is less rate determining, as evidenced by the decrease in the primary kinetic isotope effect from 6.7 to 2.3 . These results suggest that the conserved interaction between the protonated pyridine nitrogen of PLP and the negatively charged carboxylate of Asp222 is important not only for efficient C alpha-proton abstraction, but also for conformational transitions concomitant with the transamination process.

Ann Hematol, 1994 Mar, 68(3), 125 - 32
Carbohydrate-dependent binding of human myeloid leukemia cell lines to neoglycoenzymes, matrix-immobilized neoglycoproteins, and bone marrow stromal cell layers; Gabius S et al.; The presence of sugar receptors on human myeloid leukemia cells was comparatively assessed by a highly sensitive binding assay, employing a panel of 14 types of neoglycoenzymes (chemically glycosylated Escherichia coli beta-galactosidase) . The selected carbohydrate ligands mainly encompass common components of natural glycoconjugates as mono- or disaccharides . The monocytoid cells of the THP-1 line, the very young myeloblasts and the myeloblasts of the lines KG-1a and KG-1, the promyelocytes of the HL-60 line, and the early myeloblasts/erythroblasts of the K-562 line displayed a nonuniform pattern of specific binding with quantitative differences at a fixed, nonsaturating concentration of the probes . Scatchard analysis in four cases corroborated the indication of cell-type-related differences between the various cell lines . To test whether the detectable cellular sugar-binding sites can mediate adhesion to glycoligands, a rather simple model matrix of nitrocellulose-immobilized neoglycoproteins was first used . In comparison to the carbohydrate-free carrier protein significant cell adhesion was observed primarily with neoglycoproteins that exposed galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and fucose moieties among the 11 tested types of carbohydrate residue . Subsequently, human bone marrow stromal cell layers were tested as a model matrix with increased levels of physiological relevance and complexity . Mixtures of carbohydrate and neoglycoprotein were employed as inhibitors of an interaction via lectins between the stromal and the tumor cells . The carbohydrate-dependent alterations of this parameter revealed cell-type-associated properties . Tumor cell binding was significantly decreased for not more than two lines with the effective sugars, namely N-acetylgalactosamine, mannose, fucose, and sialic acid.

J Clin Pathol, 1994 Mar, 47(3), 263 - 5
CA 125 secretion by peritoneal mesothelial cells; Zeillemaker AM et al.; AIMS--To investigate the secretion of the tumour marker CA 125 by cultured human mesothelial cells; to determine if secretion of CA 125 could be observed by activating the mesothelial monolayers with different cytokines . METHODS--Mesothelial cells were isolated from human omentum and cultured to confluent monolayers on perforated polycarbonate membranes (pore size 0.4 micron) . The mesothelial monolayers were activated and the apical and basolateral secretion of CA 125 compared . To investigate the influence of cytokines, mesothelial cells were cultured and activated with recombinant interleukin-1 beta (rIL-1 beta), tumour necrosis factor-alpha (TNF-alpha) or lipopolysaccharide from Escherichia coli . The secretion of CA 125 was tested using a microparticle enzyme immunoassay . RESULTS--Mesothelial monolayers secreted CA 125 in a polarised manner with preference for the apical side . Apical polarisation occurred irrespective of the side of the inducing stimulus (p < or = 0.05) . Non-activated cultured mesothelial monolayers secreted significant quantities of CA 125, indicating constitutive production of this protein . However, CA 125 production was significantly enhanced if mesothelial cells were incubated with rIL-1 beta (p < or = 0.05), TNF-alpha (p < or = 0.05), and E coli LPS (p < or = 0.01) . CONCLUSIONS--Human mesothelial monolayers secrete CA 125 preferentially from their apical surfaces . The secretion of CA 125 can be enhanced by the inflammatory cytokines Il-1 beta, TNF-alpha, and by E coli LPS.

Appl Environ Microbiol, 1994 Mar, 60(3), 1038 - 40
Occurrence of Shiga-like toxin-producing Escherichia coli in retail fresh seafood, beef, lamb, pork, and poultry from grocery stores in Seattle, Washington; Samadpour M et al.; Fresh meat, poultry, and seafood purchased from Seattle area grocery stores were investigated for the presence of Shiga-like toxin-producing Escherichia coli by using DNA probes for Shiga-like toxin (SLT) genes I and II . Of the 294 food samples tested, 17% had colonies with sequence homology to SLT I and/or SLT II genes.

Ultramicroscopy, 1994 Mar, 53(3), 251 - 70
The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles; Penczek PA et al.; Cryo-electron microscopy of single biological particles opens up new possibilities for structure analysis: the particle can be reconstructed in its native shape and internal features are preserved . To take advantage of these possibilities we have developed new methods of data collection and image processing and we have applied them to the 70S Escherichia coli ribosome . A method of orientation search is proposed, which makes it possible to relate random-conical data sets to one another even if they are collected from low-tilt micrographs . A technique for 3D alignment of projections is described and applied to the single-micrograph 3D reconstruction.

Rinsho Ketsueki, 1994 Mar, 35(3), 296 - 9
{Infection-induced transient increase in the platelet count, and a transient remission of thrombocytopenia with high-dose intravenous gammaglobulin therapy during intercurrent pneumonia in chronic idiopathic thrombocytopenic purpura}; Katoh M et al.; A patient with chronic idiopathic thrombocytopenic purpura (ITP), who had a transient increase in platelet count during infectious episodes, and had a transient remission of thrombocytopenia with administration of high-dose intravenous gammaglobulin during intercurrent pneumonia was described . A 64-year-old Japanese woman with a 12-year history of chronic ITP was refractory to steroids and azathioprine, and the platelet count was constantly less than 10 x 10(9)/l . During both acute upper respiratory infections and chronic cystitis due to E . coli, the platelet count transiently increased to more than 40 x 10(9)/l . High-dose intravenous gammaglobulin therapy successfully induced a remarkable increase in the platelet count, which persisted for 3 months, when gammaglobulin was administered during the intercurrent pneumonia . The bleeding tendency disappeared and transient remission of ITP was obtained . In contrast, the clinical efficacy of high-dose intravenous gammaglobulin proved to be only transient and slight, when administered before the onset of pneumonia or after recovery from pneumonia . These phenomena may suggest that high-dose gammaglobulin may enhance some positive mechanisms related to platelet increment in infectious diseases accompanied with chronic ITP.

J Infect Dis, 1994 Mar, 169(3), 665 - 7
Tumor necrosis factor is involved in the appearance of interleukin-1 receptor antagonist in endotoxemia; van der Poll T et al.; To assess the role of tumor necrosis factor (TNF) in the appearance of interleukin-1 receptor antagonist (IL-1RA) in endotoxemia, 4 healthy humans were studied after a bolus intravenous injection of recombinant human TNF (50 micrograms/m2) . In addition, 8 healthy chimpanzees were investigated after a bolus intravenous injection of Escherichia coli endotoxin (4 ng/kg) with (n = 4) or without (n = 4) a simultaneous intravenous injection of a monoclonal anti-TNF antibody (15 mg/kg) . TNF induced a pronounced rise in IL-1RA concentrations, becoming apparent after 1 h and peaking after 3 h (P < .05) . The rise in IL-1RA after administration of endotoxin started 2 h later . Neutralization of the early endotoxin-induced TNF activity by anti-TNF caused a marked reduction in IL-1RA concentrations (P < .05) . These results indicate that TNF is an intermediate factor in IL-1RA release in endotoxemia.

Pharmacology, 1994 Mar, 48(3), 147 - 56
Endotoxin-mediated changes in plasma endothelin concentrations, renal endothelin receptor and renal function; Nambi P et al.; The purpose of these studies was to examine the changes in renal endothelin (ET) receptor, renal function and plasma ET (ET-1) concentration in male Sprague-Dawley rats injected with nonlethal doses of Escherichia coli endotoxin (LPS) . Prior to the injection of LPS, kidney ET receptor density was 59 +/- 5 fmol/mg protein (n = 20) . At 24 h after the injection of 1 or 3 mg/kg LPS, {125I}ET-1 binding to kidney membranes was increased by 70% in both LPS groups (p < 0.001) . Scatchard analysis of the saturation binding experiments confirmed that the increase in {125I}ET-1 binding was due to an increase in receptor density with no change in affinity (202 pmol/l at baseline and 168 pmol/l and 246 pmol/l at 24 h after the injection of 1 and 3 mg/kg LPS, respectively) . At 7 days after the injection of LPS, kidney ET-1 receptor density was still increased by 30 +/- 5% and 58 +/- 16%, respectively (p < 0.05, compared to the baseline value) . Baseline values for Na+ and K+ excretion were approximately 115 muEq/h and 214 +/- mu/Eq/h respectively, and were decreased with LPS . Maximal decreases in Na+ and K+ excretion occurred at 48 h (-85%) and 30 h (-82%), respectively, following the injection of 3 mg/kg LPS and returned to baseline levels in 7 days . Following the injection of 3 mg/kg LPS, plasma immunoreactive ET-1, as measured by radioimmunoassay, increased in a time-dependent manner: the maximal increase of 60% occurred within 1 h after the injection of LPS (p < 0.05), and thereafter returned to baseline levels . Kidney tissue levels of ET-1 increased from baseline values of 2.6 fmol/mg protein to a peak of 4.6 fmol/mg protein 1 h after the injection of LPS . Tissue ET-1 levels were still significantly elevated at 6 h but not 24 h after LPS injection . These studies suggest that ET-1, either by increases in plasma concentration and/or altered receptor density, may be involved in the LPS-induced impairment of renal function.

Mol Gen Genet, 1994 Mar, 242(6), 744 - 8
Effect of ogt expression on mutation induction by methyl-, ethyl- and propylmethanesulphonate in Escherichia coli K12 strains; Abril N et al.; We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents . We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O6-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria . Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives . Thus an ogt::kanr disruption mutation was introduced into the parental ogt+ bacteria, and the ogt::kanr mutation was then eliminated by cotransduction of ogt+ with the closely linked Tetr marker (zcj::Tn10) . The delta(ogt-fnr) deletion or ogt::kanr disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to L-arabinose resistance (Arar) . Furthermore, the number of Arar mutants increased linearly with dose, unlike the case in ogt+ bacteria, which had a threshold dose below which no mutants accumulated . Differences in mutability were even greater with propyl methanesulphonate . Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt- mutant strains and also methylmethanesulphonate mutagenesis in ada- bacteria . A sample of AB1157 obtained from the E . coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes . Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E . coli K12 strain being used is advisable.

Mol Gen Genet, 1994 Mar, 242(6), 736 - 43
The H-NS protein modulates the activation of the ilvIH operon of Escherichia coli K12 by Lrp, the leucine regulatory protein; Levinthal M et al.; The H-NS protein, the product of the hns gene, plays a central role in the cellular response of bacteria to environmental stresses such as modification of osmolarity and temperature . The leucine regulatory protein (Lrp) controls a wide array of operons both as an activator (e.g . ilvIH) and as a repressor . We demonstrate that H-NS can decrease the activity of Lrp in stationary phase and under conditions of high osmolarity . Strains containing hns mutations have higher levels of Lrp-activated ilvIH transcription, while strains carrying the hns+ allele on a pBR322 plasmid have lower activity of Lrp-directed ilvIH gene expression.

Rinsho Byori, 1994 Mar, 42(3), 242 - 8
{DNA analysis of cytochrome b positive chronic granulomatous disease (a case report)}; Azuma H; A patient was diagnosed as having chronic granulomatous disease (CGD) . This case seems to have been transmitted in an X-linked from judging from the family history . We had previously suggested that the patient's cytochrome b was normal both qualitatively and quantitatively . Thus, we thought that there might be mutation in the gp91-phox (one of the two components of cytochrome b) gene affecting electron transport but leaving other functions intact . To confirm this speculation, we performed DNA analysis . Complementary DNA (cDNA) was obtained from messenger RNA (mRNA) derived from peripheral blood lymphocytes . By using primers specific for the gp91-phox cDNA, the cDNA was amplified by polymerase chain reaction (PCR) . The amplified cDNA was then ligated into Blue Script vector and transfected into E . coli (JM109) in order to clone the cDNA of gp91-phox . Then, the cloned cDNA was sequenced . Sequence analysis showed that the nucleotides 1521-1525 were deleted and a new sequence of 8 nucleotides was substituted . This mutation converted Glu-Lys-Thr into His-Ile-Trp-Ala . To confirm that the mutated allele came from the patient's mother; we performed mismatched PCR . PCR using a mutated allele could produce approximately 250 base pair products only when the patient's cDNA was used . PCR using a wild type primer could produce 250 base pair products only when cDNA from a healthy donor was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiat Res, 1994 Mar, 137(3), 385 - 93
Electron migration along 5-bromouracil-substituted DNA irradiated in solution and in cells; Beach C et al.; Solvated electrons generated in aqueous solution after exposure to ionizing radiation can be scavenged by DNA and then transferred along the DNA molecule . This mechanism of charge transfer provides an opportunity for radiation damage to be targeted to certain regions in the DNA molecule and is a mechanism by which single-strand breaks contribute to locally multiply damaged sites to enhance cell lethality . Experiments were performed in which different amounts of 5-bromouracil (5-BrU) were substituted for thymine in Escherichia coli DNA . The amount of bromide released was assayed after quantitative reaction of radiation-induced solvated electrons with 5-BrU in DNA samples irradiated in solution and irradiated in the cellular environment . By varying the amount of 5-BrU incorporated in the DNA, the average distance between 5-BrU molecules was systematically changed and, because the number of 5-BrU/electron reactions was monitored by the amount of bromine released, the maximum average electron migration distance along the 5-BrU DNA could be estimated . Using this approach, the maximum average electron migration distance in aqueous solutions of 5-BrU DNA was about 6.5 to 10 base distances in nonhybrid 5-BrU DNA (assuming only intrastrand migration) . Similar methods revealed charge migration in 5-BrU DNA incorporated into E . coli, and the maximum average migration distance was about 5 to 6 base distances (assuming only intrastrand migration) . Only 11-16% of the electrons produced during radiolysis were scavenged by 5-BrU DNA in aqueous solution, and only 1% resulted in the release of bromide from 5-BrU-DNA inside E . coli.

J Surg Res, 1994 Mar, 56(3), 216 - 20
The limiting effect of dichloroacetate on endotoxin-induced liver damage in starved rats; Irita K et al.; Dichloroacetate has been shown to have therapeutic effects on sepsis and endotoxin shock and to reduce liver damage in rats intoxicated with ethanol or carbon tetrachloride . In this study, the effect of dichloroacetate on endotoxin hepatitis was investigated . Endotoxin hepatitis was induced by an intraperitoneal coadministration of 50 micrograms/kg lipopolysaccharide from Escherichia coli, and 200 mg/kg D-galactosamine in starved, male Wistar rats . This treatment induced the following changes within 24 hr: an increase in the serum aminotransferase activity, histological alterations of the liver including focal necrosis of liver cells and inflammatory infiltrates, an increase in blood pyruvate and alanine concentrations, and inhibition of starvation ketosis . The intraperitoneal administration of 250 mg/kg dichloroacetate 30 min after the administration of the toxins partially counteracted all of these changes . The administration of dichloroacetate might be useful in coping with hepatic damage as well as lacticemia and cardiovascular depression induced by endotoxins.

J Steroid Biochem Mol Biol, 1994 Mar, 48(4), 369 - 75
Steroid sulfation by expressed human cytosolic sulfotransferases; Falany CN et al.; The human cytosolic sulfotransferases (STs), dehydroepiandrosterone sulfotransferase (DHEA-ST) and the phenol-sulfating form of phenol sulfotransferase, (P-PST), have been expressed in bacteria and used to investigate the ability of the cloned enzymes to conjugate steroids and related compounds . DHEA-ST was capable of sulfating all of the 3-hydroxysteroids, testosterone and estrogens tested as substrates . The 3-hydroxysteroids, androsterone, epiandrosterone and androstenediol, were conjugated at 50-60% of the rate of DHEA . Of the steroids tested, P-PST was capable of conjugating only the estrogens . The catechol estrogens, 2-hydroxyestradiol, 4-hydroxyestradiol and 4-hydroxyestrone, and compounds with estrogenic activity such as 17 alpha-ethynyl-estradiol and trans-4-hydroxytamoxifen, were also tested as substrates . DHEA-ST showed little or no sulfation activity with these compounds; however, all of these compounds were sulfated by P-PST . These results indicate that the expressed human STs are valuable in analyzing the overlapping substrate specificities of these enzymes and that P-PST may have an important role in the metabolism of estrogens and estrogenic compounds in human tissues.

J Steroid Biochem Mol Biol, 1994 Mar, 48(4), 317 - 23
Nuclear extracts enhance the interaction of fusion proteins containing the DNA-binding domain of the androgen and glucocorticoid receptor with androgen and glucocorticoid response elements; De Vos P et al.; Comparable fragments of the androgen receptor (AR) (amino acids 540-607) and of the glucocorticoid receptor (GR) (amino acids 412-515) were expressed in E . coli as fusion proteins with protein A . Both fusion proteins, denoted ARF1 and GRF1, contain the DNA-binding domain and some flanking amino acids . In vitro binding assays have shown that both fusion proteins interact with androgen/glucocorticoid response elements (ARE/GREs) in an intron fragment of the C3(1) gene of the androgen-regulated rat prostatic binding protein and in the typically glucocorticoid-responsive long terminal repeat (LTR) promoter of mouse mammary tumour virus . Present results indicate that the interaction of both ARF1 and GRF1 with the C3(1) as well as the LTR fragments is enhanced in the presence of nuclear extract . The factor that gives rise to this enhancement appears to be ubiquitous and sensitive to trypsin and temperature treatment . In the C3(1) fragment, the enhancing effect requires the presence of an intact functional ARE/GRE (Core II) as well as a region spanning the ARE/GRE half-site Core I.

Clin Exp Immunol, 1994 Mar, 95(3), 530 - 5
IL-6 induces hepatic inflammation and collagen synthesis in vivo; Choi I et al.; IL-6 regulates the synthesis of a broad spectrum of acute phase proteins in the liver . Also, it is involved in the pathogenesis of many fibrogenic diseases . To study the inflammatory effects of IL-6 on the liver in vivo, human rIL-6, produced in Escherichia coli, was injected intraperitoneally into rats (25 micrograms/100 g body weight) . The major fraction of injected IL-6 was accumulated in the liver within 40 min, and the number of platelets was increased during 72 h after injection . After 5 weeks of injection, the levels of serum glutamine pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) were not changed, but they were significantly elevated at 13 weeks of treatment . Meanwhile, serum albumin levels were slightly decreased compared with those of controls . The same phenomena were observed in carbon tetrachloride-treated rats . Collagen synthesis was increased in the liver tissues and in the culture supernatants of hepatic lipocytes isolated from the rats treated with IL-6 for 13 weeks . Histological analysis correlated well with biochemical analysis . At 5 weeks of treatment, only mild pathological changes were observed, but severe hepatocyte necrosis and the accumulation of fibres in necrotic area were developed in the liver of IL-6-treated rats after 13 weeks of treatment, confirming that hepatic inflammation and fibrosis were developed . IL-6 activities in the sera and in the culture supernatants of lipocytes from IL-6-treated rats were elevated compared with those in controls . These biochemical and pathological data indicate that IL-6 can induce hepatic inflammation, and it has important roles in the pathogenesis of fibrosis and diseases of the liver in vivo . In addition, these results will provide useful information for the clinical trials of IL-6.

Clin Exp Immunol, 1994 Mar, 95(3), 390 - 6
Epitope mapping of the 52-kD Ro/SSA autoantigen; Frank MB et al.; Autoantibodies to Ro/SSA are commonly found in sera of patients with systemic lupus erythematosus (SLE) and primary Sjogren's syndrome . The presence of these antibodies is related to lymphopenia, photosensitive dermatitis, and pulmonary and renal disease, suggesting that they have an immunopathologic role {1-6} . We previously isolated a cDNA clone which encodes the 52-kD human Ro/SSA protein . In this study we have determined the number and location of epitopes recognized by SLE sera using recombinant proteins encoded by the full-length or overlapping subclones of this cDNA . An immunodominant epitope was detected using Western blots and ELISA on the NH2-terminal side of this protein's putative leucine zipper . The data suggest that 11 amino acids are critical for the recognition of this molecule by these autoantibodies . Although the titres of anti-52-kD Ro/SSA antibodies vary between different patient sera, no heterogeneity in the location of antigenic epitopes to which their autoantibodies bound was detected . This homogeneous pattern of reactivity to a single rather than multiple regions of this protein is unusual for lupus autoantigens which have been identified, and suggests that these antibodies may have arisen as by a cross-reaction to an epitope on another molecule.

Biochem J, 1994 Mar 1, 298 ( Pt 2), 427 - 33
The role of His66 and His72 in the reaction mechanism of bovine liver low-M(r) phosphotyrosine protein phosphatase; Chiarugi P et al.; Site-directed mutagenesis of a synthetic gene coding for low-M(r) phosphotyrosine protein phosphatase from bovine liver has been carried out . The two histidine residues in the enzyme have been mutated to glutamine; both single and double mutants were produced . The mutated and non-mutated sequences have been expressed in Escherichia coli as fusion proteins, in which the low-M(r) phosphotyrosine protein phosphatase was linked to the C-terminal end of the maltose-binding protein . The fusion enzymes were easily purified by single-step affinity chromatography . The mutants were studied for their kinetic properties . Both single mutants showed decreased kcat . values (30 and 7% residual activities for His66 and His72 respectively), and alterations of the Ki values relative to four-competitive inhibitors were observed . The kinetic mechanism of p-nitrophenyl phosphate hydrolysis in the presence of both single mutants was determined and compared with that of the non-mutated enzyme . The rate-determining step of the catalytic process of the His66-->Gln mutant was the same as that found for non-mutated enzyme, whereas for the His72-->Gln mutant, both the kinetic constant of the step that causes the formation of a phosphoenzyme covalent intermediate, and the kinetic constant of the step that causes the dephosphorylation of the enzyme covalent intermediate, determined the kcat . value . This observation was confirmed by phosphoenzyme covalent intermediate trapping experiments . The participation of both histidine residues (His66 and His72) at the active site is strongly suggested by the results of diethyl pyrocarbonate inactivation of both single mutants, each containing a single histidine residue . Both mutants are completely inactivated by diethyl pyrocarbonate treatment; the competitive inhibitor Pi protects both mutants from inactivation . The His66/His72 double mutant was completely inactive.

Biochem J, 1994 Mar 1, 298 ( Pt 2), 403 - 7
The gelatin-binding site of human 72 kDa type IV collagenase (gelatinase A); Banyai L et al.; To identify structures critical for gelatin-binding of 72 kDa type IV collagenase (gelatinase A), fragments of this metalloproteinase have been expressed in Escherichia coli and assayed for their gelatin affinity . Each of the three fibronectin-related type II domains was found to have affinity for gelatin . Fragments containing all three tandem type II domains had significantly stronger affinity than any of the constituent units, indicating that they co-operate to form the high-affinity gelatin-binding site . Competition experiments have also shown that gelatinase A binds more tightly to gelatin than fibronectin and can displace the latter from denatured collagen.

Biochem J, 1994 Mar 1, 298 ( Pt 2), 395 - 401
Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) using synthetic phosphopeptides; Harder KW et al.; The intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) (44 kDa) was expressed in bacteria, purified using epitope 'tagging' immunoaffinity chromatography, and characterized with respect to kinetic profile, substrate specificity and potential modulators of enzyme activity . A chromogenic assay based on the Malachite Green method was employed for the detection of inorganic phosphate (Pi) released from phosphopeptides by HPTP beta . This assay, modified so as to improve its sensitivity, was adapted to a 96-well microtitre plate format, and provided linear detection between 50 and 1000 pmol of Pi . The cytoplasmic domain of HPTP beta was strongly inhibited by vanadate, molybdate, heparin, poly(Glu, Tyr) (4:1) and zinc ions . In order to explore the substrate preferences of this PTPase, we generated 13-residue synthetic phosphotyrosine-containing peptides that corresponded to sites of physiological tyrosine phosphorylation . HPTP beta demonstrated kcat . values between 76 and 258 s-1 using four different phosphopeptides . The substrate preference of HPTP beta was in the order srcTyr-527 > PDGF-RTyr-740 > ERK1Tyr-204 >> CSF-1RTyr-708 with Km values ranging from 140 microM to greater than 10 mM . The variations in affinity were probably due to differences among the four phosphopeptides compared, particularly with respect to the character of the charged amino acids flanking the phosphotyrosine residue.

Arch Biochem Biophys, 1994 Mar, 309(2), 363 - 8
Tryptophan-free Escherichia coli F1-ATPase; Wilke-Mounts S et al.; We have engineered a mutant form of Escherichia coli F1-ATPase which is tryptophan-free and contains five mutations, namely delta W28L/alpha W513F/gamma W108Y/gamma W206Y/beta W107F . A strain carrying all five mutations grew normally by oxidative phosphorylation . Purified mutant F1-ATPase showed Vmax and Km both 65% higher than wild-type, resulting in kcat/Km the same as wild-type . The pH dependence of ATPase activity in mutant enzyme was very similar to that in wild-type . Catalytic-site nucleotide-binding characteristics were measured using the analog lin-benzo-ADP and sensitivity to inhibitors was tested using dicyclohexylcarbodiimide, azide and aurovertin . The mutant enzyme was very similar to wild-type in each of these characteristics . The fluorescence spectrum of mutant enzyme confirmed the absence of tryptophan . We have therefore established that it is possible to generate a tryptophan-free enzyme which retains normal catalytic function, oligomeric stability and in vivo assembly.

Arch Biochem Biophys, 1994 Mar, 309(2), 308 - 14
The effect of hemoglobin, hematin, and iron on neutrophil inactivation in superoxide generating systems; Kim YM et al.; When Escherichia coli was incubated with xanthine oxidase and acetaldehyde, the killing of E . coli was accelerated by iron-EDTA but inhibited by hematin or hemoglobin . On the other hand, when E . coli was incubated with human neutrophils in the presence of phorbol myristate acetate (PMA), all of these iron species at concentrations of a few micromolar accelerated the inactivation of neutrophils and in so doing protected the E . coli from being killed by the neutrophils . The inactivation of the neutrophils was accompanied by an increase in lipid peroxidation and by a decrease in viability measured with trypan blue . This inactivation was inhibited by scavengers such as deoxyribose, mannitol, or thiourea . Desferrioxamine B and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) both inhibited the inactivation mediated by iron-EDTA, but had no effect on the hematin- or hemoglobin-mediated inactivation . Vanadium (vanadyl ion), an effective Fenton reagent, behaved in the same way as iron-EDTA relative to the effects of DMPO on neutrophil inactivation . These results led us to conclude that neutrophils were inactivated during PMA stimulation by OH radicals in the presence of iron-EDTA and by some other oxidizing species when hematin or Hb is present . Ascorbate enhanced the inactivation of neutrophils mediated by these iron species . Catalase was very effective in inhibiting neutrophil inactivation . Superoxide dismutase was not as effective but the combination with catalase was most effective.

Arch Biochem Biophys, 1994 Mar, 309(2), 288 - 92
The effects of fur on the transcriptional and post-transcriptional regulation of MnSOD gene (sodA) in Escherichia coli; Schrum LW et al.; Earlier studies have shown that the Fur (ferric uptake regulation) protein acts as an anaerobic/aerobic repressor of MnSOD (sodA) expression . We found that the aerobic expression of sodA::lacZ fusion in a Fur- background to be threefold higher than in a Fur+ background . This effect of fur mutation was not seen in a strain harboring the sodA+ gene instead of the sodA::lacZ fusion . However, we observed a proportionate increase in the concentrations of sodA::lacZ and sodA+ mRNAs in response to a mutation in the fur gene . These data suggest that the formation of active MnSOD is dependent on a functional fur gene . Indeed, we found that in a fur mutant iron was incorporated into SodA in place of manganese, thus creating inactive and/or partially active forms of the enzyme (i.e., Fe2SodA and/or Mn,FeSodA, respectively), resulting in little or no increase in total MnSOD activity . Thus, Fur plays the role of a repressor at the transcriptional level, but it also plays an indirect role at the post-transcriptional level where it affects the maturation of SodA into a fully active enzyme, Mn2SodA (MnSOD).

J Immunol, 1994 Mar 1, 152(5), 2438 - 46
Platelet-activating factor antagonist TCV-309 attenuates the induction of the cytokine network in experimental endotoxemia in chimpanzees; Kuipers B et al.; Platelet-activating factor (PAF) has been postulated to play a role in the pathogenesis of sepsis . Additionally, in vitro studies have revealed tight interactions between PAF and the cytokine network, and PAF is considered to be an important stimulator of neutrophil functions . To assess the intermediate role of PAF in the induction of cytokines and neutrophil degranulation in endotoxemia in vivo, 12 healthy adult chimpanzees were i.v . injected with a bolus dose of Escherichia coli endotoxin (4 ng/kg); four animals received endotoxin alone, whereas the other chimpanzees were infused with the specific and potent PAF antagonist TCV-309 (bolus of 100 micrograms/kg, followed by either 100 micrograms/kg/h (n = 4) or 500 micrograms/kg/h (n = 4) for 5 h) . At both doses TCV-309 significantly inhibited the endotoxin-induced rise in cytokine levels . Peak TNF concentrations after injection of endotoxin alone were 366 +/- 96 pg/ml, vs 105 +/- 47 and 115 +/- 56 pg/ml after administration of endotoxin together with the lower or higher dose of TCV-309, respectively (p < 0.05) . TCV-309 also reduced the appearance of soluble TNFRs . Maximal levels of the type I soluble TNFR were diminished from 2.53 +/- 0.27 ng/ml (endotoxin alone) to 1.69 +/- 0.36 ng/ml (high dose TCV-309; p < 0.05); peak values of the type II soluble TNFR were diminished from 8.62 +/- 1.19 ng/ml to 5.76 +/- 0.92 ng/ml (p < 0.05) . Furthermore, TCV-309 attenuated the endotoxin-induced release of IL-6 (160 +/- 82 pg/ml after endotoxin alone, vs 63 +/- 30 pg/ml in the low dose TCV-309 group (p < 0.05) and 65 +/- 29 pg/ml in the high dose group (p = 0.07) as well as that of IL-8 (279 +/- 168, vs 71 +/- 15 and 46 +/- 17 pg/ml, respectively; both p < 0.05) . TCV-309 tended to reduce the endotoxin-provoked rise in serum IL-1R antagonist levels . In contrast, TCV-309 did not affect the neutrophilic leukocytosis elicited by endotoxin, nor did it inhibit endotoxin-induced neutrophil degranulation, as monitored by the plasma levels of elastase-alpha 1-antitrypsin complexes . We conclude that PAF plays a role, either directly or indirectly, in the stimulation of the cytokine network and in the shedding of soluble TNFR in endotoxemia . PAF does not seem to be an important intermediate factor in endotoxin-induced neutrophilia or neutrophil degranulation.

J Bacteriol, 1994 Mar, 176(6), 1796 - 800
Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase; Truniger V et al.; gldA, the structural gene for the NAD(+)-dependent glycerol dehydrogenase, was mapped at 89.2 min on the Escherichia coli linkage map, cotransducible with, but not adjacent to, the glpFKX operon encoding the proteins for the uptake and phosphorylation of glycerol . gldA was cloned, and its position on the physical map of E . coli was determined . The expression of gldA was induced by hydroxyacetone under stationary-phase growth conditions.

J Bacteriol, 1994 Mar, 176(6), 1793 - 5
"Protease I" of Escherichia coli functions as a thioesterase in vivo; Cho H et al.; Escherichia coli protease I is assayed as an esterase active with certain synthetic model chymotrypsin substrates . However, the gene encoding protease I has the same DNA sequence and genomic location as tesA, a gene that encodes E . coli thioesterase I . We report that both hydrolase activities utilize the same active site and demonstrate that the protein functions as a thioesterase in vivo.

J Bacteriol, 1994 Mar, 176(6), 1767 - 72
Role of the purine repressor hinge sequence in repressor function; Choi KY et al.; A protease-hypersensitive hinge sequence in Escherichia coli purine repressor (PurR) connects an N-terminal DNA-binding domain with a contiguous corepressor-binding domain . Binding of one molecule of dimeric repressor to operator DNA protects the hinge against proteolytic cleavage . Mutations in the hinge region impair repressor function in vivo . Several nonfunctional hinge mutants were defective in low-affinity binding to operator DNA in the absence of corepressor as well as in high-affinity corepressor-dependent binding to operator DNA, although binding of corepressor was similar to binding of the wild-type repressor . These results establish a role for the hinge region in operator binding and lead to a proposal for two routes to form the holoPurR-operator complex.

J Bacteriol, 1994 Mar, 176(6), 1673 - 82
DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting; Brikun I et al.; Derivatives of Escherichia coli K-12 of known ancestry were characterized by random amplified polymorphic DNA (RAPD) fingerprinting to better understand genome evolution in this family of closely related strains . This sensitive method entails PCR amplification with arbitrary primers at low stringency and yields arrays of anonymous DNA fragments that are strain specific . Among 150 fragments scored, eight were polymorphic in that they were produced from some but not all strains . Seven polymorphic bands were chromosomal, and one was from the F-factor plasmid . Five of the six mapped polymorphic chromosomal bands came from just 7% of the genome, a 340-kb segment that includes the terminus of replication . Two of these were from the cryptic Rac prophage, and the inability to amplify them from strains was attributable to deletion (excision) or to rearrangement of Rac . Two other terminus-region segments that resulted in polymorphic bands appeared to have sustained point mutations that affected the ability to amplify them . Control experiments showed that RAPD bands from the 340-kb terminus-region segment and also from two plasmids (P1 and F) were represented in approximate proportion to their size . Optimization experiments showed that the concentration of thermostable polymerase strongly affected the arrays of RAPD products obtained . Comparison of RAPD polymorphisms and positions of strains exhibiting them in the pedigree suggests that many sequence changes occurred in these historic E . coli strains during their storage . We propose that the clustering of such mutations near the terminus reflects errors during completion of chromosome replication, possibly during slow growth in the stab cultures that were often used to store E . coli strains in the early years of bacterial genetics.

J Bacteriol, 1994 Mar, 176(6), 1609 - 15
Cell cycle-dependent transcription from the gid and mioC promoters of Escherichia coli; Ogawa T et al.; Transcription from the gid and mioC promoters, which neighbor the origin of replication of the Escherichia coli chromosome (oriC), has been implicated in the control of initiation of replication of minichromosomes . The amounts of transcripts from these two promoters on the chromosome were quantified at various times in a synchronized culture of a temperature-sensitive dnaC mutant strain . Transcription from the gid promoter was most active before the initiation of replication and was inhibited after initiation, during the time corresponding to the period of sequestration of the oriC region from the dam methyltransferase . On the other hand, transcription from the mioC promoter was inhibited before initiation and the inhibition was relieved after initiation prior to the recovery of gid transcription . The strict regulation of transcription from the gid and mioC promoters may be involved in positive and negative control of chromosomal replication, respectively, as has been suggested for minichromosome replication . The DnaA protein was involved in repression of mioC transcription, indicating that the activity of the DnaA protein changes during the cell cycle.

J Bacteriol, 1994 Mar, 176(6), 1598 - 608
Treponema pallidum rare outer membrane proteins: analysis of mobility by freeze-fracture electron microscopy; Bourell KW et al.; Freeze-fracture and deep-etch electron microscopy were used to investigate the molecular architecture of the Treponema pallidum outer membrane (OM) . Freeze-fracture electron microscopy of treponemes freshly harvested from rabbit testes revealed that the intramembranous particles (IMPs) in both the concave and convex OM leaflets were distributed into alternating areas of relatively high and low particle density; in many OM fractures, IMPs formed rows that ran either parallel to or obliquely across the fracture faces . Statistical analysis (runs test) confirmed that the IMPs were nonrandomly distributed in both OM leaflets . Examination of deep-etched specimens revealed that the particles observed in freeze-fractured OMs also were surface exposed . Combined analysis of deep-etched and cross-fractured treponemes revealed that the OM particles were located in regions of the OM away from the endoflagella and closely apposed to the cytoplasmic membrane-peptidoglycan complex . When treponemes were incubated for extended periods with heat-inactivated immune rabbit syphilitic serum, no alteration in the distribution of OM IMPs was detected . In further experiments, approximately 1:1 mixtures of T . pallidum and Escherichia coli or separate suspensions of the nonpathogenic Treponema phagedenis biotype Reiter were fixed at 34 degrees C or after cooling to 0 degree C (to induce lateral phase separations that would aggregate IMPs) . Only particles in the T . pallidum OM failed to aggregate in cells fixed at the lower temperature . The combined data suggest that the mobility of T . pallidum rare OM proteins is limited, perhaps as a result of interactions between their periplasmic domains and components of the peptidoglycan-cytoplasmic membrane complex.

J Bacteriol, 1994 Mar, 176(6), 1570 - 7
Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli; Ryder L et al.; The formation of recombinants in Hfr crosses was studied in Escherichia coli strains carrying combinations of genes known to affect recombination and DNA repair . Mutations in ruv and recG eliminate activities that have been shown to process Holliday junction intermediates by nuclease cleavage and/or branch migration . Strains carrying null mutations in both ruv and recG produce few recombinants in Hfr crosses and are extremely sensitive to UV light . The introduction of additional mutations in recF, recJ, recO, recQ, or recR is shown to increase the yield of recombinants by 6- to 20-fold via a mechanism that depends on recBC . The products of these genes have been linked with the initiation of recombination . We propose that mutation of recF, recJ, recO, recQ, or recR redirects recombination to events initiated by the RecBCD enzyme . The strains constructed were also tested for sensitivity to UV light . Addition of recF, recJ, recN, recO, recQ, or recR mutations had no effect on the survival of ruv recG strains . The implications of these findings are discussed in relation to molecular models for recombination and DNA repair that invoke different roles for the branch migration activities of the RuvAB and RecG proteins.

EMBO J, 1994 Mar 1, 13(5), 1226 - 34
In vivo assembly of active maltose binding protein from independently exported protein fragments; Betton JM et al.; The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides . It is synthesized in the cytoplasm with an N-terminal signal peptide that is cleaved upon export . We examined whether active MBP could assemble into an active protein in bacteria, from N- and COOH-terminal complementary protein fragments encoded by distinct, engineered segments of its structural gene . We found export and functional periplasmic assembly of MBP fragments, despite the complex polypeptide chain topology of this protein, if two conditions were satisfied . First, each of the two fragments must carry a signal peptide . Second, the boundaries between the two fragments must correspond to a permissive site within the protein . Functional assembly of active MBP occurred in five cases where these conditions were met: sites after residues 133, 161, 206, 285 and 303; but not in three other cases where the break junction corresponded to a non-permissive site: after residues 31, 120 and 339 . Thus, permissive sites which were initially characterized because they could accept extensive genetic insertion/deletion modifications without loss of most biological properties provide a means of defining complementing protein fragments . This observation opens a way to study genetically the relationships between protein export and folding into the periplasm.

EMBO J, 1994 Mar 1, 13(5), 1028 - 38
Electron transfer from plastocyanin to photosystem I; Haehnel W et al.; Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e . the thylakoid-targeting domain that acts as a bacterial export signal . The identity of the purified plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry . The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes . The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87 . The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin . We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+ . The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin . This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule . Plastocyanin (Y83L) expressed in either E . coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein . The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.

Toxicol Appl Pharmacol, 1994 Mar, 125(1), 17 - 26
Effects of atmospheric ammonia on pulmonary hemodynamics and vascular permeability in pigs: interaction with endotoxins; Gustin P et al.; The influence of atmospheric ammonia on the somatic growth, the plasma cortisol and ammonia concentrations, and cell blood counts was investigated in pigs exposed to four concentrations (0, 25, 50, and 100 ppm) for 6 days in a specifically designed air-pollutants exposure chamber . The effects of this gas on pulmonary vascular hemodynamics and permeability and on the endotoxin-induced vascular response were also assessed using an isolated perfused lung preparation . The total pulmonary blood flow resistance (Rt) was partitioned into four components: arterial (Ra), pre-(Ra') and post-(Rv') capillary and venous (Rv) . The capillary filtration coefficient (Kf,c) was evaluated by using a gravimetric technique . None of the concentrations of ammonia significantly modified the plasma cortisol and ammonia concentrations or the differential leukocyte percentages and total white blood cell count, suggesting an absence of stress related to ammonia . In exposed animals, lethargy and a concentration-related depression of the somatic growth were observed . The equation of the regression line plotted relating the mean values of the changes in body weight gain recorded over the exposure period expressed as percentages of the initial body weight (y) and ammonia concentrations (x) was: y = 3.204 - 0.177x + 0.001x2 (r = 0.99; p < or = 0.013) . Endotoxin infused in the perfusion liquid of lungs from unexposed animals for 180 min induced a significant 208% increase in Rt (p < 0.001) which can be ascribed to a 338 and 180% increase in Ra' and Rv', respectively . Endotoxin infusion also induced a 62% (p < or = 0.001) increase in the Kf,c . Exposure of pigs to ammonia at any concentration did not modify the baseline values of any hemodynamic or permeability parameters . However, the hemodynamic response to endotoxins in lungs from pigs exposed to 100 ppm was significantly altered . The increase in Rt, Ra', and Rv' observed in unexposed pigs was completely abolished as shown by the limited changes in Rt (+34.9%) . An intermediate reaction (+131.7%) was obtained in pigs exposed to 50 ppm . This inhibiting effect of ammonia was closely correlated with gas concentration by a linear regression (r = 0.99; p < or = 0.037) . The changes in the Kf,c recorded in the control group were not modified by exposure to ammonia . It was concluded that exposure of pigs to aerial ammonia concentrations from 0 to 100 ppm for 6 days has no direct effect on the pulmonary microvascular hemodynamics and permeability and induces no stress response.(ABSTRACT TRUNCATED AT 400 WORDS)

Surgery, 1994 Mar, 115(3), 310 - 24
Splanchnic and systemic hemodynamic responses to portal vein endotoxin after resuscitation from hemorrhagic shock; Gavin TJ et al.; BACKGROUND . Hemorrhagic shock and sepsis are usually studied separately or in rodents . This study combined the two insults in a large animal model . METHODS . Anesthetized pigs were bled, held in shock for 1 hour, and then resuscitated with fluid . After 3 days, Escherichia coli endotoxin LPS was infused into the portal vein (150 micrograms/kg x 30 min) to mimic the effect of enteric substances breaching the mucosal barrier . Systemic and splanchnic hemodynamics, circulating leukocytes, and plasma levels of tumor necrosis factor (TNF) were measured in five groups: 40% hemorrhage plus fluid only resuscitation, 40% hemorrhage plus fluid-blood resuscitation, 50% hemorrhage plus fluid-blood resuscitation, sham, or sham plus 5 micrograms/kg LPS priming dose instead of hemorrhage . RESULTS . On day 4 before infusion of LPS, there were no differences between groups in hemodynamics or O2 utilization, but systemic O2 delivery and O2 consumption were each reduced in the hemorrhaged groups because of the hemodilution associated with resuscitation . For 3 hours after infusion of LPS, all animals received aggressive fluid and respiratory support, but arterial blood pressure decreased, and systemic O2 utilization, splanchnic O2 utilization, and arterial lactate level increased; there were no differences between groups . In the 50% group compared with sham, LPS-evoked decreases in cardiac index and stroke index were eliminated, and LPS-evoked tachycardia, pulmonary and systemic vasoconstriction, and increases in hepatic and portal vein lactate levels were blunted . Despite similar leukocyte counts before infusion of LPS and similar leukopenia after LPS infusion, plasma LPS level was higher in the 50% group compared with sham . Furthermore, LPS evoked increases in portal and hepatic vein plasma TNF in the shams, but those changes were reduced in the 50% group . CONCLUSIONS . Most responses to LPS were similar after hemorrhagic shock or a sham operation, which is inconsistent with the concept of "priming." LPS-evoked increases in plasma TNF were blunted after shock, probably because of trauma-induced immune dysfunction . A combined shock plus septic challenge in a large animal model may be valuable for investigating the pathogenic mechanism in human beings.

Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1922 - 6
Differential roles of the transposon termini in IS91 transposition; Mendiola MV et al.; Insertion sequence 91 (IS91) inserts specifically at GTTC or CTTG target sequences without duplication of the target . After insertion, the right inverted repeat (IRR) lies adjacent to the 3' end of the target sequences (or 5' to the complementary sequence CAAG or GAAC) . We have analyzed the effects of alteration of each terminus of IS91 on transposition activity in Escherichia coli . IRR is absolutely required for transposition . Deletion analysis indicates that a 14-bp segment is not sufficient, but an 81-bp sequence within the IRR region is sufficient . Furthermore, the GTTC/CTTG target site is also required . The left inverted repeat (IRL) of IS91 is dispensable . Plasmid fusions originated by one-ended transposition of IS91 derivatives lacking IRL occur at about the same frequency as cointegrate formation observed for the wild-type element . In the one-ended-type fusions, the inserted fragment of donor DNA is flanked at one end (constant end) by IRR and at the other end by a GTTC or CTTG sequence present in the donor (variable end) in a way that usually results in multiple tandem insertions of the donor plasmid in the target site . These results are easily accommodated by a rolling-circle replicative transposition mechanism . This model also draws support from the finding that the IS91 transposase is related in sequence to the superfamily of rolling-circle replication proteins and the observation that IRR shows some conservation in sequence and secondary structure with the origins of replication of some rolling-circle replication plasmids.

Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1917 - 21
Temperature acclimation and competitive fitness: an experimental test of the beneficial acclimation assumption; Leroi AM et al.; Phenotypic acclimation is generally assumed to confer an advantage in the environment that stimulates the response . To test this beneficial acclimation assumption explicitly, we investigated the consequences of temperature acclimation for the fitness of Escherichia coli at two temperatures, 32 degrees C and 41.5 degrees C . Both temperatures permit growth and long-term persistence of the genotypes in serial culture . We found that prior acclimation to 32 degrees C, relative to acclimation to 41.5 degrees C, enhanced fitness at 32 degrees C, consistent with the assumption . But prior acclimation to 41.5 degrees C actually reduced fitness at 41.5 degrees C, relative to acclimation to 32 degrees C . Hence, the assumption that acclimation always confers an advantage is demonstrated to be false . Acclimation to 41.5 degrees C did, however, improve survival at 50 degrees C, a lethal temperature . This protective response has been shown to be associated with the induction of stress proteins . The reduced competitive fitness caused by acclimation at 41.5 degrees C may reflect a physiological burden associated with expression of stress proteins when they are not needed to prevent lethal damage . Whatever the cause, acclimation to the higher temperature decreased competitive fitness at that temperature.

Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1731 - 5
KAP: a dual specificity phosphatase that interacts with cyclin-dependent kinases; Hannon GJ et al.; The cyclin-dependent kinases are key cell cycle regulators whose activation is required for passage from one cell cycle phase to the next . In mammalian cells, CDK2 has been implicated in control of the G1 and S phases . We have used a two-hybrid protein interaction screen to identify cDNAs encoding proteins that can interact with CDK2 . Among those identified was a protein (KAP), which contained the HCXX-XXGR motif characteristic of protein tyrosine phosphatases . KAP showed phosphatase activity toward substrates containing either phosphotyrosine or phosphoserine residues . Since KAP is not significantly similar to known phosphatases beyond the catalytic core motif, it represents an additional class of dual specificity phosphatase . KAP interacted with cdc2 and CDK2 in yeast . In mammalian cells, KAP also associated with cdc2 and CDK2 but showed a preference for cdc2 . The ability of KAP to bind multiple cyclin-dependent kinases suggests that it may play a role in cell cycle regulation.

Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1721 - 5
Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex; Pan CQ et al.; The Escherichia coli Fis (factor for inversion stimulation) protein functions in many diverse biological systems including recombination, transcription, and DNA replication . Although Fis is a site-specific DNA-binding protein, it lacks a well-defined consensus recognition sequence . The electrophoretic mobility of Fis-DNA complexes, along with considerations of the Fis crystal structure, indicates that significant deformation of DNA occurs upon Fis binding . To investigate the structure of Fis-DNA complexes, the chemical nuclease 1,10-phenanthroline-copper complex (OP-Cu) has been linked to four specific sites within the Fis DNA-binding domain . Two of these Fis-OP derivatives were active in cleaving DNA . The scission patterns obtained on four different Fis binding sites indicate that Fis positions itself on these highly divergent DNA sequences in a very similar fashion . The patterns of cleavage of a derivative at Asn-98 generally support a model of a Fis-DNA complex that contains specific bends within the core-recognition sequence . Data from a second Fis-OP derivative at Asn-73 provides evidence for greater wrapping of flanking DNA around the sides of the Fis protein than was previously postulated . The cleavage efficiency of flanking segments varies, suggesting that the extent of DNA wrapping is sequence dependent . Specific amino acids on Fis are implicated in promoting this DNA wrapping.

Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1657 - 61
Osmotic regulation of gene action; Douzou P; Most reactions involved in gene translation systems are ionic-dependent and may be explained in electrostatic terms . However, a number of observations of equilibria and rate processes making up the overall reactions clearly indicate that there is still an enormous gap between the rough picture of the mechanism of ionic regulation and the detailed behavior of reactions at the molecular level that hold the key to specific mechanisms . The present paper deals with possible osmotic contributions arising from the gel state of gene systems that are complementary to, and interdependent of, electrostatic contributions . This treatment, although still oversimplified, explains many previous observations by relating them to a general osmotic mechanism and suggests experimental approaches to studying the mechanisms of gene regulation in organelle-free and intact systems.

Invest Ophthalmol Vis Sci, 1994 Mar, 35(3), 924 - 30
Cytokine mRNA levels in rat ocular tissues after systemic endotoxin treatment; Planck SR et al.; PURPOSE . An intertwined cascading network of cytokines is believed to direct the development of endotoxin-induced uveitis (EIU) . This study investigated mRNA levels of interleukin (IL) 1 alpha, IL-1 beta, IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta 1, and the helper T lymphocyte marker, CD4, during the course of EIU in rats . METHODS . Reverse transcription followed by polymerase chain reaction amplification was used to determine relative mRNA levels in four ocular tissues (iris/ciliary body, cornea, lens, and neuroretina) at 0, 1, 3, 6, 24, and 48 hours after subcutaneous injection of 200 micrograms of Escherichia coli endotoxin . RESULTS . Four general patterns of mRNA expression were observed: (1) constitutively expressed and unaffected by endotoxin; (2) constitutively expressed but further induced by endotoxin, reaching peak levels at 3 hours postinjection; (3) initially undetectable or marginally detectable and induced by endotoxin, with peak levels occurring 3 hours postinjection; and (4) never present at appreciable levels . The most dramatic responses were seen in the mRNA levels of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma in iris/ciliary body . Lesser mRNA level responses were found for IL-1 beta and IL-6 in cornea and for IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma in neuroretina . Little or no changes in mRNA levels were observed for CD4 or TGF-beta 1 in any tissue or for any mRNA examined in lens . CONCLUSIONS . These data show that subcutaneous endotoxin induces cytokine mRNA expression differentially in ocular tissues . These data support the hypothesis that induction of cytokine expression in iris/ciliary body plays a major role in the development of EIU.

Eur J Biochem, 1994 Mar 1, 220(2), 623 - 31
Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis; Haruki M et al.; The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the alpha V helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis . Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E . coli strain MIC3001 . Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo . All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability . Among them, only the mutant proteins {D134H}RNase H and {D134N}RNase H were shown to have considerable ribonuclease H activities . Determination of the kinetic parameters revealed that replacement of Asp134 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding . Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure . The protein stability was determined from the thermal denaturation curves . All mutant proteins were more stable than the wild-type protein . Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the alpha V helix . These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its delta 1 position impacts catalysis.

Eur J Biochem, 1994 Mar 1, 220(2), 607 - 14
Cloning and functional expression of poly(ADP-ribose) polymerase cDNA from Sarcophaga peregrina; Masutani M et al.; A cDNA spanning the entire coding region for poly(ADP-ribose) polymerase (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequence was determined . The longest open reading frame encodes a polypeptide of 996 amino acid residues with a molecular mass of 113,033 Da . The similarities to the human PARP in amino acid sequence were relatively low in the DNA-binding and auto-modification domains, but very high in the C-terminal catalytic domain: identity of amino acids is 34% in the N-terminal DNA-binding domain (residues 1-369), 27% in the auto-modification domain (residues 370-507), and 56% in the C-terminal NAD-binding domain (residues 508-996) . Two zinc-fingers (C-X2-C-X28-H-X2-C and C-X2-C-X31-H-X2-C)2 and a basic region in the N-terminal DNA-binding domain recognized in other PARP are conserved . Downstream of the basic region, another cysteine-rich motif (C-X2-C-X13-C-X9-C), a putative zinc-finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human . A leucine-zipper motif (L-X6-L-X6-L-X6-L) which was found in the auto-modification domain of Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second leucine is replaced by proline, and the third leucine by valine . Full-length cDNA for Sarcophaga PARP was cloned into an expression plasmid and expressed in Escherichia coli . A lysate of E . coli cells containing expressed protein reacted with antibody against Sarcophaga PARP, and PARP activity was detected . Thus, we conclude that isolated cDNA encodes a functional Sarcophaga PARP cDNA.

Eur J Biochem, 1994 Mar 1, 220(2), 551 - 8
Disruption of the gene encoding the NADH-binding subunit of NADH: ubiquinone oxidoreductase in Neurospora crassa . Formation of a partially assembled enzyme without FMN and the iron-sulphur cluster N-3; Fecke W et al.; In this study, the gene of the 51-kDa NADH-binding subunit of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) in Neurospora crassa was inactivated by homologous replacement with a defective gene copy . The resulting mutant, nuo51, lacks the 51-kDa subunit and shows no complex I activity but still grows at one third of the wild-type growth rate . The enzyme activity of the alternative NADH:ubiquinone oxidoreductase(s) is increased twofold while the activities of the other mitochondrial respiratory enzymes are normal . Complex I is almost completely assembled except for the NADH-binding subunit and still possesses three out of the four EPR-detectable iron-sulphur clusters . Since the deleted subunit contains the sequence motif for one tetranuclear iron-sulphur cluster, the missing cluster N-3 is considered to be bound to this subunit.

Eur J Biochem, 1994 Mar 1, 220(2), 543 - 9
Characterization of aromatic aminotransferases from the hyperthermophilic archaeon Thermococcus litoralis; Andreotti G et al.; The hyperthermophilic archaeon (formerly archaebacterium) Thermococcus litoralis grows at temperatures up to 98 degrees C using peptides and proteins as the sole sources of carbon and nitrogen . Cell-free extracts of the organism contained two distinct types of aromatic aminotransferases (EC 2.6.1.57) which were separated and purified to electrophoretic homogeneity . Both enzymes are homodimers with subunit masses of approximately 47 kDa and 45 kDa . Using 2-oxoglutarate as the amino acceptor, each catalyzed the pyridoxal-5'-phosphate-dependent transamination of the three aromatic amino acids but showed virtually no activity towards aspartic acid, alanine, valine or isoleucine . From the determination of Km and kcat values using 2-oxoglutarate, phenylalanine, tyrosine and tryptophan as substrates, both enzymes were shown to be highly efficient at transaminating phenylalanine (kcat/Km approximately 400 s-1 mM-1); the 47-kDa enzyme showed more activity towards tyrosine and tryptophan compared to the 45-kDa one . Kinetic analyses indicated a two-step mechanism with a pyridoxamine intermediate . Both enzymes were virtually inactive at 30 degrees C and exhibited maximal activity between 95-100 degrees C . They showed no N-terminal sequence similarity with each other (approximately 30 residues), nor with the complete amino acid sequences of aromatic aminotransferases from Escherichia coli and rat liver . The catalytic properties of the two enzymes are distinct from bacterial aminotransferases, which have broad substrate specificities, but are analogous to two aromatic aminotransferases which play a biosynthetic role in a methanogenic archaeon . In contrast, it is proposed that one or both play a catabolic role in proteolytic T . litoralis in which they generate glutamate and an arylpyruvate . These serve as substrates for glutamate dehydrogenase and indolepyruvate ferredoxin oxidoreductase in a novel pathway for the utilization of aromatic amino acids.

Eur J Biochem, 1994 Mar 1, 220(2), 447 - 54
Tandem overproduction and characterisation of the nuclease domain of colicin E9 and its cognate inhibitor protein Im9; Wallis R et al.; We report the overproduction of the non-specific endonuclease domain of the bacterial toxin colicin E9 and its preliminary characterisation in vitro . The enzymatic colicins (61 kDa) are normally released from producing cells in a complex with their cognate inhibitors, known as the immunity proteins (9.5 kDa) . Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N-terminal and central regions of the toxin and a heterodimer comprising the catalytically active C-terminal domain of the colicin bound to its intact immunity protein, Im9 . N-terminal amino acid sequencing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heterogeneous, thus making subsequent mechanistic and structural analysis difficult . This problem was circumvented by selectively overexpressing the C-terminal 15-kDa nuclease domain of colicin E9 in tandem with its cognate inhibitor in Escherichia coli . This tandem overexpression strategy allowed high-level production of a 25-kDa protein complex comprising the C-terminal DNase domain of colicin E9 tightly bound to its specific inhibitor Im9, thus masking the anticipated toxicity of the nuclease . The DNase domain was then separated from Im9 under denaturing conditions, refolded by removal of the denaturant and the renatured protein shown to possess both endonuclease and Im9 binding activity . These results describe a novel method for the overproduction of a nuclease in bacteria by co-expressing its specific inhibitor and lay the foundations for a full mechanistic, biophysical and structural characterization of the isolated DNase domain of the colicin E9 toxin.

Eur J Biochem, 1994 Mar 1, 220(2), 409 - 13
Spectrochemical evidence for the presence of a tyrosine residue in the allosteric site of glucosamine-6-phosphate deaminase from Escherichia coli; Altamirano MM et al.; The interaction of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli with its allosteric activator, N-acetyl-D-glucosamine 6-phosphate, was studied by different spectrophotometric methods . Analysis of the circular-dichroism differential spectra produced by the binding of the allosteric activator or the competitive inhibitor 2-amino-2-deoxy-D-glucitol 6-phosphate (a homotropic ligand displacing the allosteric equilibrium to the R conformer), strongly suggests the presence of tyrosine residues at or near the allosteric site, although a conformational effect cannot be ruled out . The involvement of a single tyrosine residue in the N-acetyl-D-glucosamine-6-phosphate binding site of glucosamine-6-phosphate deaminase was supported by spectrophotometric pH titrations performed in the presence or absence of the homotropic and heterotropic ligand . In these experiments, a single titrated tyrosine residue is completely protected by saturation with the allosteric activator; this group is considerably acidic (pK 8.75) . The analysis of the amino acid sequence of the deaminase using a set of indices for the prediction of surface accessibility of amino acid residues, suggests that the involved residue may be Tyr121 or Tyr254.

Eur J Biochem, 1994 Mar 1, 220(2), 377 - 84
Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C-terminal processing at Arg537; Rossmann R et al.; Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel-containing polypeptide, which is subject to C-terminal proteolytic processing . This processing reaction could be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel . N-terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C-terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination . Nickel-containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (delta hycH) in the presence of the reducing agent dithiothreitol . Using 2-mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing . This conclusion was supported by the finding that HYCE precursor from strain HD708 (delta hycH) chromatographed with radioactivity from 83Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from strain BEF314 (delta hypB-E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.

Eur J Biochem, 1994 Mar 1, 220(2), 349 - 57
Enhancement by lipoprotein-free plasma of the inhibitory effect of endotoxin on endocytotic catabolism of low density lipoproteins in Hep G2 cells; Liao W et al.; We previously showed that the presence of microgram levels of endotoxin inhibited low-density lipoprotein (LDL) uptake and degradation in Hep G2 cells . We also showed that both the polysaccharide and lipid A parts of endotoxins are needed for the inhibitory effects of endotoxins on cellular LDL uptake . The current study was carried out by inclusion of lipoprotein-free plasma (LFP) in tissue culture medium to observe the modulatory influence of non-lipoprotein factor(s) on endotoxin-induced inhibition of endocytotic catabolism of LDL in Hep G2 cells . We found that LFP dramatically promotes the inhibitory effect of endotoxins with a complete polysaccharide, but has no influence on the effect of the Re mutant endotoxin (from S . minnesota Re595), which lacks polysaccharide . By using gel-filtration chromatography, agarose electrophoresis and agarose isoelectric focusing, we further showed that in the presence of LFP, both the endotoxins with a complete polysaccharide and the Re mutant endotoxin complex with and anionize LDL, while in the absence of LFP, these endotoxins poorly interact with LDL . Thus, endotoxin inhibits cellular endocytotic catabolism of LDL by forming LDL-endotoxin complexes, and LFP enhances endotoxin-induced inhibition of endocytotic catabolism of LDL by promoting the interaction between endotoxin and LDL . In addition, our finding that the Re mutant endotoxin also interacts with LDL to form LDL-endotoxin complexes, but has no significant effect on LDL uptake and degradation, further supports the notion that both the polysaccharide and lipid A parts of endotoxins are needed for the inhibitory effects of endotoxins on cellular LDL uptake.

Eur J Biochem, 1994 Mar 1, 220(2), 331 - 8
Triose-phosphate isomerase of Leishmania mexicana mexicana . Cloning and characterization of the gene, overexpression in Escherichia coli and analysis of the protein; Kohl L et al.; The gene of triose-phosphate isomerase in Leishmania mexicana has been cloned and characterized . The gene encodes a polypeptide of 251 amino acids, with a calculated molecular mass of 27,561 Da and a net charge of +2 . Only one gene could be detected, although the enzyme is present in two different compartments of the cell, in microbody-like organelles called glycosomes and in the cytosol . The primary structure of the enzyme has many features in common with that of triose-phosphate isomerase in the related organism Trypanosoma brucei . Their sequences are 68% identical . The residues constituting the subunit interface are highly conserved between the enzyme of L . mexicana and T . brucei, but are mostly different from those in the enzyme of other organisms . One major substitution was detected in the interface region of the L . mexicana protein: a glutamate was found at position 66, instead of glutamine in all other available 20 sequences . The glutamine is thought to be important for the stability of the dimeric enzyme . L . mexicana triose-phosphate isomerase has been overexpressed in Escherichia coli . Growth conditions were established to obtain high levels of soluble and active protein . The enzyme has been purified to near homogeneity . It appears a stable dimeric protein with a specific activity of 5500 units/mg protein, a subunit mass of 28 kDa and an isoelectric point of 9.0 . The enzyme has also been partially purified from glycosomes of cultured L . mexicana promastigotes . Some kinetic properties of the recombinant protein have been compared with those of the promastigote enzyme and with the values previously reported for the T . brucei enzyme . The kinetics of the different enzyme preparations were very similar . For the recombinant enzyme the following values were measured: with glyceraldehyde 3-phosphate as substrate Km = 0.30 +/- 0.05 mM and kcat = 2.5 x 10(5) min-1; with dihydroxyacetone phosphate as substrate Km = 1.3 +/- 0.3 mM and kcat = 2.8 x 10(4) min-1.

Crit Care Med, 1994 Mar, 22(3), 499 - 505
In vivo 31P nuclear magnetic resonance spectroscopy of skeletal muscle energetics in endotoxemic rats: a prospective, randomized study; Gilles RJ et al.; OBJECTIVE: To identify possible alterations in the skeletal muscle high-energy phosphate metabolism at the early phase of endotoxic shock in rats . DESIGN: A prospective, randomized study of skeletal muscle energetics in endotoxemic and in control rats, by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy at rest, under regional ischemia, and during reperfusion . SETTING: Biochemical NMR laboratory equipped for surgery and hemodynamic monitoring . SUBJECTS: Wistar rats were randomized to different groups . Eight rats were injected with Escherichia coli endotoxin (15 mg/kg, survival time 19 +/- 4 hrs) intraperitoneally . Seven other rats served as controls . The additional nine rats were studied for the saturation recovery pulse sequence . INTERVENTIONS: In the treatment group, endotoxin was injected 8 hrs before NMR spectroscopy . The right hind limbs were studied under anaesthesia using a surface coil NMR probe . Their high-energy phosphate contents and intracellular pH were determined by 31P NMR spectroscopy . After baseline measurements, an ischemia-reperfusion challenge was imposed on the muscle by transient clamping of the abdominal aorta . Contralateral femoral artery pressure was constantly monitored . MEASUREMENTS AND MAIN RESULTS: During the baseline period, the endotoxin-treated muscles did not show any difference in the distribution of the high-energy phosphate compounds or in intracellular pH, as compared with the controls . Ischemia resulted in a significantly faster decline of the inorganic phosphate/creatine phosphate ratio in the endotoxin-treated rats (1.35 +/- 0.17 vs . 0.51 +/- 0.06 at the end of the 38-min ischemic period) . Skeletal muscle acidosis developed earlier and was deeper in the endotoxemic animals (pH: 6.94 +/- 0.02 vs . 7.02 +/- 0.03 at the end of ischemia) . During reperfusion, the calculated time constants of recovery of inorganic phosphate to phosphocreatine ratios were identical between groups . CONCLUSIONS: Resting nonischemic muscles of endotoxin-treated rats show no evidence of alterations in high-energy phosphate metabolism . However, under ischemic conditions, high-energy phosphate metabolism deteriorates faster in the skeletal muscles of treated animals . These data support the hypothesis of a greater mismatch during perfusion at very low pressure between residual oxygen availability and oxygen needs in the endotoxin-treated muscle cell.

Virology, 1994 Mar, 199(2), 448 - 52
The open reading frames 1, 2, 71, and 75 are nonessential for the replication of equine herpesvirus type 1 in vitro; Sun Y et al.; Equine herpesvirus type 1 (EHV-1) strain Ab4 has five genes, 1, 2, 67, 71, and 75, which have no homologues in any of the herpesviruses sequenced to date; i.e., they are unique to EHV-1 . The functions of these unique genes are not known . To study their role in the virus life cycle, we have constructed four independent mutants in which the majority of the coding sequences of genes 1, 2, 71, and 75 has been deleted and replaced by the Escherichia coli lacZ gene . Mutant ED1 has a deletion of 508 bp within the 608-bp gene 1 open reading frame (ORF); mutant ED2 has a 400-bp deletion within the 617-bp gene 2 ORF; mutant ED71 has a 1811-bp deletion within the 2393-bp gene 71 ORF, whereas mutant ED75 lacks 189 bp of the 392-bp gene 75 ORF . Mutants ED1, ED2, and ED75 display growth characteristics indistinguishable from those of wild-type virus in cell culture . The growth in vitro of ED71 is marginally impaired compared to that of wild-type virus . Our results demonstrate that genes 1, 2, 71, and 75 are not essential for EHV-1 growth in tissue culture . The isolation of a deletion mutant in gene 67 was unsuccessful, indicating that gene 67 is probably essential for virus growth in tissue culture.

Virology, 1994 Mar, 199(2), 439 - 47
Replicase-mediated resistance to cucumber mosaic virus in transgenic plants involves suppression of both virus replication in the inoculated leaves and long-distance movement; Carr JP et al.; Tobacco plants transformed with a gene encoding a truncated cucumber mosaic virus (CMV) 2a replicase protein are resistant to systemic CMV disease . Experiments using protoplasts derived from plants of two R2-generation CMV-resistant transgenic plant lines (lines R2-2 and R2-5) showed that resistance operates at the single cell level . Low levels of CMV-specific RNAs were detected in CMV-inoculated protoplasts obtained from both R2-2 and R2-5 plants indicating that resistance is due at least in large part to a marked but incomplete suppression of virus replication . Leaves of immature plants belonging to line R2-2 occasionally exhibited local chlorosis when inoculated with high concentrations of CMV . Areas of local chlorosis were sites of low but detectable levels of CMV RNA, CMV virions, and CMV replicase activity, but did not act as foci for subsequent systemic disease . An antiserum raised against the CMV 2a replicase protein overexpressed in Escherichia coli was used to detect the presence of trace amounts of the truncated CMV 2a replicase protein in CMV-resistant transgenic tobacco plants . It was concluded that expression of the transgene, potentially as protein, engenders resistance primarily by suppressing virus replication but may also, to a lesser extent, do so by inhibiting systemic movement of the virus.

Virology, 1994 Mar, 199(2), 366 - 75
Isolation and characterization of mutants of pseudorabies virus with deletion in the immediate-early regulatory gene; Yamada S et al.; Pseudorabies virus (PrV) immediate-early regulatory protein (IE180) is a potent and promiscuous activator of viral and cellular gene transcription . A mutant with deletion in the IE180 gene is expected to be useful for elucidating the functions of IE180 . In this study, we constructed such mutants . Initially, transformants that express IE180 were established from PK-15 and Vero cells by transfection with the IE180 gene of the wild-type PrV . Two types of the deletion mutant could be obtained by using those transformants . One type was the deletion mutant, represented by AY64, in which the IE180 gene was replaced by insertion of the modified Escherichia coli lacZ gene, which was inserted under the control of the IE180 promoter . The other type, represented by AY1030, was a mutant produced from the former type by subsequent deletion of the lacZ gene . The deletion mutants of both types could replicate in the transformants with the IE180 gene, but not in the parent cells . On the other hand, the lacZ gene in AY64 was functional under the control of the IE180 promoter and could express beta-galactosidase (beta-gal) in not only the transformants but also the parent cells, indicating that the IE180 promoter of the mutants can fulfill the intended function in inoculated cells . The parent cells infected with the AY64 expressed only beta-gal, but neither infectious virus nor viral products relating to PrV . The deletion mutants constructed in this study appeared to provide a helpful model for investigating the functions of the IE180-homologs of other herpesviruses as well as IE180 itself, since PrV has only one IE gene . The applicability of the deletion mutants as a useful and safe vector for the introduction of foreign genes into various cells and animals was discussed.

Hepatology, 1994 Mar, 19(3), 758 - 63
Blood-to-lymph migration of small lymphocytes through the liver of the sheep; Young AJ et al.; The process of lymphocyte migration is required for the systemic dissemination of immunological memory and immune surveillance . We report here experiments to quantitate the normal traffic of lymphocytes that occurs from blood to lymph through the liver and hepatic node in the sheep . Comparisons were made with known lymphocyte homing pools . Individual afferent hepatic lymphatics had cell outputs of 1.4 +/- 0.1 x 10(6) cells/hr, suggesting that the total combined lymphocyte output from the liver was no greater than about 1 x 10(7) cells/hr . The lymphocyte output in efferent hepatic lymph was 6.2 +/- 0.4 x 10(7) cells/hr, comparable to the cell outputs recorded from other lymph nodes of similar size . When the specificity of lymphocytes homing through the liver or hepatic node was examined, we found similarities to both the peripheral lymph node and intestinal lymph node homing patterns . Migration into afferent hepatic lymph was found to be different from that into intestinal or subcutaneous efferent lymph, and the kinetics of migration into hepatic afferent lymph was faster than that observed into efferent compartments . Intravenously injected endotoxin was found to alter the normal lymph flow through the liver tissue and the hepatic node; it appeared to enhance the migration of macrophages out of the liver by way of the afferent lymph . These studies suggest unique features of lymphocyte traffic through the liver and the need for further experiments on hepatic lymphocyte traffic, particularly in pathological states with substantial mononuclear cell infiltration.

Endocrinology, 1994 Mar, 134(3), 1082 - 8
Recombinant bovine somatotropin blunts plasma tumor necrosis factor-alpha, cortisol, and thromboxane-B2 responses to endotoxin in vivo; Elsasser TH et al.; In vivo studies determined the effects of recombinant bovine somatotropin (bST; sometribove) administration (0.1 mg/kg.day, im) to calves on the increases in plasma immunoreactive tumor necrosis factor-alpha (TNF alpha), prostacyclin {6-keto-prostaglandin F1 alpha (6KP)}, thromboxane-B2 (TXB), and cortisol (C) that occurred after endotoxin challenge (ET) . Two ETs were administered 5 days apart to test the effect of bST on the natural attenuation of hormone and cytokine responses that occurs after repeated challenge with endotoxin . Calves (n = 6) were treated with bST for 5 days . On day 6, the first ET was administered (Escherichia coli; 055:B5; 0.2 microgram/kg, i.v.) . Blood was sampled before and hourly after ET through 6 h . For the next 4 days, bST injections continued, and ET was repeated 1 day later . Six additional calves were treated with bicarbonate buffer as contemporary controls for the bST and were similarly challenged with endotoxin . Plasma TNF alpha, C, 6KP, and TXB were significantly increased after each ET . The increases in TNF alpha, C, and TXB were blunted after the second Et compared to those after the first in both untreated and bST-treated animals . The increases in plasma TNF alpha and C and peak plasma TXB and TXB/6KP ratios were smaller in bST-treated than in untreated after the first ET . TNF alpha receptor binding was studied in hepatic microsomal fractions from three bST-treated and three untreated calves . Microsomal fractions from bST-treated calves bound 40% less TNF alpha (5.97 vs . 9.96 pmol/mg) than similar fractions from controls . The data indicate that bST decreases TNF alpha, TXB, and C responses to ET and reduces the TNF alpha-binding capacity of hepatic membranes, suggesting a multiplicity of sites where bST might affect the physiological response to endotoxin.

Carcinogenesis, 1994 Mar, 15(3), 533 - 7
3T3 NIH murine fibroblasts and B78 murine melanoma cells expressing the Escherichia coli N3-methyladenine-DNA glycosylase I do not become resistant to alkylating agents; Imperatori L et al.; The role of alkylation of the N3 position of adenine in the cytotoxicity of alkylating agents in mammalian cells is still undefined . By co-transfecting NIH3T3 murine fibroblast and murine B78 H1 melanoma cells with pSG5tag and pSV2neo, we obtained clones expressing the mRNA of the bacterial tag gene coding for N3-methyladenine-DNA glycosylase I (Gly I), which specifically repairs N3-methyladenine . The levels of Gly I were 400 times higher in NIH3T3 pSG5tag (clone 3.9.4) and 12-33 times higher in B78 H1 tag clones (2A4, 2A6, 2C3 and 2D1) than in the respective control cells . The sensitivity to alkylating agents was evaluated in tag-expressing cells in comparison with pSG5, pSV2neo co-transfected control cells . As regards the cytotoxic activity of methylating agents (N-methylnitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, dimethylsulfate and temozolomide) and other alkylators with different structure and different interactions with DNA such as CC-1065 and FCE-24517 (minor groove binders known to bind to N3 of adenine), 4-{bis(2-chloroethyl)amino}-L-phenylalanine and cis-diamminedichloroplatinum II, cytotoxicity was the same for tag-expressing and non-expressing cells . These results suggest that the increased expression of N3-methyladenine-DNA glycosylase is not necessarily a crucial mechanism for the resistance of cells to alkylating agents.

Carcinogenesis, 1994 Mar, 15(3), 425 - 9
Fpg protein protects Escherichia coli K-12 from mutation induction by the carcinogen 4-nitroquinoline 1-oxide; Ruiz-Laguna J et al.; This paper investigates the role of the fpg gene product in protecting Escherichia coli cells against the lethal and mutagenic effects of 4-nitroquinoline 1-oxide (4NQO) . To this end, the araD81 mutation which make the cells sensitive to L-arabinose was combined with an fpg-1::Knr allele, in either an uvrA+ or uvrA-, umuC+ or umuC-, genetic background . Mutation induction was monitored by selecting forward mutations to L-arabinose resistance (Arar) . The formamidopyrimidine-DNA glycosylase (Fpg protein) protected bacteria from 4NQO-induced mutagenesis since Fpg- defective cells showed greater Arar mutation induction than fpg+ bacteria did . This was confirmed since the increased sensitivity of the fpg- cells to mutagenesis by 4NQO was suppressed when the Fpg protein was overproduced by placing the fpg gene in a multicopy plasmid vector . The fpg- mutation had no detectable influence on 4NQO mutagenesis in a uvrA- genetic background, but its effect was magnified in umuC- cells . No influence on cell survival was observed after 4NQO treatment . Our data suggest that 8-hydroxyguanine, a non-lethal, non-bulky and directly miscoding lesion, might be responsible for the detected influence of Fpg protein expression on mutation induction by 4NQO . This is in agreement with the reported in vivo formation of 8-hydroxyguanine in cellular DNA after 4NQO exposure . The increased 4NQO-induction of GC to TA transversions on fpg- bacteria further support such a possibility . This work reinforces the role of Fpg protein in the bacterial defense against the mutagenicity by genotoxic agents.

Blood, 1994 Mar 1, 83(5), 1436 - 41
Expression and biochemical characterization of human glucose-6-phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes; Tang TK et al.; We have developed a system to characterize normal and mutated glucose-6-phosphate dehydrogenase (G6PD) enzymes in vitro . Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli . When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes . This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->G; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain . Our results show that the G6PD activities of these three mutants were greatly reduced . No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations . However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation . This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P . In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese . In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.

Biochemistry, 1994 Mar 1, 33(8), 2279 - 84
Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis; Xu X et al.; In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0-A resolution, His-372 was found only 3.8 A away from the zinc and forms a hydrogen-bonding interaction with Asp-327, a bidentate ligand of the zinc at the M1 site . However, His-372 does not directly interact with the zinc atom at the M1 site . In order to investigate the role of the side chain of His-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine . The fact that the His-372-->Ala enzyme has similar zinc binding affinity as the wild-type enzyme indicates that His-372 is not involved in zinc binding at the M1 site . However, the altered kinetic behavior of the mutant enzyme compared to the wild-type enzyme suggests that the imidazole ring of His-372 plays an indirect role in the catalytic mechanism of the enzyme . The hydrolysis activity of the His-372-->Ala enzyme at pH 8.0 is 10-fold lower than that of the wild-type enzyme . In the presence of a phosphate acceptor at pH 8.0, the mutant enzyme is approximately 80% as active as the wild-type enzyme . Therefore, the His-372-->Ala mutation selectively enhances the transphosphorylation activity of the enzyme . The His-372-->Ala enzyme also exhibits 4- and 30-fold decreases in Km as compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris, buffer at pH 8.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Mar 1, 33(8), 2269 - 78
N5-carboxyaminoimidazole ribonucleotide: evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli; Mueller EJ et al.; Conversion of aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins, PurE and PurK, previously thought to be subunits of a single enzyme, AIR carboxylase . Past studies revealing an ATP requirement for this reaction (Meyer et al., 1992), in conjunction with present studies, reveal that PurE and PurK possess independent catalytic activities . PurK is shown, by NMR spectroscopy, to catalyze the conversion of AIR in the presence of HCO3- and ATP to ADP, P(i), and the carbamate of AIR (designated N5-CAIR) . PurE has been shown by NMR spectroscopy and kinetic analysis, to catalyze the reversible conversion of N5-CAIR and CAIR . N5-CAIR has a half-life of 0.9 min at pH 7.8 and 30 degrees C . Thus, two new enzymatic activities and a new intermediate have been discovered in the de novo purine biosynthetic pathway of E . coli.

Biochemistry, 1994 Mar 1, 33(8), 2262 - 8
Interactions of transcription inhibitors with the Escherichia coli RNA polymerase-lacUV5 promoter open complex; Mazumder A et al.; The interactions of transcription inhibitors with the open complex composed of Escherichia coli RNA polymerase and the lacUV5 promoter have been studied using gel retardation, the chemical nuclease activity of the cuprous complexes of 1,10-phenanthroline (OP) and its derivatives, and steady-state kinetics . Gel retardation shows that two inhibitors, the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex {(2,9-Me2OP)2Cu+} and rifampicin, bind stably to the open-complex . (2,9-Me2OP)2Cu+ blocks scission by the chemical nuclease by interfering with the binding of its redox-active isosteres . Rifampicin does not block scission by the cuprous complexes of 3,4,7,8-tetramethyl-OP, 4-phenyl-OP, and OP but does perturb scission by the cuprous complex of 5-phenyl-OP . Organic ligands including intercalating agents and groove binders (e.g., daunomycin, di(amidinophenyl)indole (DAPI), actinomycin D, distamycin, 9-aminoacridine, mithramycin, and chromomycin A3), which bind to free DNA with high affinity, do not form stable ternary complexes with the open-complex . Gel retardation experiments demonstrate that they promote dissociation of the enzyme from the promoter . The greater sensitivity of enzymatic catalysis to inhibitor concentration relative to polymerase binding suggests that these ligands form metastable, catalytically inactive ternary complexes with RNA polymerase and the promoter.

Biochemistry, 1994 Mar 1, 33(8), 2255 - 61
In vitro methylation of Escherichia coli 16S rRNA by tRNA (m5U54)-methyltransferase; Gu X et al.; 16S rRNA, isolated from Escherichia coli or synthesized in vitro, is methylated by tRNA (m5U54)-methyltransferase (RUMT) and S-adenosyl-L-methionine to give ribothymidine (m5U) . By methylation studies of 16S rRNA fragments, nearest-neighbor analysis, and nuclease protection experiments, the site of methylation was identified as U788 . We have previously shown that the substrate consensus sequence for the T-arm of tRNA consists of a 2-5 base-pair stem and a 7-base loop, with certain constraints on base substitutions within the loop, and in the first two bases which close the loop {Gu, X., & Santi, D . V . (1991) Biochemistry 30, 2999-3002} . U788 of 16S rRNA is within a 9-base loop of a predicted stem-loop structure of 16S rRNA . If Ado substitution is allowed at the third and seventh positions of the loop and the first and ninth bases of the loop form an A-C base pair, the resulting stem-loop falls within the RUMT consensus sequence of the T-arm of tRNA . Individual mutants of the tRNA T-arm at these positions confirm that the substitutions are allowable, and expand the previous consensus sequence . Further, prevention of 7-base loop formation by requiring C-C base-pair formation at the loop closure abolishes substrate activity . RUMT forms a complex with Syn 16S rRNA which can be isolated on nitrocellulose filters or by SDS-PAGE electrophoresis . The enzyme also catalyzes exchange of tritium of {3H}Ura-16S rRNA for protons of water . By analogy with studies with tRNA {Gu, X., & Santi, D . V . (1991) Biochemistry 31, 10295-10302}, the mechanism of methylation is proposed to involve formation of a covalent, albeit reversible, Michael adduct with the target U788 of 16S rRNA.

Biochemistry, 1994 Mar 1, 33(8), 2171 - 7
Expression of house fly CYP6A1 and NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-metabolizing P450 system; Andersen JF et al.; The house fly (Musca domestica) cytochrome P450 gene CYP6A1 was expressed in Escherichia coli . The native protein was produced at a level of 0.25-0.34 mumol/L (15-20 mg/L) of culture with approximately 50% of the P450 being associated with the membrane fraction . The CYP6A1 protein was characterized spectrally and purified by a combination of hydrophobic interaction and hydroxyapatite chromatography . The house fly NADPH-cytochrome P450 reductase gene was also expressed in E . coli . Expression of a cytoplasmically directed reductase resulted in a protein that reduced cytochrome c but did not support P450 monooxygenase reactions . However, a periplasmically directed reductase was found to support monooxygenase reactions with CYP6A1 in a reconstituted system . The reconstituted system was effective in the epoxidation of the cyclodiene insecticides aldrin and heptachlor, with turnover rates of 12 and 34 min-1, respectively . The enzyme showed little detectable activity in the O-dealkylation and N-dealkylation of various compounds that are metabolized by house fly microsomes . Incubation with polyclonal antisera raised against purified CYP6A1 inhibited the microsomal epoxidation of heptachlor by 65% . Under the same conditions, the metabolism of 7-methoxy-4-methylcoumarin was inhibited only slightly . The results suggest that CYP6A1 is a major cyclodiene epoxidase in the house fly and that multiple P450 forms are responsible for the elevated monooxygenase activities in insecticide-resistant flies.

Biochemistry, 1994 Mar 1, 33(8), 2042 - 7
GTPase activation of ATP sulfurylase: the mechanism; Liu C et al.; ATP sulfurylase from Escherichia coli K12 catalyzes two, coupled reactions: the hydrolysis of GTP and the formation of activated sulfate (APS) . At saturating levels of GTP, the initial rate of APS formation is stimulated 116-fold . The mechanism of this activation has been investigated using isotope trapping, mass spectrometry, and initial velocity kinetic techniques . In the presence of GTP, APS formation proceeds via nucleophilic attack of sulfate at the alpha-phosphoryl group of ATP . Isotope-trapping experiments demonstrate productive, random binding of ATP and GTP . ATP is hydrolyzed to yield AMP and PPi . AMP production requires GTP and is suppressible by sulfate, suggesting GTP-dependent formation of an E*AMP intermediate in the synthesis of APS . Studies using the hydrolysis-resistant nucleotide analogues AMPCPP and GMPPNP demonstrate that GTP hydrolysis precedes scision of the alpha-beta bond of ATP . Product inhibition studies indicate that PPi release occurs prior to the addition of sulfate and APS formation . These results are used to construct a proposed mechanism for the GTP-activated synthesis of APS.

Biochemistry, 1994 Mar 1, 33(8), 2033 - 41
Characterization of an Arabidopsis calmodulin-like domain protein kinase purified from Escherichia coli using an affinity sandwich technique; Binder BM et al.; A full-length cDNA encoding a calcium-dependent protein kinase with a calmodulin-like domain from Arabidopsis thaliana (AK-1 for Arabidopsis kinase-1) has been expressed as a fusion protein (called AK-1-6H) in Escherichia coli and purified to near homogeneity with high specific activity (typically 2000 nmol min-1 mg-1) using an "affinity sandwich" technique . AK-1-6H protein phosphorylation activity using histone as substrate was stimulated up to 50-fold by the addition of calcium alone or up to 5-fold by the addition of specific phospholipids alone; together calcium and these lipids acted synergistically to give up to 100-fold stimulation . We earlier reported that, of a wide array of lipids tested, only phosphatidylinositol and lysophosphatidylcholine stimulated histone phosphorylation by AK-1 {Harper, J . F., Binder, B . M., & Sussman M . R . (1993) Biochemistry 32, 3282-3290} . The properties of lipid stimulation were further explored by testing the effects of lipids on autophosphorylation and on other catalytic properties of the kinase . Although phosphatidylinositol stimulated autophosphorylation up to 11-fold, lysophosphatidylcholine was inactive . Basic peptides such as polylysine (average M(r) approximately 37,100) were potent, mixed-type inhibitors of AK-1-6H with an IC50 of 2 nM . In the presence of phosphatidylinositol, the inhibition was reduced and the IC50 for polylysine was increased to 341 nM . As with autophosphorylation, lysophosphatidylcholine was inactive in alleviating the basic peptide inhibition, which suggests that this lipid's stimulatory effects using exogenous substrate are distinct from those of phosphatidylinositol . These results are consistent with a model in which phosphoinositides directly interact with the kinase protein and alleviate a catalytic block caused by basic charges.

Biochemistry, 1994 Mar 1, 33(8), 2011 - 20
An anion binding site in human aldose reductase: mechanistic implications for the binding of citrate, cacodylate, and glucose 6-phosphate; Harrison DH et al.; Aldose reductase is a NADPH-dependent aldo-keto reductase involved in the pathogenesis of some diabetic and galactosemic complications . The published crystal structure of human aldose reductase {Wilson et al . (1992) Science 257, 81-84} contains a hitherto unexplained electron density positioned within the active site pocket facing the nicotinamide ring of the NADPH and other key active site residues (Tyr48, His110, and Cys298) . In this paper we identify the electron density as citrate, which is present in the crystallization buffer (pH 5.0), and provide confirmatory evidence by both kinetic and crystallographic experiments . Citrate is an uncompetitive inhibitor in the forward reaction with respect to aldehyde (reduction of aldehyde), while it is a competitive inhibitor with respect to alcohol in the backward reaction (oxidation of alcohol), indicating that it interacts with the enzyme-NADP(+)-product complex . Citrate can be replaced in the crystalline enzyme complex by cacodylate or glucose 6-phosphate; the structure of each of these complexes shows the specific molecule bound in the active site . All of the structures have been determined to a nominal resolution of 1.76 A and refined to R-factors below 18% . While cacodylate can be bound within the active site under the crystallization conditions, it does not inhibit the wild-type enzyme in solution . Glucose 6-phosphate, however, is a substrate for aldose reductase . The similar location of the negative charges of citrate, cacodylate, and glucose 6-phosphate within the active site suggests an anion-binding site delineated by the C4N of nicotinamide, the OH of Tyr48, and the N epsilon of His110 . The location of citrate binding in the active site leads to a plausible catalytic mechanism for aldose reductase.

Biochemistry, 1994 Mar 1, 33(8), 2004 - 10
Human immunodeficiency virus type 1 protease inhibitors: evaluation of resistance engendered by amino acid substitutions in the enzyme's substrate binding site; Sardana VV et al.; The human immunodeficiency virus type 1 (HIV-1) protease is a homodimeric aspartyl endopeptidase that is required for virus replication . A number of specific, active-site inhibitors for this enzyme have been described . Many of the inhibitors exhibit significant differences in activity against the HIV-1 and HIV type 2 (HIV-2) enzymes . An initial study was conducted to ascertain the HIV-1 protease's potential to lose sensitivity to several test inhibitors while retaining full enzymatic activity . The substrate binding sites of the HIV-1 and HIV-2 enzymes are almost fully conserved, except for four amino acid residues at positions 32, 47, 76, and 82 . Accordingly, recombinant mutant type 1 proteases were constructed that contained the cognate type 2 residue at each of these four positions . The substitution at position 32 resulted in a significant adverse effect on inhibitor potency . However, this substitution also mediated a noted increase in the Km of the substrate . Individual substitutions at the remaining three positions, as well as a combination of all four substitutions, had very little effect on enzyme activity or inhibitor susceptibility . Hence, the four studied active site residues are insufficient to be responsible for differences in inhibitor sensitivity between the HIV-1 and HIV-2 proteases and are unlikely to contribute to the generation of inhibitor-resistant mutant HIV-1 protease.

Biochemistry, 1994 Mar 1, 33(8), 1994 - 2003
Site-directed mutagenesis of a catalytic antibody: an arginine and a histidine residue play key roles; Stewart JD et al.; Individual residues important for ligand binding and catalytic activity were identified by computer modeling and investigated by site-directed mutagenesis for catalytic antibody 43C9, which accelerates amide hydrolysis by a factor of 10(6) . On the basis of a computer model, Tyr L32, His L91, Arg L96, His H35, and Tyr H95 were chosen for replacement by site-directed mutagenesis . To facilitate these studies, an expression system was developed in which properly folded 43C9 single-chain antibody was secreted from an engineered Escherichia coli host . Substitution of His L91 by Gln produced a mutant with no catalytic activity, but whose affinities for ligands were nearly the same as those of the wild-type, identifying His L91 as the nucleophile that forms the acyl intermediate implicated by previous kinetic studies . Arg L96 is also critical for catalytic activity and appears to function as a oxyanion hole for the tetrahedral transition states . Two substitutions for His H35 resulted in mutant proteins with no catalytic activity as well as altered affinities for ligands, indicating an important structural role for this residue . Substitutions for Tyr L32 and Tyr H95 were made in an attempt to improve the catalytic efficiency of 43C9 . The results of these mutations allow us to propose a mechanism for 43C9-catalyzed hydrolysis: Substrate binding to 43C9 orients the scissile carbonyl group adjacent to both the His L91 and Arg L96 side chains . The imidazole of His L91 acts as a nucleophile, forming an acyl-antibody intermediate that breaks down by hydroxide attack to afford the products and regenerate the catalyst.

Mol Cell Biol, 1994 Mar, 14(3), 2140 - 6
Large exon size does not limit splicing in vivo; Chen IT et al.; Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases . It has been proposed that this limitation may derive from the exon definition model of splice site recognition . In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon . This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact . To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced . DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus . PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides . A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution . Central exons as large as 1,400 bases could be spliced into mRNA . We also tested individual plasmid clones containing exon inserts of defined sizes . The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons . We conclude that a limitation in exon size is not part of the exon definition mechanism.

Mol Cell Biol, 1994 Mar, 14(3), 1669 - 79
Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system; Resnitzky D et al.; Conditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor . After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition . We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts . We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity . Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence . However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition . Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E . These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence.

J Exp Med, 1994 Mar 1, 179(3), 993 - 8
A diarrheal pathogen, enteropathogenic Escherichia coli (EPEC), triggers a flux of inositol phosphates in infected epithelial cells; Foubister V et al.; Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes diarrhea in infants by adhering to intestinal epithelial cells . EPEC induces host cell protein phosphorylation and increases intracellular calcium levels that may function to initiate cytoskeletal rearrangement . We found that EPEC triggers the release of inositol phosphates (IPs) after adherence of bacteria to cultured epithelial cells . We also demonstrated that the EPEC-induced flux of IPs precedes actin rearrangement and bacterial invasion . EPEC mutants and tyrosine protein kinase inhibitors were used to establish that formation of IPs is dependent on tyrosine phosphorylation of a 90-kD HeLa protein . Collectively these results suggest that EPEC-induced tyrosine phosphorylation of a host cell substrate(s) leads to release of IPs, which may then trigger cytoskeletal rearrangement.

J Bacteriol, 1994 Mar, 176(5), 1468 - 74
Isolation of anaerobic respiratory mutants of Shewannella putrefaciens and genetic analysis of mutants deficient in anaerobic growth on Fe3+; DiChristina TJ et al.; A genetic approach was used to study (dissimilatory) ferric iron (Fe3+) reduction in Shewanella putrefaciens 200 . Chemical mutagenesis procedures and two rapid plate assays were developed to facilitate the screening of Fe3+ reduction-deficient mutants . Sixty-two putative Fe3+ reduction-deficient mutants were identified, and each was subsequently tested for its ability to grow anaerobically on various compounds as sole terminal electron acceptors, including Fe3+, nitrate (NO3-), nitrite (NO2-), manganese oxide (Mn4+), sulfite (SO3(2-)), thiosulfate (S2O3(2-)), trimethylamine N-oxide, and fumarate . A broad spectrum of mutants deficient in anaerobic growth on one or more electron acceptors was identified . Nine of the 62 mutants (designated Fer mutants) were deficient only in anaerobic growth on Fe3+ and retained the ability to grow on all other electron acceptors . These results suggest that S . putrefaciens expresses at least one terminal Fe3+ reductase that is distinct from other terminal reductases coupled to anaerobic growth . The nine Fer mutants were conjugally mated with an S . putrefaciens genomic library harbored in Escherichia coli S17-1 . Complemented S . putrefaciens transconjugants were identified by the acquired ability to grow anaerobically on Fe3+ as the sole terminal electron acceptor . All recombinant cosmids that conferred the Fer+ phenotype appeared to carry a common internal region.

J Bacteriol, 1994 Mar, 176(5), 1383 - 9
Role of the delta subunit in enhancing proton conduction through the F0 of the Escherichia coli F1F0 ATPase; Monticello RA et al.; We studied the effect of the delta subunit of the Escherichia coli F1 ATPase on the proton permeability of the F0 proton channel synthesized and assembled in vivo . Membranes isolated from an unc deletion strain carrying a plasmid containing the genes for the F0 subunits and the delta subunit were significantly more permeable to protons than membranes isolated from the same strain carrying a plasmid containing the genes for the F0 subunits alone . This increased proton permeability could be blocked by treatment with either dicyclohexyl-carbodiimide or purified F1, both of which block proton conduction through the F0 . After reconstitution with purified F1 in vitro, both membrane preparations could couple proton pumping to ATP hydrolysis . These results demonstrate that an interaction between the delta subunit and the F0 during synthesis and assembly produces a significant change in the proton permeability of the F0 proton channel.

J Bacteriol, 1994 Mar, 176(5), 1332 - 8
Inversions and deletions generated by a mini-gamma delta (Tn1000) transposon; Wang G et al.; Intramolecular transposition by an engineered derivative of the transposon gamma delta (Tn1000) is described . This 1-kb element contains inverted repeats of the 40 bp of the delta end of gamma delta, bracketing a kan gene, but it contains no resolution site . Transposition was analyzed in two plasmids; one contained two contraselectable (conditional lethal) genes (thyA and sacB) adjacent to the mini-gamma delta element in a 13.0-kb pBR322/pUC-based two-component plasmid (a heterodimer), and the other contained a different contraselectable gene (strA {rpsL}) in a 13.2-kb three-component plasmid (a heterotrimer) . Selection for loss of function of a single contraselectable gene yielded inversions and deletions . Each inversion plasmid was 1 kb larger than the parent plasmid: it had a second copy of mini-gamma delta inserted in the contraselected gene, with that copy plus the intervening segment inverted, and the 5-bp target site duplicated . Each deletion plasmid was smaller than the parent plasmid and had a deletion that extended from one transposon end into or through the contraselected gene for distances of up to 9.4 kb . The frequencies of deletions versus inversions ending in a single target gene were similar, although overall, deletions outnumbered inversions because deletions, but not inversions, into sites beyond the contraselected gene inactivate it . This work also demonstrates that thyA (which encodes thymidylate synthetase) is a useful contraselectable marker.

J Bacteriol, 1994 Mar, 176(5), 1309 - 15
A distant upstream site involved in the negative regulation of the Escherichia coli ompF gene; Huang KJ et al.; The two-component regulatory system, OmpR-EnvZ, of Escherichia coli K-12 regulates the expression of the major outer membrane porin protein, OmpF . OmpR is a DNA-binding protein which acts as both an activator and a repressor to control ompF transcription . In this article, we describe a new OmpR-binding site that is located between 384 to 351 bp upstream from the ompF start point of transcription . Inactivation of this site by insertion of a 22-bp fragment prevents the repression of ompF expression conferred by the dominant negative mutation, envZ473 . On the basis of the location of this binding site, the presence of bent DNA in the ompF regulatory region (T . Mizuno, Gene 54:57-64, 1987), and the fact that mutations altering integration host factor result in constitutive ompF expression (P . Tsui, V . Helu, and M . Freundlich, J . Bacteriol . 170:4950-4953, 1988), we propose that the negative regulation of ompF involves a DNA loop structure.

J Bacteriol, 1994 Mar, 176(5), 1297 - 302
The delta (argF-lacZ)205(U169) deletion greatly enhances resistance to hydrogen peroxide in stationary-phase Escherichia coli; Volkert MR et al.; In this study, we demonstrate that a strain bearing the delta (argF-lacZ)205(U169) deletion exhibits a high level of resistance to hydrogen peroxide compared with its undeleted parent . Our initial investigation of the mechanism behind the observed differences in peroxide resistance when parent and mutant strains are compared indicates that the parent strain carries a region near argF that is responsible for the H2O2-sensitive phenotype, which we have named katC . The H2O2 resistance phenotype of the delta katC {delta (argF-lacZ)205(U169)} mutant strain can be duplicated by Tn9 insertion in a specific locus (katC5::Tn9) which maps near argF . The increased H2O2 resistance of the delta katC and katC5::Tn9 mutant strains can be seen only when cells are grown to stationary phase; exponential-phase cells are unaffected by the presence or absence of katC . This H2O2 resistance mechanism requires functional katE and katF genes, which suggests that the mechanism of H2O2 resistance may involve the activity of the stationary-phase-specific catalase HPII . Cloning, DNA sequencing, and analysis of the katC5::Tn9 insertion allele in comparison with its parent allele implicate two insertion elements, IS1B and IS30B, and suggest that their presence sensitizes parent cells to H2O2.

Infect Immun, 1994 Mar, 62(3), 961 - 7
Extracellular and cytoplasmic CuZn superoxide dismutases from Brugia lymphatic filarial nematode parasites; Tang L et al.; We have isolated full-length cDNAs encoding two distinct types of CuZn superoxide dismutases (SODs) from the filarial nematode parasite Brugia pahangi . The derived amino acid sequences suggested that one class of cDNAs represented a cytoplasmic form of SOD and the second class represented an extracellular (EC) variant . The predicted proteins were highly homologous to each other, but the sequence of the latter contained an additional 43 residues at the N terminus, the first 16 of which were markedly hydrophobic, and four potential sites for N-linked glycosylation . Western blotting (immunoblotting) with an antiserum to a partial SOD expressed in Escherichia coli revealed two proteins with estimated molecular masses of 19 and 29 kDa . Digestion with N-glycanase indicated that the latter protein corresponded to the EC form, as it possessed N-linked oligosaccharide chains at three sites, leaving a peptide backbone with an estimated molecular mass of 22 kDa, which was consistent with the additional 27 amino acids predicted from the cDNA sequence . Gel filtration indicated that both enzymes were dimeric in their native forms, in contrast to the human EC-SOD, which is tetrameric . Comparison of the primary structure of the parasite EC-SOD with that of the human EC enzyme revealed two major differences: the N-terminal extension of the parasite enzyme was shorter by 25 residues, and it also lacked the C-terminal charged extension which mediates binding to cell surface sulfated proteoglycans . Lavage of Mongolian jirds infected intraperitoneally with Brugia malayi resulted in the recovery of filarial CuZn SODs, principally the EC form, indicating that this form of SOD is secreted in vivo . This EC enzyme may contribute to parasite persistence by neutralizing superoxide generated by activated leukocytes, thus acting as both an antioxidant and an anti-inflammatory factor.

Infect Immun, 1994 Mar, 62(3), 936 - 42
Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus; Moser M et al.; A 32-kDa nonglycosylated alkaline protease (EC 3.4.1.14) with elastolytic activity, secreted by the opportunistic pathogen Aspergillus fumigatus ATCC 42202, is suggested to be a virulence factor of this fungus . The enzyme is a serine protease of the subtilisin family, and its cDNA nucleotide sequence has recently been reported . We have cloned the cDNA encoding the mature protease into a high-level Escherichia coli expression plasmid and produced the recombinant protease as a fusion protein with a six-adjacent-histidine affinity tag at the carboxy terminus . Subsequently, the recombinant protease was purified to homogeneity, with affinity chromatography yielding 30 to 40 mg of recombinant protease per liter of E . coli culture . Refolded recombinant protease, in comparison with native protease, demonstrated weak enzymatic activity but similar immunochemical characteristics as analyzed by antigen-specific enzyme-linked immunosorbent assay (ELISA), competition ELISA, and immunoblotting assays . To assess the allergenic potential of the protease, sera from patients with allergic bronchopulmonary aspergillosis and sera from healthy control individuals were analyzed by ELISA and immunoblotting techniques . Sera from patients with allergic bronchopulmonary aspergillosis did not have protease-specific immunoglobulin E (IgE) antibodies and, remarkably, did not show significantly elevated protease-specific IgG antibody levels compared with those in sera from healthy control individuals . This suggests that the alkaline protease from A . fumigatus does not elicit IgE antibodies and has weak immunogenicity, a property which may explain fungus persistence in allergic individuals.

Infect Immun, 1994 Mar, 62(3), 904 - 9
Cloning, sequencing, and expression of the gene coding for an antigenic 120-kilodalton protein of Rickettsia conorii; Schuenke KW et al.; Several high-molecular-mass (above 100 kDa) antigens are recognized by sera from humans infected with spotted fever group rickettsiae and may be important stimulators of the host immune response . Molecular cloning techniques were used to make genomic Rickettsia conorii (Malish 7 strain) libraries in expression vector lambda gt11 . The 120-kDa R . conorii antigen was identified by monospecific antibodies to the recombinant protein expressed on construct lambda 4-7 . The entire gene DNA sequence was obtained by using this construct and two other overlapping constructs . An open reading frame of 3,068 bp with a calculated molecular mass of approximately 112 kDa was identified . Promoters and a ribosome-binding site were identified on the basis of their DNA sequence homology to other rickettsial genes and their relative positions in the sequence . The DNA coding region shares no significant homology with other spotted fever group rickettsial antigen genes (i.e., the R . rickettsii 190-, 135-, and 17-kDa antigen-encoding genes) . The PCR technique was used to amplify the gene from eight species of spotted fever group rickettsiae . A 75-kDa portion of the 120-kDa antigen was overexpressed in and purified from Escherichia coli . This polypeptide was recognized by antirickettsial antibodies and may be a useful diagnostic reagent for spotted fever group rickettsioses.

Infect Immun, 1994 Mar, 62(3), 874 - 9
Vaccine-specific T cells in human peripheral blood after oral immunization with an inactivated enterotoxigenic Escherichia coli vaccine; Wenneras C et al.; We have examined whether oral immunization of adult Swedish volunteers with a prototype enterotoxigenic Escherichia coli vaccine would induce antigen-specific T-cell responses in blood . Volunteers were given one to three doses of the whole-cell component of the vaccine, which consisted of formalin-inactivated bacteria expressing the fimbrial colonization factor antigens I and II . Following immunization, in vitro stimulation of blood mononuclear cells with the colonization factor antigens resulted in modest proliferative responses which were accounted for mainly by CD4+ T cells and, to a lesser extent, by CD8+ T cells . A main finding of this study was that a majority of the orally immunized volunteers had circulating T cells capable of producing large quantities of gamma interferon following in vitro exposure to either of the colonization factor antigens . No interleukin 2 production could be detected in the cell cultures . These results suggest that oral immunization of humans induces the migration of specific mucosal T immunocytes from the intestine into peripheral blood.

Infect Immun, 1994 Mar, 62(3), 1046 - 51
Cloning, sequencing, and expression of the Escherichia coli cytolethal distending toxin genes; Pickett CL et al.; A limited number of Escherichia coli isolates which produce an apparently novel toxin, termed cytolethal distending toxin (CDT), have been reported . The toxic activity produced by these strains causes certain cultured cell lines to become slowly distended and then disintegrate . DNA was isolated from the CDT-producing E . coli strain, 9142-88, and cloned into a cosmid vector . Plasmid DNA from a toxin-positive transductant was further subcloned until a plasmid with a 4-kb insert which still encoded the toxin activity was obtained . Nucleotide sequencing of a portion of this insert revealed the presence of three adjacent open reading frames . Further subcloning and deletion analysis suggested that the products of all three open reading frames may be required for toxin activity . Minicell experiments identified the products of all three open reading frames . The three proteins had predicted sizes of 27,753,29,531, and 19,938 Da, and all three appeared to have strong consensus leader sequences . None of the three predicted proteins had significant homology to known proteins.

Biochim Biophys Acta, 1994 Mar 1, 1217(2), 147 - 55
Mosquito large subunit ribosomal RNA: simultaneous alignment of primary and secondary structure; Kjer KM et al.; We report the sequence and propose a secondary structure for the cytoplasmic large subunit (5.8S and 28S) ribosomal RNA of the mosquito, Aedes albopictus, in an aligned format that incorporates secondary structure comparisons with Homo sapiens, Drosophila melanogaster, and Escherichia coli ribosomal RNAs . This format facilitates comparison of subtle differences between models, allowing nucleotide by nucleotide analysis at each position of discrepancy . Comparison of the A . albopictus large subunit ribosomal RNA gene with those from other species revealed new compensatory base changes . The aligned format focuses attention to the specific contribution of the A . albopictus sequence by facilitating comparison with the sequence of another dipteran, D . melanogaster . This is the second report of a complete large subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a member of the lower Diptera (Nematocera).

J Virol, 1994 Mar, 68(3), 1468 - 74
Activities of the feline immunodeficiency virus integrase protein produced in Escherichia coli; Vink C et al.; Retroviral DNA integration requires the activity of at least one viral protein, the integrase (IN) protein . We cloned and expressed the integrase gene of feline immunodeficiency virus (FIV) in Escherichia coli as a fusion to the malE gene and purified the IN fusion protein by affinity chromatography . The protein is active in site-specific cleavage of the viral DNA ends, DNA strand transfer, and disintegration . FIV IN has a relaxed viral DNA substrate requirement: it cleaves and integrates FIV DNA termini, human immunodeficiency virus DNA ends, and Moloney murine leukemia virus DNA ends with high efficiencies . In the cleavage reaction, IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack . In vitro, either H2O, glycerol, or the 3' OH group of the viral DNA terminus can serve as nucleophile in this reaction . We found that FIV IN preferentially uses the 3' OH ends of the viral DNA as nucleophile, whereas HIV IN protein preferentially uses H2O and glycerol as nucleophiles.

Chin Med Sci J, 1994 Mar, 9(1), 1 - 7
Molecular mutagenesis induced by glycidyl methacrylate; Gao H et al.; Glycidyl methacrylate (GMA) is a recently recognized mutagen . In order to explore the mutagenicity and mechanism of GMA, plasmid pBR322 was used for in vitro binding, mutant screening, restriction enzyme mapping, and DNA sequencing . To explore the mechanism by which an initial premutational event is converted into a stable heritable mutation, pBR322 and GMA-bound pBR322 were transformed into E . coli HB101, and the following results were obtained: 1) GMA-bound pBR322 induced phenotype changes in competent cells . Two stable and heritable mutants were isolated (ApRTcS and ApSTcR) . 2) When restriction enzyme mapping was used to analyze the mutant ApRTcS, four of seven maps showed changes, but no large DNA insertion or deletion were observed . 3) The frequency of deletion and insertion forms counted about 10% . Sequence specificity and hot spot regions were evident in the sequence analysis of mutated plasmid . The above results indicate that the premutagenic lesions of plasmid induced by GMA can be converted into point mutations in vivo.

Dev Comp Immunol, 1994 Mar-Apr, 18(2), 147 - 53
High in vitro endotoxin responsiveness of macrophages from an endotoxin-resistant wild rodent species, Sigmodon hispidus; Dabbert CB et al.; It has been reported that macrophages primarily mediate endotoxin shock and cell death by synthesizing and releasing cytokines, largely tumor necrosis factor (TNF) and interleukin-1 (IL-1) . However, macrophages from some laboratory mouse strains such as C3h/HeN are unresponsive to endotoxin both in vivo and in vitro . We found members of a wild rodent species, Sigmodon hispidus, to also be extremely resistant to bacterial endotoxin challenge . Intravenous administration of up to 100,000 micrograms/kg body mass of Escherichia coli O26:B6 endotoxin did not cause lethality in adult S . hispidus . In contrast to the endotoxin-resistant mouse strain, peritoneal macrophages derived from S . hispidus were responsive to in vitro endotoxin challenge as measured by high levels of TNF and IL-1 activity in supernatants of macrophage cultures . Thus, in vitro macrophage responsiveness to endotoxin does not always indicate high host sensitivity to endotoxin challenge.

Braz J Med Biol Res, 1994 Mar, 27(3), 627 - 36
Periplasmic trehalase from Escherichia coli--characterization and immobilization on spherisorb; Tourinho-dos-Santos CF et al.; 1 . Trehalase was partially purified from Escherichia coli and characterized . The Km for trehalose was 0.78 mM, the pH optimum 5.5 and the temperature optimum 30 degrees C . 2 . Trehalase represented approximately 50% of the total protein released by osmotic shock . The preparation was free of nonspecific carbohydrate hydrolases, which act on sucrose, galactose and maltose, permitting trehalose determination in biological samples, such as insect hemolymph and free cell extracts among others . 3 . The enzyme was stable in 50 mM maleate buffer, pH 6.2, at -8 degrees C for at least 6 months and could be used to determine trehalose in the range of 6 to 30 nmol . 4 . Immobilization of the enzyme was achieved by covalent linkage to spherisorb-5NH2 (spherical silica gel) . Retention of total catalytic activity averaged 32% . 5 . The reactor, stored for one month at -5 degrees C, retained 98% of its initial immobilized activity . 6 . This immobilized form of the enzyme could be used routinely for specific determinations of trehalose.

Agents Actions, 1994 Mar, 41(1-2), 111 - 3
Groups I, II and III extracellular phospholipases A2: selective inhibition of group II enzymes by indomethacin but not other NSAIDs; Lobo IB et al.; The three types (groups I, II and III) of stable extracellular 14 kDa phospholipase A2 enzymes differ in their primary amino acid sequences and their properties . It may thus be possible to design low-molecular weight inhibitors targeted to the secretory form of mammalian PLA2 . This enzyme has been implicated in inflammatory disorders . We have studied the inhibition of four distinct PLA2 enzymes by a range of NSAIDs, using 3H-oleate release from prelabelled membranes of E . coli for assay . The enzymes used were cobra venom PLA2 (Naja naja, a group I enzyme), bee venom PLA2 (Apis mellifera, group III), recombinant human synovial PLA2 (group II) and rat peritoneal PLA2 (group II) . Under the conditions of the 3H-oleate E . coli assay, 1 mM concentrations of aspirin, sodium salicylate, paracetamol (acetaminophen), oxphenbutazone, ibuprofen, flurbiprofen and nabumetone failed to inhibit significantly any of the four enzymes . However, indomethacin inhibited all four enzymes, although effects were greatest on the two group II enzymes (rat peritoneal and human synovial PLA2) . Approximate IC50 values were 28 and 35 microM, respectively . Inhibition by indomethacin was not time dependent and was greater at micromolar rather than millimolar levels of calcium . We conclude that indomethacin but not the other tested classes of NSAID inhibits the group II PLA2 enzyme in a selective manner and suggest that this may be relevant both to its clinical spectrum and to the design of novel pharmaceutical leads.

Mol Biochem Parasitol, 1994 Mar, 64(1), 75 - 86
A homologous recombination strategy to analyze the vinblastine resistance property of the V-circle in Leishmania; Wong AK et al.; The generation of extrachromosomal DNA elements in Leishmania sp . can occur naturally or during in vitro selection with drugs . Previously, we had established a strong association between V-circle amplification and drug resistance in L . enriettii stepwise selected with increasing concentrations of vinblastine . Further, we demonstrated the presence of the lemdr1 gene in the amplified V-circle and subsequent functional analysis by transfection of this gene alone confirmed its role in conferring a drug-resistant phenotype, but the level of resistance was significantly lower than in cell lines obtained from stepwise drug selection . The aim of this work was to determine if other genes either on the V-circle or elsewhere in the genome were necessary for expression of vinblastine resistance . We report here the development of a homologous recombination method to convert the entire V-circle from the LeV160 cell line into a shuttle vector and further the targeted disruption of specific sites within the V-circle . Our results clearly demonstrate that the V-circle alone is sufficient to confer full vinblastine resistance and the disruption of the lemdr1 locus destroys the ability of the V-circle to confer vinblastine resistance.

Mol Biochem Parasitol, 1994 Mar, 64(1), 43 - 54
Pyruvate kinase of Leishmania mexicana mexicana . Cloning and analysis of the gene, overexpression in Escherichia coli and characterization of the enzyme; Ernest I et al.; Leishmania mexicana mexicana contains two tandemly arranged genes for pyruvate kinase (PYK) . The 5' located gene codes for a polypeptide with a molecular mass of 54,370 . The calculated net charge and isoelectric point of the polypeptide are -6 and 6.5, respectively . Its amino-acid sequence is 73.7% identical to that of the Trypanosoma brucei PYK and 46.4-49.8% to the enzyme of mammalian cells . The second gene appears not to be functional, because its 5' and 3' extremities have undergone recombinations . L . m . mexicana PYK has been overexpressed in Escherichia coli, using a T7 expression system . Approximately 30% of the protein was detected in the soluble cell fraction . It has been highly purified by chromatography over DEAE-Sephacel and Affigel Blue . From a 1-1 culture 6 mg enzyme was obtained with a specific activity of 224 units mg-1 . The protein has a subunit molecular mass of 59,000, as determined by SDS/PAGE, and an isoelectric point of 5.9 . Some kinetic properties of the enzyme have been measured and compared with those reported for the T . brucei enzyme . The kinetics of both enzymes are very similar, the most important aspect being their activation by fructose 2,6-bisphosphate . Nevertheless, some differences were observed; the T . brucei enzyme is activated by the effector in a cooperative manner, whereas the activation of the L . m . mexicana enzyme is not cooperative.

Mol Biochem Parasitol, 1994 Mar, 64(1), 135 - 44
Characterization of the complete protein disulfide isomerase gene of Schistosoma mansoni and identification of the tissues of its expression; Finken M et al.; cDNA and genomic clones of Schistosoma mansoni containing the complete sequence of a homolog of protein disulfide isomerase have been identified . The protein disulfide isomerase gene in schistosomes is a single copy gene having a genomic structure that is very similar to that of man . The C-terminus of the deduced protein is KDEL which in mammals functions as a signal sequence for retention of luminal proteins in the endoplasmic reticulum . Immunohistology and in situ hybridization identify the gastrodermis of the gut, the wall cells of the protonephridia, and the sustentacular cells of the testes to be the major tissues of protein disulfide isomerase gene expression . The protein disulfide isomerase of schistosomes, produced in an expression vector in Escherichia coli, catalyzes disulfide/sulfhydryl isomerization in vitro.

Mol Biochem Parasitol, 1994 Mar, 64(1), 11 - 23
Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction; Heussler VT et al.; Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica . Five of the amplified F . hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type . Southern blot analysis suggests that some members of this protease gene family are present in multiple copies . Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases . The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe . The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli . Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F . hepatica . In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa . In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1 . In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.

Virus Genes, 1994 Mar, 8(2), 165 - 7
Nucleotide sequence of the coat protein coding region of tulip breaking virus; Ohira K et al.; cDNA of tulip breaking virus-tulip (TBV-tulip) RNA was synthesized and cloned in E . coli . One clone that contains a 4.5 kb insert was identified by restriction enzyme analysis, dot immunobinding assay (DIBA), and partial sequencing . Then 1479 nucleotides of the 3'-terminus of the clone were sequenced and revealed that the sequence contains one open reading frame (ORF), followed by an untranslated region of 255 nucleotides and a poly(A) tract . The deduced amino acid sequence was found to include the C terminus of the predicted RNA-dependent RNA polymerase and the coat protein . A glutamine-alanine dipeptide was identified as a putative NIa protease cleavage site at the N terminus of the coat protein.

Zentralbl Bakteriol, 1994 Mar, 280(4), 526 - 33
A recombinant immunoblot and ELISA for detection of acute parvovirus B19 infection; Schwarz TF et al.; Laboratory diagnosis of parvovirus B19 (B19) infection has been hampered by the limited availability of B19 virus . Recombinant viral proteins are now available for use as antigen in serological assays . We compared detection of anti-B19 IgM by "mu-capture assay" using viral B19 particles to a recombinant (rec.) immunoblot and a rec . enzyme-immunoassay (ELISA) using viral structural proteins as antigens expressed in E . coli . The rec . immunoblot was 94.3% sensitive and 96.4% specific for anti-B19 IgM, and the sensitivity of the rec . ELISA was 94.3% and the specificity, only 72.7% . There was an agreement between the "mu-capture assay" and the rec . immunoblot in 87.8% and the rec . ELISA in only 74.4% . For detection of anti-B19 IgG in patients with acute B19 infection, the rec . immunoblot was 94.3% and the rec . ELISA 85.7% sensitive . The rec . immunoblot is more reliable for detection of acute B19 infection than the rec . ELISA.

J Biochem (Tokyo), 1994 Mar, 115(3), 568 - 77
Construction of aminotransferase chimeras and analysis of their substrate specificity; Miyazawa K et al.; Escherichia coli aspartate aminotransferase (AspAT) and E . coli aromatic amino acid aminotransferase (AroAT) have almost identical and high activities toward acidic amino acid substrates . AroAT also has high activity toward aromatic amino acid substrates . The two proteins have 44% amino acid sequence homology . In order to study the mechanism responsible for the different substrate specificities of these aminotransferases, chimeric enzymes of AspAT and AroAT were constructed using homologous recombination in E . coli cells . Five chimeric enzymes were obtained, even though the nucleotide sequence homology between the two parent enzymes was as low as about 50% . The yields of the legitimate chimeric genes were related to the lengths of the homologous region between the two parent genes . Homologous recombination occurred in the region where more than eight nucleotides out of ten were identical . The substrate specificity of the chimeric enzymes suggest that not only the amino acid residues in the active site but also those distant from the active site contribute to the substrate specificity of the parental aminotransferases.

J Biochem (Tokyo), 1994 Mar, 115(3), 545 - 51
Overproduction, purification, and characterization of ferrochelatase from Escherichia coli; Miyamoto K et al.; To establish a system for overproduction of the ferrochelatase {EC 4.99.1.1} from Escherichia coli, a plasmid designated pFC3 was constructed . The 35-kDa protein was accumulated in E . coli DH5 alpha cells that harbored pFC3 to a level equal to approximately 9% of the total protein (roughly 50 mg/liter) upon thermal induction . This 35-kDa protein was identified as the ferrochelatase of E . coli by Western blotting and amino-terminal amino acid sequence analysis . The protein with ferrochelatase activity was purified from the cells by three simple steps with a yield of 17% . The optimum pH of the purified enzyme was around 8.0 . The molecular weight of the enzyme was estimated to be 35-kDa from column chromatography on Sephacryl S-300, a value consistent with that estimated from SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a monomer . The isoelectric point of the enzyme was approximately 4.7 . Determination of the far-ultraviolet circular dichroism spectrum allowed us to calculate the alpha-helix and beta-sheet contents of the enzyme as 10 +/- 0.2 and 39 +/- 0.2%, respectively . High-level production of the ferrochelatase from E . coli will greatly facilitate detailed structural analysis of this protein.

Mol Biol Cell, 1994 Mar, 5(3), 297 - 312
A role for autophosphorylation revealed by activated alleles of FUS3, the yeast MAP kinase homolog; Brill JA et al.; We have isolated dominant gain-of-function (gf) mutations in FUS3, a Saccharomyces cerevisiae mitogen-activated protein (MAP) kinase homolog, that constitutively activate the yeast mating signal transduction pathway and confer hypersensitivity to mating pheromone . Surprisingly, the phenotypes of dominant FUS3gf mutations require the two protein kinases, STE7 and STE11 . FUS3gf kinases are hyperphosphorylated in yeast independently of STE7 . Consistent with this, FUS3gf kinases expressed in Escherichia coli exhibit an increased ability to autophosphorylate on tyrosine in vivo . FUS3gf mutations suppress the signal transduction defect of a severely catalytically impaired allele of STE7 . This finding suggests that the tyrosine-phosphorylated form of FUS3 is a better substrate for activation by STE7 . Furthermore, these results imply that the degree of autophosphorylation of a MAP kinase determines its threshold of sensitivity to upstream signals.

J Physiol Pharmacol, 1994 Mar, 45(1), 69 - 80
Origin of circulating acute phase cytokines: modified proteins may trigger IL-6 production by macrophages . Preliminary report; Koj A et al.; Human peripheral blood monocytes isolated by centrifugation with Mono-Poly resolving medium, and human alveolar macrophages obtained by lung lavage during fiberoscopic bronchoscopy, were cultured in RPMI containing 2% foetal calf serum . The cultures were exposed to modified human proteins: alpha-1-antitrypsin cleaved with papain, fibrinogen degradation products (fraction D) purified from plasmin digest, and non-enzymatically glycosylated (glycated) serum albumin . Conditioned macrophage media were tested for the contents of acute phase cytokines by bioassay with hepatoma cells, and the concentration of interleukin-6 was determined with ELISA . Modified proteins stimulated macrophages to produce acute phase cytokines and the response was not abrogated by polymyxin B in distinction to stimulation of macrophages by endotoxin . Our data indicate that some proteolytically damaged proteins or the end glycosylation products formed in pathological states (acute inflammation, diabetes) may be responsible for the appearance of cytokines in the circulation.

J Biochem Biophys Methods, 1994 Mar, 28(2), 155 - 9
Rapid purification of antiserum against Mycoplasma hyopneumoniae by an efficient absorption method; Ro LH et al.; A simple and efficient method for the removal of unwanted cross-reactive antibodies has been developed . The antiserum purification method was based on treatment of the antiserum with both sonicated extracts and boiling extracts of the Escherichia coli host cells used in immunoscreening the lambda EMBL3 library . We have demonstrated unambiguously that through this simple treatment, the rabbit anti-Mycoplasma hyopneumoniae antiserum can be effectively purified so that the amount of antibodies cross-reacted with Escherichia coli lysate proteins is drastically reduced . Compared with the traditional absorption methods, which require the chemical coupling of an absorbing agent to an insoluble support, and affinity purification methods, which have harsh denaturing condition, this method should greatly facilitate a successful immunoscreening experiment.

Biochem Mol Biol Int, 1994 Mar, 32(4), 641 - 50
Active DNA topoisomerase II with minimum molecular mass from regenerating rat liver; Park SH et al.; We have purified the type II DNA topoisomerase from regenerating rat liver . The purified topoisomerase II migrated as two bands with molecular masses of 70 kDa and 55 kDa on SDS-PAGE . Immunoblotting analysis using antiserum against rat topoisomerase II gene product expressed in Escherichia coli suggested that the two bands on SDS-gel are proteolytic products of the intact 173 kDa form . However, these products retained the enzyme activities such as catenation and relaxation of supercoiled circular duplex monomer DNA and unknotting of knotted phage P4 DNA . These results suggest that DNA topoisomerase II consists of different functional domains and that the whole enzyme is not required for its activity . The activities of the purified enzyme were completely inhibited by 1 mM novobiocin, a bacterial gyrase inhibitor . However, no inhibitory effect was observed when another gyrase inhibitor, nalidixic acid was used.

Biochem Mol Biol Int, 1994 Mar, 32(4), 623 - 32
Interaction of EcoP1 modification methylase with S-adenosyl-L-methionine: a UV-crosslinking study; Krishnamurthy V et al.; EcoP1 modification methylase was radioactively labeled when incubated with S-adenosyl-L-{methyl-3H}methionine in the presence of ultraviolet light . Crosslinking of the enzyme as detected by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel followed by fluorography and autoradiography, was shown to be specific by a number of criteria . More importantly, EcoP1 modification methylase was also radioactively labeled with S-adenosyl-L-{carboxyl-14C}methionine demonstrating that labeling involved binding of the entire AdoMet molecule rather than methylation of the protein . Further, c2 EcoP1 mutant DNA modification methylases which show negligible or very little methylation activity, correspondingly formed a weak or no adduct upon crosslinking . These results suggest that photolabeling of EcoP1 DNA modification methylase occurs at the AdoMet binding site.

Actas Urol Esp, 1994 Mar, 18(3), 200 - 3
{Perirenal abscess following lithotripsy}; Martinez Sarmiento M et al.; Presentation of one case of perirenal abscess secondary to lithotrity on a calcified cyst simulating a pyelic lithiasis . A pathogenic hypothesis and the importance of urography in the confirmation diagnosis of the lithiasic disease is raised.

J Pharm Sci Technol, 1994 Mar-Apr, 48(2), 59 - 63
Destruction of challenged endotoxin in a dry heat oven; Nakata T; The amount and pyrogenicity of challenged endotoxin (derived from E . coli 055:B5, 10,000 endotoxin units, EU) processed in a dry heat oven at 200 degrees and 250 degrees C was measured and compared with the predicted amount and pyrogenicity calculated from destruction kinetics . The values for the assayed amount and pyrogenicity of the endotoxin processed in the dry heat oven at 200 degrees and 250 degrees C were fairly close to the predicted values . When the challenged endotoxin was exposed for 60 min in a dry heat oven at 200 degrees C, the amount of destruction did not reach a 3 log cycle reduction, although the pyrogenicity of the endotoxin was sufficiently reduced . However, at 250 degrees C for 30 min in the dry heat oven, the amount of endotoxin destroyed quickly reached a 3 log cycle reduction, and the pyrogenicity was quickly reduced to a sufficient level.

Biochem Mol Biol Int, 1994 Mar, 32(3), 537 - 43
Enhancement of expression of human granulocyte-macrophage colony stimulating factor by argU gene product in Escherichia coli; Hua Z et al.; Human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned into expression vector pIN III-ompA1 and expressed in Escherichia coli JA221 . When supplementation with a minor tRNA(AGA/AGG)Arg encoded by the E . coli argU gene, the expression level of hGM-CSF was raised about 3-4-fold, although there is only one rare AGG codon in hGM-CSF cDNA gene.

Bioconjug Chem, 1994 Mar-Apr, 5(2), 158 - 61
Synthesis of 2,6-diazido-9-(beta-D-ribofuranosyl)purine 3',5'-bisphosphate: incorporation into transfer RNA and photochemical labeling of Escherichia coli ribosomes; Wower J et al.; 2,6-Diazido-9-(beta-D-ribofuranosyl)purine was prepared by the reaction of 2,6-dichloro-9-(beta-D-ribofuranosyl)purine with sodium azide . The nucleoside was bisphosphorylated with pyrophosphoryl chloride to form 2,6-diazido-9-(beta-D-ribofuranosyl)purine 3',5'-bisphosphate . This product was labeled with 32P using T4 polynucleotide kinase to exchange the 5' phosphate with the gamma phosphate of {gamma-32P}ATP . When yeast tRNA(Phe) containing 2,6-diazido-9-(beta-D-ribofuranosyl)purine at the 3' terminus was bound to the P site of the Escherichia coli ribosome in the presence of poly(U) and irradiated with 300-nm light, the photoreactive tRNA derivative became cross-linked exclusively to the 50S subunit . The label was attached to proteins L27 and L33 as well as to the 23S rRNA.

Bioconjug Chem, 1994 Mar-Apr, 5(2), 126 - 32
Cysteine analogs of recombinant barley ribosome inactivating protein form antibody conjugates with enhanced stability and potency in vitro; Bernhard SL et al.; Antibody immunoconjugates were made with native and recombinant forms of the type-I ribosome inactivating protein from barley (BRIP) and with three recombinant BRIP (rBRIP) analogs engineered to contain a unique cysteine residue near the C terminus (at amino acid 256, 270, or 277) . rBRIP and all three cysteine analogs (rBRIPc256, rBRIPc270, and rBRIPc277) were produced in E . coli, with yields of soluble protein as high as 1 g/L, and were as active as native BRIP in inhibiting protein synthesis in vitro . Interestingly, the position of the engineered cysteine influenced not only the efficiency of conjugation to antibody but also the efficacy and disulfide bond stability of the immunoconjugates . Anti-CD5 antibody conjugates prepared with native and rBRIP were relatively inactive against antigen-positive target cells, while the conjugate made with rBRIPc277 was 5-fold more cytotoxic . Anti-CD7 antibody conjugates made with rBRIPc277 or rBRIPc270 also exhibited improved potency and stability compared to the conjugate with native BRIP . These results indicate that engineering a cysteine residue into selected positions near the C-terminus of a type-IRIP such as BRIP can improve immunoconjugate yield, disulfide bond stability, and potency.

Plasmid, 1994 Mar, 31(2), 158 - 65
Entry exclusion of the IncN plasmid pKM101 is mediated by a single hydrophilic protein containing a lipid attachment motif; Pohlman RF et al.; The eex gene(s) of pKM101, which mediates that plasmid's entry exclusion phenotype, is located within a cluster of tra genes that are thought to encode a mating bridge . We have determined the DNA sequence of the region containing eex, including the points of insertion of two flanking tra::Tn5 insertions . Sequence analysis of this interval strongly suggests that it contains only one gene . This gene is transcribed in a clockwise direction on the circular map of pKM101 and appears to be translationally coupled to neighboring tra genes . A Tn5 insertion that causes a null eex mutation disrupts this ORF . Computer analysis of the predicted eex protein suggests that it may be exported and covalently modified by the addition of lipid moieties . eex was amplified by polymerase chain reaction, subcloned, and fused to the Escherichia coli lac promoter . The resulting plasmid was sufficient to mediate entry exclusion . The degree of exclusion was greatly enhanced by overexpression of this gene.

Plasmid, 1994 Mar, 31(2), 111 - 20
Nucleotide sequence and replication characteristics of RepHI1B: a replicon specific to the IncHI1 plasmids; Gabant P et al.; The IncHI1 plasmids are multireplicon plasmids . They contain at least three autoreplicative regions, one of which is closely related to the RepFIA replicon of F . Two other IncHI1-specific replicons, RepHI1A and RepHI1B, have been recently isolated and mapped on the R27 (IncHI1) genome (P . Gabant, P . Newnham, D . Taylor, and M . Couturier, J . Bacteriol . 175, 7697-7701, 1993) . In the present work, the DNA sequence of RepHI1B was determined . It reveals DNA repeats of 17 base pairs located upstream and downstream from a gene coding for a 32 kilodalton protein (RepA) required for replication . Interestingly, RepA presents significant homology with other Rep proteins encoded by plasmids belonging to different incompatibility groups: P1 (IncY), Rts1 (IncT), RepFIB (IncFI) and RepHI1A (IncHI1) . All these results provide strong evidence that the RepHI1B replicon of the IncHI1 subgroup belongs to the group of plasmids which control their copy number by an iteron mechanism.

Circ Shock, 1994 Mar, 42(3), 147 - 53
Elevations in circulating calcitonin gene-related peptide correlate with hemodynamic deterioration during endotoxic shock in pigs; Arden WA et al.; Calcitonin gene-related peptide (CGRP) is a potent vasodilatory neuropeptide, which may play a role in vascular dysfunction during septic shock . Sixteen pigs (25-50 kg) were anesthetized with ketamine and isoflurane in O2, and administered 100 micrograms/kg Escherichia coli lipopolysaccharide i.v . (LPS; n = 8) or saline vehicle (n = 8) . Pigs were instrumented for hemodynamic determinations and blood sampling for CGRP assay (pg/ml) from the portal vein (PV) and the pulmonary (PA) and carotid (CA) arteries . Blood samples were collected into EDTA and aprotinin before (baseline) and at 60, 120, and 180 min after LPS administration . LPS caused significant deterioration in indices of hemodynamic function and a significant increase in plasma CGRP concentration at all sampling sites by 120 min (P < 0.01) . No significant difference between sampling sites was recorded at any time . Plasma CGRP concentrations displayed significant negative correlations with mean arterial pressure, cardiac index, and left ventricular stroke work . These data confirm our previous findings of CGRP elevations in endotoxemic rats, and indicate that 1) LPS is a potent stimulus for the systemic release of CGRP, 2) increasing plasma CGRP concentrations temporally correlates with cardiovascular deterioration during LPS shock, and 3) there is little evidence that the portal circulation is a major source of circulating CGRP levels during LPS shock . Vasoactive neuropeptides, such as CGRP, may interact with other documented mediators of vascular dysfunction in the pathogenesis of septic shock.

Circ Shock, 1994 Mar, 42(3), 115 - 20
Increased sinusoidal efflux of reduced and oxidized glutathione in rats with endotoxin/D-galactosamine hepatitis; Irita K et al.; The changes in the concentrations of reduced (GSH) and oxidized glutathione (GSSG) in the plasma as well as in the liver were investigated in rats with endotoxin hepatitis . Hepatitis was induced by intraperitoneal co-administration of small doses of Escherichia coli endotoxin and D-galactosamine . In the liver, the concentration of GSH decreased and that of GSSG increased 12 hr later . In the plasma taken from the right atrium, the concentration of both GSH and GSSG increased . The GSH/GSSG ratio in the plasma decreased, as it did in the liver . The net sinusoidal efflux of GSH and GSSG from the liver was calculated by subtracting their concentrations in plasma of the infrahepatic, suprarenal inferior vena cava from those of the suprahepatic inferior vena cava . The efflux started to increase as early as 2-4 hr after the injection of the toxins . In contrast, a leakage of alanine aminotransferase, an elongation of prothrombin time, an inhibition of starvation ketosis, and an increase in serum concentration of total bilirubin were detected as late as 6-8 hr after the injection . We conclude that endotoxin/D-galactosamine hepatitis induced an increase in plasma concentrations of GSH as well as GSSG by increasing the efflux of these peptides from the liver, and that changes in plasma glutathione status might be useful and sensitive markers for liver damage.

Biomed Environ Sci, 1994 Mar, 7(1), 25 - 34
Spectrum of glycidyl methacrylate-induced mutation in plasmid-Escherichia coli system; Gao HL et al.; In order to characterize the spectrum of mutation induced by glycidyl methacrylate (GMA), the plasmid pBR322 was modified with this mutagen in vitro, transfected into appropriate Escherichia coli host HB101 . The mutants were then screened and defined by DNA sequencing . Sequence analysis reveals that GMA induces two classes of mutations: deletion of the mono-, di- or tetra-base or the insertion of mono- or di-base . Both types of mutations, with about 10% frequency, occur predominantly at C-G runs and at 5'-CNCCN-3' sequence, which are hotspots for GMA damage and may cause frameshift mutation.

Mol Microbiol, 1994 Mar, 11(6), 999 - 1007
Structural dynamics of translating ribosomes: 16S ribosomal RNA bases that may move twice during translocation; Laughrea M; Recent footprinting, sedimentation and neutron-scattering results obtained in vivo or on pre-translocation and post-translocation ribosomal complexes are integrated with cross-linking and immunoelectron microscopy information . It is proposed that the 30S subunit pulses during translocation and that its pre- and post-translocation structures are not necessarily identical . Accordingly, translocation is characterized by three consecutive conformational states of the 30S and 50S subunits . State 1 (the pre-translocation state) lasts until the elongation factor EF-G.GTP complex binds to the ribosome or adopts the GTPase conformation . State 2 (the translocation state, or the peak or plateau of the pulse) follows and lasts until EF-G adopts a subsequent conformation or is released from the ribosome . State 3 (the post-translocation state) ensues and lasts until A (aminoacyl) site binding of aminoacyl-tRNA . In state 2, 16S RNA hairpins 26 and 33-33A, located in the platform and the head of the 30S subunit, respectively, become kinked or twisted, and residue A1503, near the decoding site, becomes exposed . A platform twist is associated with P (peptide) to E (exit) site tRNA movements and a head twist with pivoting of the peptidyl-tRNA elbow from the A to the P site, around a (retractable?) S19 domain . These twists result in an unlocking of the platform and the head from the 50S subunit . Exposure of A1503 is tentatively associated with movements of mRNA or tRNA anticodon stem-loops . These twisted or otherwise-exposed residues readopt their previous setting upon completion of translocation, i.e . states 1 and 3 of 16S RNA differ more from state 2 than from each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1994 Mar, 11(6), 983 - 90
Rho and RNA: models for recognition and response; Platt T; Escherichia coli Rho factor is required for termination of transcription at certain sites by RNA polymerase . Binding to unstructured cytosine-containing RNA target sites, subsequent RNA-dependent ATP hydrolysis, and an RNA-DNA helicase activity that presumably facilitates termination, are considered essential for Rho function . Yet the RNA recognition elements have remained elusive, the parameters relating RNA binding to ATPase activation have been obscure, and the mechanistic steps that integrate Rho's characteristics with its termination function in vitro and in vivo have been largely undefined . Recent work offers new insights into these interactions with results that are both surprising and satisfying in the context of Rho's emerging structure . These include the requirements for binding and ATPase activation by a variety of RNA substrates, dynamic analyses of Rho tracking, helicase and termination activity, and the participation of a new factor (NusG) that interacts with Rho . Models for Rho function are considered in the light of these recent revelations.

Mol Microbiol, 1994 Mar, 11(6), 1169 - 79
TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon; Mejean V et al.; The trimethylamine N-oxide (TMAO) respiratory system is subject to a strict positive control by the substrate . This property was exploited in the performance of miniMu replicon-mediated in vivo cloning of the promoter region of gene(s) positively regulated by TMAO . This region, located at 22 min on the chromosome, was shown to control the expression of a transcription unit composed of three open reading frames, designated torC, torA and torD, respectively . The presence of five putative c-type haem-binding sites within the TorC sequence, as well as the specific biochemical characterization, indicated that torC encodes a 43,300 Da c-type cytochrome . The second open reading frame, torA, was identified as the structural gene for TMAO reductase . A comparison of the predicted amino-terminal sequence of the torA gene product to that of the purified TMAO reductase indicated cleavage of a 39 amino acid signal peptide, which is in agreement with the periplasmic location of the enzyme . The predicted TorA protein contains the five molybdenum cofactor-binding motifs found in other molybdoproteins and displays extensive sequence homology with BisC and DmsA proteins . As expected, insertions in torA led to the loss of TMAO reductase . The 22,500 Da polypeptides encoded by the third open reading frame does not share any similarity with proteins listed in data banks.

Mol Microbiol, 1994 Mar, 11(6), 1159 - 68
Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent insertion element, IS 186; Craig JE et al.; Escherichia coli is known to contain more than one active transport system for nucleoside uptake . In the present study we report the sequence of a gene encoding a second nucleoside transport system, nupC (in addition to nupG) . An open reading frame (ORF) of 1200 bp was identified that codes for a hydrophobic polypeptide of 43,560 Da and an NupC fusion protein was shown to be membrane associated . The native NupC protein is also identified, following over-expression . NupC exhibits short regions of homology to several membrane-associated proteins, including LacY and Cyd . Analysis of the nupC promoter region revealed the presence of at least two putative CRP-binding sites, centred at -40bp and -89bp, which probably flank a CytR-binding site . In addition, an adjacent IS 186 element was identified and found to reside within a putative terminator structure, downstream from the nupC ORF . This arrangement is shown to reflect the previously established gene order on the E . coli chromosome.

Mol Microbiol, 1994 Mar, 11(6), 1059 - 71
Characterization of the carbon starvation-inducible and stationary phase-inducible gene slp encoding an outer membrane lipoprotein in Escherichia coli; Alexander DM et al.; Escherichia coli induces the expression of more than 50 proteins in response to starvation for a carbon source . Strains MC7 (csi7::phoA) and MC19 (csi19::phoA) contain fusions of a signal peptide-deficient phoA reporter sequence to a csi (carbon starvation-inducible) gene . PhoA expression increased when these strains were deprived of a carbon source or entered stationary phase but did not when the cells were deprived of a nitrogen source or subjected to osmotic, oxidative or thermal stress . Mapping and sequence analysis of the cloned phoA fusions in strains MC7 and MC19 indicated that they had occurred in different locations within the same previously unidentified gene . The wild-type allele of this gene was cloned and the encoded protein was found to be a new lipoprotein . Therefore we propose to call this locus slp (starvation lipoprotein) . The 22 kDa Slp protein is associated with the outer membrane fraction . The slp gene was located at 78.6 centisomes on the E . coli genetic map . The -10 and -35 regions upstream of the mRNA start site were characteristic of a sigma 70 promoter . The major transcript from this promoter was sufficiently large to contain slp sequences but not the downstream open reading frame . Induction of beta-galactosidase activity from a slp::lacZ translational fusion during carbon starvation or stationary phase was independent of cAMP, RpoS (KatF) and DnaK, all of which are known to affect the expression of certain starvation-inducible or stationary phase-inducible proteins.

Mol Microbiol, 1994 Mar, 11(6), 1029 - 43
Starvation-induced expression of SspA and SspB: the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation; Williams MD et al.; Maxicell labelling and two-dimensional gel electrophoresis (2-D PAGE) have identified the proteins encoded by sspA and sspB (SspA, SspB) as proteins D27.1 and A25.8, respectively, in the Escherichia coli gene-protein database . SspA expression increases with decreasing growth rate and is induced by glucose, nitrogen, phosphate or amino acid starvation . The promoter, Pssp, is similar to gearbox promoters . Inactivation of SspA (sspA::neo) blocks sspB expression . {35S}-methionine-labelled proteins synthesized during growth and during stationary phase are different in delta sspA strains compared to sspA+ strains . This difference is enhanced during extended stationary phase (24-72 h) . Long-term (10 d) viability of arginine-starved isogenic strains shows that sspA+ cultures remain viable significantly longer than delta sspA mutants . 2-D PAGE of proteins expressed during exponential growth shows that expression of at least 11 proteins is altered in delta sspA strains . A functional relA gene is required for sspA to affect protein synthesis.

Mol Microbiol, 1994 Mar, 11(6), 1019 - 28
Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS(katF) gene; Chang YY et al.; The activity of Escherichia coli pyruvate oxidase (PoxB) was shown to be growth-phase dependent; the enzyme activity reaches a maximum at early stationary phase . We report that PoxB activity is dependent on a functional rpoS(katF) gene which encodes a sigma factor required to transcribe a number of stationary-phase-induced genes . PoxB activity as well as the beta-galactosidase encoded by a poxB::lacZ protein fusion was completely abolished in a strain containing a defective rpoS gene . Northern and primer extension analyses showed that poxB expression was regulated at the transcriptional level and was transcribed from a single promoter; the 5' end of the mRNA being located 27 bp upstream of the translational initiation codon of poxB . The poxB gene was expressed at decreased levels under anaerobiosis; however, the anaerobic regulatory genes arcA, arcB or fnr were not involved in anaerobic poxB gene expression . Expression of the rpoS(katF) gene has been reported to be affected by acetate, the product of PoxB reaction . However, we found that poxB null mutations had no effect on rpoS(katF) expression . Inactivation of two genes involved in acetate metabolism, ackA and pta, had no effect on either poxB or rpoS(katF) expression.

Mol Microbiol, 1994 Mar, 11(6), 1009 - 17
The growth phase-dependent synthesis of cyclopropane fatty acids in Escherichia coli is the result of an RpoS(KatF)-dependent promoter plus enzyme instability; Wang AY et al.; The formation of cyclopropane fatty acids (CFAs) in Escherichia coli is a post-synthetic modification of the phospholipid bilayer that occurs predominantly as cultures enter the stationary phase of growth . The mechanism of this growth phase-dependent regulation of CFA synthesis was unclear, since log-phase and stationary-phase cultures had been reported to contain similar levels of the enzyme catalysing the reaction (CFA synthase) . We report that the timing of CFA synthesis can be explained by two unusual features . Fist, the gene encoding CFA synthase (cfa) was found to be transcribed from two promoters and the 5' ends of both transcripts were mapped by primer extension . One of the promoters was active only during the log-to-stationary phase transition and depended on the putative sigma factor encoded by the rpoS(katF) gene whereas the other promoter had a standard sigma 70 promoter consensus sequence and was expressed throughout the growth curve . Second, CFA synthase activity was shown to be unstable in vivo and a Cfa fusion protein was found to have a half life of < 5 min . The combination of these factors meant that, although CFA synthase was synthesized throughout the growth curve, a large increase in activity occurred during the log-to-stationary phase transition . As stationary phase progressed, the increased CFA synthase activity rapidly declined to the basal level . This transient increase in CFA synthase activity coupled with the cessation of net phospholipid synthesis in stationary phase provides an explanation for the unusual time course of CFA synthesis.

Mol Microbiol, 1994 Mar, 11(5), 965 - 82
Isolation and characterization of hypophosphite--resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter; Suppmann B et al.; Hypophosphite was used as a toxic analogue to identify genes whose products have a putative function in the transport of formate . Two Tn10-derived insertion mutants were identified that exhibited increased resistance to high concentrations of hypophosphite in the culture medium . The transposon was located in the identical position in the focA (formate channel; previously termed orf) gene of the pfl operon in both mutants . A defined chromosomal focA nonsense mutant, which showed minimal polarity effects on pfl gene expression, had the same phenotype as the insertion mutants . Results obtained using a hycA-lacZ fusion to monitor changes in the intracellular formate concentration in a focA mutant indicated that the level of formate inside the cell was elevated compared with the wild type . Moreover, it could be shown that there was a corresponding reduction of approximately 50% in the amount of formate excreted by a focA mutant into the culture medium . Taken together, these results indicate that formate accumulates in anaerobic cells which do not have a functional focA gene product and that one function of FocA may be to export formate from the cell . A further significant result was that hypophosphite could substitute for formate in activating hycA gene expression . This hypophosphite-dependent activation of hycA gene expression was reduced 10-fold in a focA null mutant, suggesting that hypophosphite must first enter the cell before it can act as a signal to activate hycA expression . By analogy, these data suggest that focA may also be functional in the import of formate into anaerobic Escherichia coli cells . Site-specific mutagenesis identified the translation initiation codon of focA as a GUG . Therefore, the FocA polypeptide has a molecular weight of 30,958 . FocA shows significant similarity at both the primary and secondary structural levels with the NirC protein of E . coli and the FdhC protein of Methanobacterium formicicum . All three proteins are predicted to be integral membrane proteins . A detailed in vivo TnphoA mutagenesis study predicted that FocA has six membrane-spanning segments.

Mol Microbiol, 1994 Mar, 11(5), 955 - 64
Anaerobic activation of arcA transcription in Escherichia coli: roles of Fnr and ArcA; Compan I et al.; The ArcA and Fnr regulators of Escherichia coli, both of which are activated in anaerobic conditions, negatively regulate the sodA gene (coding for manganese superoxide dismutase), but Fnr has no effect on anaerobic sodA expression in a delta arcA delta fnr background (Compan and Touati, 1993) . We show here that the sdh gene (coding for succinate dehydrogenase) is also negatively regulated by Fnr, but again Fnr exerts no control in a delta arcA background . One interpretation of these results is that Fnr activates arcA transcription . Using arcA-lac transcriptional and translational fusions, we show that arcA expression increases (about fourfold) in anaerobiosis and that both Fnr and ArcA are required for full expression . In a delta fnr background, there is no autoactivation, suggesting that ArcA enhances activation by Fnr . Transcript and sequence analyses reveal that the arcA upstream regulatory region lies within a 530 bp non-coding DNA fragment, which contains five putative promoter sequences and a putative Fnr-binding site . Identification of the transcription start sites indicates that transcription occurs in aerobiosis from three constitutive upstream promoters (Pe, Pd, Pc) . In anaerobiosis an additional completely Fnr-dependent transcript starting at Pa is present; expression from Pa is reduced in the absence of ArcA, and Fnr activation at Pa blocks the weak anaerobic-dependent expression from Pb . Fnr activation of arcA transcription may play an important role in the co-ordination of expression of genes associated with aerobic and anaerobic metabolism during environmental changes.

Mol Microbiol, 1994 Mar, 11(5), 913 - 8
Escherichia coli cad operon functions as a supplier of carbon dioxide; Takayama M et al.; We examined the gene expression of the Escherichia coli cad operon, which consisted of the genes cadB and cadA (lysine decarboxylase), using cells possessing cadB-lacZ fusion gene . The cad operon was expressed when O2 was limited, and the expression was optimal at pH 6.3 . The beta-galactosidase activity was lowered by the addition of sodium carbonate to the medium . The expression of the cad operon was reduced in cells containing the plasmid-encoding ornithine decarboxylase, which produced carbon dioxide, indicating that the gene expression of the cad operon was regulated by carbon dioxide (or its derivatives) . It is known that the Krebs cycle is a major pathway for producing carbon dioxide, and that its activity is repressed when O2 is limited . Thus, our present results suggested that the physiological role of the cad operon is to supply carbon dioxide when its internal level is lowered under O2-limiting conditions at a low pH.

Mol Microbiol, 1994 Mar, 11(5), 903 - 11
Open complex formation by DnaA initiation protein at the Escherichia coli chromosomal origin requires the 13-mers precisely spaced relative to the 9-mers; Hsu J et al.; The 245 bp chromosomal origin, oriC, of Escherichia coli contains two iterated motifs . Three 13-mers tandemly repeated at one end of the origin and four 9-mers in a nearby segment of oriC are highly conserved in enteric bacteria, as is the distance separating these two sequence clusters . Mutant origins were constructed with altered spacing of the 9-mers relative to the 13-mers . Loss or addition of even a single base drastically reduced replication, both in vivo and in vitro . Spacing mutant origins bound effectively to DnaA protein but failed to support efficient open complex formation . These results suggest that interaction with the 9-mers positions at least one subunit of DnaA to recognize directly the nearest 13-mer for DNA melting.






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